Sage Journals: Discover world-class research

Abstract

Background: Aluminum phosphide (AlP) poisoning is prevalent in numerous countries, resulting in high mortality rates. Phosphine gas, the primary agent responsible for AlP poisoning, exerts detrimental effects on various organs, notably the heart, liver and kidneys. Numerous studies have documented the advantageous impact of Coenzyme Q₁₀ (CoQ₁₀) in mitigating hepatic injuries. The objective of this investigation is to explore the potential protective efficacy of CoQ₁₀ against hepatic toxicity arising from AlP poisoning. Method: The study encompassed distinct groups receiving almond oil, normal saline, exclusive CoQ₁₀ (at a dosage of 100 mg/kg), AlP at 12 mg/kg; LD₅₀ (lethal dose for 50%), and four groups subjected to AlP along with CoQ₁₀ administration (post-AlP gavage). CoQ₁₀ was administered at 10, 50, and 100 mg/kg doses via Intraparietal (ip) injections. After 24 h, liver tissue specimens were scrutinized for mitochondrial complex activities, oxidative stress parameters, and apoptosis as well as biomarkers such as aspartate transaminase (AST) and alanine transaminase (ALT). Results: AlP induced a significant decrease in the activity of mitochondrial complexes I and IV, as well as a reduction in catalase activity, Ferric Reducing Antioxidant Power (FRAP), and Thiol levels. Additionally, AlP significantly elevated oxidative stress levels, indicated by elevated reactive oxygen species (ROS) production, and resulted in the increment of hepatic biomarkers such as AST and ALT. Administration of CoQ₁₀ led to a substantial improvement in the aforementioned biochemical markers. Furthermore, phosphine exposure resulted in a significant reduction in viable hepatocytes and an increase in apoptosis. Co-treatment with CoQ₁₀ exhibited a dose-dependent reversal of these observed alterations. Conclusion: CoQ₁₀ preserved mitochondrial function, consequently mitigating oxidative damage. This preventive action impeded the progression of heart cells toward apoptosis.

Keywords

phosphine mitochondrial dysfunction oxidative stress apoptosis

Introduction

AlP is a powerful solid insecticide that is widely used in the protection of stored grains. Although this insecticide is a dangerous agent for non-target organisms including humans, but its benefits such as cost-effectiveness and very good effectiveness on all life stages of insects outweigh its risks and it is widely used by farmers until today. Easy access to this substance in the Asian market has made it a widely used agent in suicide .¹ The cause of death of more than 70% of AlP-poisoned people is the toxic effects of this chemical on vital organs such as heart, liver and kidney. Notably, liver failure emerges as the predominant reason for mortality within the critical timeframe of 24 to 72 h post-AlP intoxication.² AlP releases very deadly phosphine gas under acidic condition or water in the stomach. The definite mechanism of phosphine toxicity is not known, but what is certain is that phosphine causes mitochondrial dysfunction and leads to damaged oxidative phosphorylation and a decrease in cellular energy level (ATP) and oxidative stress.³

Following previous research, phosphine gas (PH₃) elicits an elevation in ROS by impeding the electron transport chain, a primary source of free radicals. Furthermore, disruption of mitochondria induces the inhibition of complex IV and a reduction in the activities of complex I and II.^4,5 Additionally, phosphine contributes to increased oxidative stress by elevating ROS and diminishing catalase activity, FRAP, and Thiol levels. Some phenomena such as cytochrome c release from the mitochondrial intermembrane space into the cell cytoplasm medium are as a result of mitochondrial dysfunction.^6,7 These agents play a role in initiating the apoptosis pathway. Additionally, there exists a robust association between free radicals and the initiation of both inflammation and apoptosis.⁸

CoQ₁₀, a benzoquinone compound commonly referred to as ubiquinone, is widely distributed in plants, animals, and various body tissues, existing as a lipophilic agent. It is notably abundant in tissues such as muscles, liver, kidney, and heart, owing to their heightened energy demands.⁹ Within the realm of metabolism and mitochondria, CoQ₁₀ serves as an electron carrier during the electron transport chain, facilitating oxidative phosphorylation and adenosine triphosphate (ATP) production. Beyond the confines of the mitochondria, CoQ₁₀ functions as a potent lipophilic antioxidant, bestowing cellular protection against the generation of free radicals, and complementing the actions of other antioxidants.^10,11 Also, the presence of CoQ₁₀ fosters cell growth and hinders apoptosis.¹² Additionally, the outcomes of prior research highlighting some beneficial effects of CoQ₁₀ as antioxidant, anti-inflammatory agent, preventive of cell apoptosis processes, and other cytoprotective properties¹³ suggest the probability to counteract the mechanisms underlying liver disorder induced by AlP. Consequently, the primary objective of this study is to elucidate the favorable impacts of CoQ₁₀ on liver toxicity due to AlP effect using experimental models. It is noteworthy to say that, to the best of our knowledge, this investigation represents the first study focusing on the assessment of the effects of CoQ₁₀ on the AlP-induced hepatotoxicity.

Materials and methods

Ethics

Following Animal Care regulations, all procedures involving animals were conducted in accordance to the guidelines established by the Research Committee at AJA University of Medical Science, identified by the reference number IR.AJAUMS.REC.1402.023.

Chemicals

AlP tablets, known by their IUPAC name as alumanylidynephosphane, were prepared from Samiran Pesticide Formulating Co. in Iran. CoQ₁₀ was acquired from Sigma-Aldrich in Munich, Germany. ELISA kits for evaluating catalase activity were procured from Abcam in the USA. Kit for mitochondria isolation was sourced from BioChain Inc. in the USA. ELISA Kits for measuring AST and ALT were acquired from Pars Azmoon Inc. in Iran. The ApoFlowEx® fluorescein isothiocyanate (FITC) kit for the flow cytometry assay was supplied by Exbio in Vestec, Czech Republic. Various other chemicals utilized in the study were obtained from Merck or Sigma-Aldrich.

Animals

Male adult Albino Wistar rats weighing between 210 and 240 g were housed in polycarbonate cages in typical room settings, which included 20°C–24°C temperatures and 50–55% humidity. The rats were subjected to a 12 h dark/light cycle and provided with a standard rat diet along with access to water. The AJA ethics committee approved all experimental procedures, which were conducted in strict accordance with guidelines for animal care.

AlP LD50 calculation

The 50% lethal dose (LD50) of AlP has been reported to vary between 8.7 and 12.5 mg/kg in previous studies.^3,14–16 In the current study, an effort was made to determine the exact LD₅₀ dose of AlP. To this end, multiple doses of AlP, ranging from 6 to 14 mg/kg, were analyzed. The AlP tablets were crushed, and different amounts were dissolved in 1.5 mL of almond oil. Subsequently, the solutions were orally administered to rats in each cage, with six rats per cage. The rats were monitored for a duration of 24 h post-exposure, and the total number of deaths was recorded. Through the application of the Probit test, the LD₅₀ was computed as 12 mg/kg.

Treatment strategy

The 72 rats used in the animal study methodology were divided into six groups of twelve rats each, which were assigned at random to the following: Group 1 received only almond oil by gavage (Control); Group 2 received CoQ₁₀ alone at a dosage of 100 mg/kg via intraperitoneal injection (CoQ₁₀(100 mg)); Group 3 received AlP with LD₅₀ dose via gavage (AlPLD₅₀); Group 4 received AlP plus 10 mg/kg of CoQ₁₀ (AlP+CoQ₁₀(10 mg)); Group 5 received AlP plus 50 mg/kg of CoQ₁₀ (AlP+CoQ₁₀(50 mg)); and Group 6 received AlP plus 100 mg/kg of CoQ₁₀ (AlP+CoQ₁₀(100)). The rats in all groups were monitored for 24 h, with subsequent analysis focusing on six surviving rats. In the treatment groups, almond oil served as the solvent for both CoQ₁₀ and AlP. CoQ₁₀ was injected 30 min after aluminum gavage. The doses of AlP and CoQ₁₀, as well as the administration strategy for rat treatments, were determined based on a comprehensive literature review^17–20 and preliminary investigations conducted in our pilot study.

Serum and liver tissue sampling

After a 24 h period, six surviving treated rats were euthanized for sample collection. In the current study, blood samples were obtained directly from the heart using a syringe and transferred into heparinized tubes for subsequent analysis. The samples underwent immediate centrifugation at 3000 g for 5 min to separate and isolate the serum. Simultaneously, liver tissue was surgically isolated, thoroughly rinsed with refrigerated PBS, and then divided into multiple segments. With the exception of samples specifically designated for the analysis using flow cytometry technique, all collected specimens were promptly frozen at −80°C to ensure preservation until further analysis.

Hepatic serum enzyme evaluation

The levels of serum ALP and ALT were determined using the spectrophotometric method on a Hitachi 902 Automatic Analyzer in accordance with the protocols provided by the respective kits. The results are presented in international units per liter (IU/L).^21,22

Measurement of catalase activities

Catalase activities were evaluated by measuring the reaction of a probe with unconsumed H₂O₂ through a colorimetric assay,²³ with absorbance readings at 570 nm following the kit using instruction. Subsequently, catalase activities in the liver samples were assessed by employing a standard curve and subsequently expressed as units per milligram of tissue protein.²⁴

Measurement of ROS

The assessment of ROS levels involved the oxidation of DCFH-DA into DCF, employing a methodology previously outlined by Chen et al.²⁵ The quantification of ROS levels was performed utilizing a standard curve and reported as units per milligram of tissue protein.

Determination of FRAP

The FRAP (Ferric Reducing Antioxidant Power) test assesses the antioxidant capability of plasma based on its ability to reduce Fe³⁺ to Fe²⁺. The reagents utilized in this test include 300 mM acetate buffer (pH 3.6) containing 16 mL acetic acid per liter of the buffer solution, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mM HCl, and 20 mM FeCl₃. To prepare the working FRAP reagent, 25 mL of acetate buffer, 2.5 mL of TPTZ solution, and 2.5 mL of FeCl₃ solution are combined as required. Subsequently, 10 µl of the diluted sample in H₂O is freshly added to 300 mL of the reagent, preheated to 37°C. The formation of a complex between TPTZ and Fe²⁺ results in a blue color, and the absorbance is measured at 593 nm.²⁶

Total thiol measurement

Total Thiol (SH) was measured by mixing 200 µl of the sample with 600 µl of Tris-EDTA buffer (containing Tris base [0.25 M], EDTA [20 mM], pH 8.2) in a 10-ml test tube, then blending this mixture with 40 mL of DTNB (10 mM) in methanol. Then 3.16 mL of methanol was added to make a final volume of 4.0 mL. After that, the capped test tube was centrifuged at 3000 g for 10 min at room temperature. After 15–20 min of incubation, the color appeared, and the supernatant’s absorbance was measured at 412 nm.²⁷

Assessment of liver mitochondrial function

Assessment of mitochondrial complex I activity

The determination of Complex I activity was performed through spectrophotometry at 340 nm. This involved monitoring the rotenone-sensitive oxidation of NADH to NAD⁺ in the presence and absence of rotenone. The decrease in absorbance of NADH at 340 nm, reflecting the overall activity of Complex I, was measured over a 3 min duration using a spectrophotometer from BioTek® Instruments, Inc. (Winooski, USA). Enzyme activity was computed using an extinction coefficient of 6.22 mM⁻¹ cm⁻¹ at 340 nm and reported as μM NADH/min/mg mitochondrial protein.²⁸

Assessment of mitochondrial Complex II activity

The specific activity of Complex II was evaluated by measuring the reduction of 2,6-Dichlorophenolindophenol (DCPIP) through spectrophotometric analysis at 600 nm. Mitochondria were preincubated in a solution comprising potassium phosphate buffer, MgCl₂, and succinate. After the addition of antimycin A, rotenone, KCN, and DCPIP, the baseline was recorded for 3 min. The reaction was initiated with ubiquinone, and the enzyme-dependent reduction of DCPIP was measured for an additional 3–5 min at 600 nm. The activity of Complex II was then calculated using a DCPIP standard curve and expressed as μM DCIP/min/mg of mitochondrial protein.²⁹

Assessment of mitochondrial Complex IV activity

The reduction of cytochrome c was initiated by the addition of sufficient sodium hydrosulfite, followed by the introduction of mitochondrial protein and Lubrol-PX in potassium phosphate buffer. This sequence of additions initiated the reaction. Utilizing a previously established spectrophotometric method, the reduction of cytochrome c was monitored by measuring the decrease in optical absorption at 550 nm for a period of 3–6 min. The data were reported as the first-order rate constant (k) in min/mg of mitochondrial protein and were displayed as the natural logarithm of the absorbance divided by time.³⁰

Utilizing flow cytometry to examine necrosis and apoptosis

The liver tissue was first perfused with normal saline, and then hepatic cells were isolated using a previously described method. The single cells obtained were then stained using the kit’s instructions (annexin V-FITC for phosphatidylserine assessment in apoptotic cells, and propidium iodide [PI] for necrotic cells). Flow cytometry analysis was performed to examine the various cell populations.^31,32

Statistical analysis

All data are presented as the mean ± standard error of the mean (SEM). The one-way ANOVA test was used to determine statistical significance, and the post-hoc Tukey test was used for multiple comparisons. A p-value of less than 0.05 was deemed statistically significant.

Results

Oxidative stress parameters in liver tissue

Exposure to AlP resulted in a notable elevation in the generation of oxygen free radicals (ROS) within the liver tissue. According to the obtained findings, the administration of CoQ₁₀ led to a substantial reduction in the level of oxygen free radicals compared to the AlP group. Specifically, the ROS level significantly improved at the 50 mg/kg CoQ₁₀ dose compared to the AlP group, and at the 100 mg/kg dose, it exhibited no significant difference from the control group.

The catalase enzyme is recognized as a crucial antioxidant in the body for mitigating risks associated with oxidative stress. In animals from the AlP group, the activity of this enzyme significantly decreased in comparison to the control group that received only almond oil. Simultaneous administration of various CoQ₁₀ doses after AlP poisoning induction significantly enhanced catalase enzyme activity. Notably, at the 100 mg/kg CoQ₁₀ dose, catalase enzyme activity was comparable to that of the control group.

The level of Thiol in the liver tissue exhibited a significant decrease following exposure to AlP compared to the control group. However, in groups receiving different CoQ₁₀ doses after AlP poisoning induction, tissue Thiol demonstrated a dose-dependent increase compared to the AlP group. At the 100 mg/kg CoQ₁₀ dose, Thiol significantly improved compared to the AlP group but did not reach the level observed in the control group.

AlP exposure in the AlP group led to a reduction in the tissue antioxidant power index compared to the control groups (almond oil). Treatment with different CoQ₁₀ doses showed a notable difference compared to the AlP group, as CoQ₁₀ increased the FRAP in a dose-dependent and significant manner. The improvement in FRAP was particularly significant at various CoQ₁₀ doses, with the highest dose resulting in a significant enhancement compared to the AlP group, aligning closely with the levels observed in the control group (Figure 1).

Figure 1.

Changes oxidative stress parameters; ROS (A), Catalase activity (B), FRAP (C) and Total Thiol (D) in liver tissue. The data presented represent the mean ± SEM six animals in each group. The control group was administered almond oil. The Q₁₀ (100 mg) group received Q₁₀ at a dose of 100 mg/kg. The AlP (LD₅₀) group was administered AlP at a dose of 12 mg/kg. The AlP+Q₁₀ (10 mg) group received a combination of AlP and Q10 at doses of 10 mg/kg each. The AlP+Q10 (50 mg) group received a combination of AlP and Q₁₀ at doses of 50 mg/kg each. The AlP+Q₁₀ (100 mg) group received a combination of AlP and Q₁₀ at doses of 100 mg/kg. ^a Significantly different from control groups at p < 0.05. ^b Significantly different from AlP group at p < 0.05.

Mitochondrial liver activity

The analysis of the results revealed a significant reduction in the activity level of complexes I in the group exposed to AlP compared to the control group. CoQ₁₀ effectively counteracted this reduction, indicating that simultaneous consumption of CoQ₁₀ with AlP resulted in a diminished inhibitory effect of AlP on the activity of complex I in the respiratory chain. This was evident as the reduction in the inhibitory effect of AlP in the AlP+Q₁₀ (50 mg) and AlP+Q₁₀ (100 mg) groups was significantly different from the AlP group, although it remained significantly different from the Control group as well. Additionally, the administration of CoQ₁₀ alone did not exhibit a significant impact on the activity of complex I.

Regarding the activity of Complex II, the results showed that AlP did not exert a significant effect on its activity at different times. Furthermore, it was observed that CoQ₁₀, whether administered alone or in conjunction with AlP, did not influence the activity of complex II at the specified time points. In terms of the activity of Complex IV, exposure to AlP led to a notable decrease in its activity within the mitochondrial respiratory chain. This reduction was significant, indicating a considerable departure from the proportion observed in the control group. CoQ₁₀, however, acted as a preventive measure against the reduction in complex IV activity. The effect of increasing the activity of complex IV was particularly significant in the AlP+Q₁₀ (50 mg) and AlP+Q₁₀ (100 mg) groups compared to the AlP group. Interestingly, the administration of Q₁₀ (100 mg) alone did not yield a significant effect on the activity of complex IV (Table 1).

Table 1.

Impact of different treatments on the activity of mitochondrial complexes in liver tissue.

	Time (h)	Control	Q₁₀(100_mg)	AlP(LD₅₀)	AlP+Q₁₀(10_mg)	AlP+Q₁₀(50_mg)	AlP+Q₁₀(100_mg)
Complex I (nmol/min/mg)	24	329.72 ± 4.53	332.56 ± 5.82	207.22 ± 10.67	219.79 ± 7.97^a	244.49 ± 7.59^a,b	297.26 ± 7.68^a,b
Complex II (nmol/min/mg)	24	76.22 ± 4.81	77.31 ± 2.62	68.14 ± 8.41	71.47 ± 5.63	73.24 ± 6.86	75.97 ± 4.70
Complex IV (K/min/mg)	24	421.57 ± 4.13	425.62 ± 4.33	284.05 ± 10.33^a	310.96 ± 8.85^a	370.37 ± 6.19^a	403.10 ± 8.18^a,b

The data presented represent the mean ± SEM six animals in each group. The control group was administered almond oil. The Q₁₀ (100 mg) group received Q₁₀ at a dose of 100 mg/kg. The AlP (LD₅₀) group was administered AlP at a dose of 12 mg/kg. The AlP+Q₁₀ (10 mg) group received a combination of AlP and Q10 at doses of 10 mg/kg each. The AlP+Q10 (50 mg) group received a combination of AlP and Q₁₀ at doses of 50 mg/kg each. The AlP+Q₁₀ (100 mg) group received a combination of AlP and Q₁₀ at doses of 100 mg/kg.

^aSignificantly different from control groups at p < .05.

^bSignificantly different from AlP group at p < .05.

Apoptosis and necrosis detection using flow cytometry technique

The evaluation of hepatocyte live cells, necrotic cells and late apoptotic cells, through flow cytometry involved annexin V-FITC/PI staining. Flow cytometry data analysis for the control group revealed percentages of live cells (annexin V−/PI−) at 92.8%, late apoptotic cells (annexin V+/PI+) at 2.32%, and necrotic cells (annexin V−/PI+) at 4.23%. Conversely, phosphine significantly reduced hepatocyte viability to 77.3% and increased the rates of late apoptosis and necrosis to 12.6% and 6.29%, respectively. Co-administration of CoQ10 mitigated the aforementioned alterations. Co-treatment with CoQ10 at doses of 50 mg/kg and 100 mg/kg significantly increased live cells and markedly decreased late apoptotic cells. Early apoptosis in these groups was not significantly altered (data not shown) (Figure 2).

Figure 2.

Analysis of the cell viability with annexin-V/PI staining using by flow cytometry. The data presented represent the mean ± SEM six animals in each group. The control group was administered almond oil. The Q₁₀ (100 mg) group received Q₁₀ at a dose of 100 mg/kg. The AlP (LD₅₀) group was administered AlP at a dose of 12 mg/kg. The AlP+Q₁₀ (10 mg) group received a combination of AlP and Q10 at doses of 10 mg/kg each. The AlP+Q10 (50 mg) group received a combination of AlP and Q₁₀ at doses of 50 mg/kg each. The AlP+Q₁₀ (100 mg) group received a combination of AlP and Q₁₀ at doses of 100 mg/kg. ^a Significantly different from control groups at p < 0.05. ^b Significantly different from AlP group at p < 0.05.

Levels of AST and ALT in serum

ALT, as a specific liver enzyme, increases only in liver diseases. However AST level increases in liver parenchyma damage. Simultaneous measurement of AST and ALT is used to diagnose cardiac and muscle damage from liver damage. AST/ALT ratio is used in differential diagnosis of liver diseases. This study showed that phosphine significantly increases the level of these biomarkers compared to the control group. Simultaneous treatment with coenzyme CoQ₁₀ in 50mgkg and 100 mg/kg significantly reduced the increase of these biomarkers in the serum. Remarkably, at the dosage of 100 mg/kg CoQ₁₀, the decrease in AST levels was comparable to that observed in the control group (Figure 3).

Figure 3.

Effects of treatments on AST and ALT as liver Biomarkers. The data presented represent the mean ± SEM six animals in each group. The control group was administered almond oil. The Q₁₀ (100 mg) group received Q₁₀ at a dose of 100 mg/kg. The AlP (LD₅₀) group was administered AlP at a dose of 12 mg/kg. The AlP+Q₁₀ (10 mg) group received a combination of AlP and Q10 at doses of 10 mg/kg each. The AlP+Q10 (50 mg) group received a combination of AlP and Q₁₀ at doses of 50 mg/kg each. The AlP+Q₁₀ (100 mg) group received a combination of AlP and Q₁₀ at doses of 100 mg/kg. Serum AST levels (A) and ALT levels (B) were measured. ^a Significantly different from control groups at p < 0.05. ^b Significantly different from AlP group at p < 0.05.

Discussion

As highlighted in a literature review, hepatic injury stands out as a significant factor contributing to mortality in cases of phosphine intoxication.⁷ Phosphine is an extremely toxic gas emanating from metal phosphides such as AlP and zinc phosphide.³³ For this investigation, AlP was selected as the origin of this toxic gas, given that AlP poisoning is a prevalent cause of death globally, particularly in developing countries.³⁴ While phosphine toxicity has been extensively studied for its impact on acute cardiovascular injuries, limited research has delved into its toxic effects on the liver. Consequently, information is scarce regarding the mechanisms underlying its hepatic toxicity.

This current study aims to elucidate the mechanisms underlying phosphine-induced hepatotoxicity and investigate the potential protective role of CoQ₁₀ in a rat model. We relied on reputable articles in similar fields to select the sample size.^2,35 However, we did not perform a separate calculation for determining the sample size, which could be considered one of the limitations of this study. The LD₅₀ of AlP in the present study was determined to be 12 mg/kg. It is noteworthy that the continuous decomposition of AlP can have implications for its shelf life. Consequently, the LD₅₀ of this toxic chemical should be evaluated separately for each test due to potential variations influenced by factors such as storage conditions and duration. The mechanisms of hepatotoxicity induced by phosphine in this study are succinctly outlined in Figure 4.

Figure 4.

The mechanisms involved in PH₃ (phosphine) hepatotoxicity. Phosphine inhibited the activity of mitochondrial complexes I and IV, elevated ROS, and apoptosis, and decreased FRAP, Catalase activity, and Total Thiol and also increased AST and ALT level. ROS: reactive oxygen species; FRAP: Ferric Reducing Antioxidant Power; AST: aspartate transaminase; ALT: alanine transaminase.

Results of the present study reveal that AlP administration triggers hepatotoxicity by instigating oxidative stress, and apoptosis. Oxidative stress is characterized by a decline in antioxidant agents that counteract free radicals and an uncontrolled increase in the levels of free radicals within cells.^36,37 The majority of these free radicals are recognized as key contributors to hepatotoxicity.³⁸ The findings of the current study reveal that phosphine induces an upsurge in ROS levels, concomitant with a reduction in catalase activity, FRAP, and total thiol content, all of which serve as markers of oxidative stress in hepatic tissue. Furthermore, it is observed that these alterations are mitigated by CoQ₁₀. Previous studies have emphasized the pivotal role of oxidative stress in phosphine-induced toxicity across various tissues.³⁹ Free radicals have the capability to interact with cellular molecules, causing disruptions in cells and organelles, particularly in mitochondria. This interaction can initiate pathways such as apoptosis.⁴⁰ Under normal conditions, cells generate various types of oxygen free radicals, especially during processes such as oxygen utilization and metabolism within mitochondria.^41,42 The production of ROS in mitochondria takes place through the electron transport chain during oxidative phosphorylation. Mitochondrial respiratory chain complexes I and III are primarily responsible for ROS production. Uncoupling of the electron transport chain has been reported to significantly enhance the production of ROS.⁴³ Previous studies have provided evidence that one mechanism contributing to the elevation of ROS induced by phosphine involves a malfunction in certain mitochondrial respiratory chain complexes.^44,45 The study results indicated a significant increase in the production of ROS in the liver tissue of animals exposed to AlP compared to the control group. However, administration of CoQ₁₀ led to a notable reduction in ROS levels compared to the AlP group. Specifically, at a dose of 50 mg/kg, the ROS level exhibited a significant improvement compared to the AlP group, and at a dose of 100 mg/kg, the ROS level was not significantly different from the control group. Catalase, which is known as an important antioxidant enzyme of the body to deal with reducing the risks caused by oxidative stress, a widely distributed enzyme in living organisms and tissues, plays a crucial role in breaking down hydrogen peroxide (H₂O₂) into water and oxygen.⁴⁶ This enzyme is highly efficient, capable of degrading millions of H₂O₂ molecules in just one second.⁴⁷ The current study notes a decrease in catalase activity in hepatic tissue 24 h after exposure to phosphine. These results align with previous reports indicating that phosphine diminishes catalase activity.¹⁴ The suggested mechanism entails the phosphine and metal ion co-factors located at the active site of the catalase enzyme interactions. Simultaneous administration of different doses of CoQ₁₀ after induction of AlP poisoning significantly increased catalase enzyme activity. The augmentation observed was substantial to the extent that at a dosage of 100 mg/kg of CoQ₁₀, the catalase enzyme activity did not exhibit a significant variance compared to the control group.

Thiol compounds, characterized by sulphydryl groups, play a protective role against hepatic injury by interacting with RNS and ROS. Thiol compounds undergo one-electron oxidation, producing thiol radicals, thereby demonstrating their antioxidant properties through the trap of free radicals, metal ions chelators, and participation in the thiol/disulfide component of the redox buffer.⁴⁸ Both in vitro and in vivo evidence corroborates the utilization of thiols in oxidative stress situations. In this study, hepatic tissue thiol levels in animals exhibited a significant decrease following exposure to AlP when compared to the control group. However, in groups that received varying doses of CoQ₁₀ after the induction of AlP poisoning, tissue thiol levels showed a notable dose-dependent increase compared to the AlP-treated group. Specifically, at a dosage of 100 mg/kg, the tissue thiol levels exhibited a significant improvement compared to the AlP group. Nevertheless, despite this improvement, the thiol levels did not fully reach those observed in the control group by the conclusion of the study.

Research has indicated that complex IV serves as the primary site of phosphine interference with the electron transport chain (ETC), leading to the disruption of ATP levels and the cell energy requirement.⁴⁹ Phosphine appears to react with every enzyme and macromolecule containing a heme group, functioning as a non-specific inhibitor of cytochrome. The reduction of the heme structure in hemoglobin leads to the formation of methemoglobinemia, as evidenced by some previous findings. However, it is noteworthy that in the case of AlP poisoning, there was no notable decrease in complex II activity, as reported in previous studies.^50,51 Meanwhile, complex I and IV activity is notably diminished in the liver tissue of rats subjected to AlP poisoning.² Interestingly, the decrease in activities of complex I and IV was not observed in animals treated with CoQ₁₀. Moreover, these effects were reversed by CoQ₁₀ supplementation. The activity of complex I exhibited a parallel increase with the elevation of CoQ₁₀ concentration. Specifically, CoQ₁₀ at a dosage of 100 mg/kg led to a significant rise in complex I activity compared to the AlP-treated groups. Notably, CoQ₁₀ with 50 and 100 mg/kg doses increases the activity of complex IV in rats poisoned with AlP. During previous studies, we also found that CoQ₁₀ modulates mitochondrial function and biogenesis and increases mitochondrial complex activities.^13,52 Taken together, CoQ10 emerges as a mitochondrial-targeted therapeutic agent that offers robust protection against pathological conditions.⁵³

Apoptosis, a tightly regulated form of programmed cell death, serves a pivotal role in maintaining cellular homeostasis and ensuring cell viability.⁵⁴ This highly programmed process is physiologically crucial for shaping organs and tissues, as well as controlling cell numbers. Conversely, any aberrations in this process may contribute to tissue disorders.⁵⁵ Numerous studies have indicated that phosphine exposure enhances hepatocytes apoptosis. However, CoQ₁₀ exerts cytoprotective effects by mitigating apoptosis in various cellular contexts, including hepatocytes.⁵⁶ These findings align with other research indicating that phosphine induces apoptosis in various organs.⁵⁷ Furthermore, CoQ₁₀ has been demonstrated to mitigate apoptosis in several disorders, potentially through its antioxidant properties, anti-apoptotic effects, and its ability to alleviate mitochondrial dysfunction.⁵⁸

Furthermore, a flow cytometry technique was employed to evaluate the populations of survived, apoptotic, and necrotic cells in liver tissues across various experimental groups. Our investigation unveiled a noteworthy decrease in the percentage of viable liver cells and a concurrent increase in apoptotic cells subsequent to exposure to AlP. Intriguingly, the administration of CoQ₁₀ ameliorated the reduction in cell viability induced by AlP exposure. These findings suggest that CoQ₁₀ may exert inhibitory effects on the release of cytochrome c and other apoptotic agents, although these aspects were not specifically addressed in the current study. The anti-apoptotic effects of CoQ₁₀ could potentially manifest through various pathways. Previous research has indicated that CoQ₁₀ is implicated in the up-regulation of anti-apoptotic agents, such as Bclxl, Bcl-2,⁵⁹ and survivin, along with the down-regulation of proapoptotic agents including Bax, Bid, and Fas.⁵⁸ CoQ₁₀ also elevated the expression of survival proteins, thereby promoting cell survival through its effective antioxidant properties.⁶⁰

AST and ALT are enzymes predominantly concentrated within hepatic tissue, albeit they are also present in red blood cells, cardiac myocytes, skeletal muscle fibers, and various other organs including the pancreas and kidneys. Elevated serum levels of AST or ALT serve as pivotal biomarkers, especially in the diagnostic evaluation of hepatic disorders. These enzymes are released into the bloodstream following cellular damage, particularly in the context of hepatocellular injury, where their elevated levels provide valuable insights into liver function and pathology.⁶¹ The concentration of these enzymes in the bloodstream correlates directly with the extent of tissue damage.⁶² In a conducted study, treatment with CoQ₁₀ demonstrated a significant reduction in liver tissue damage induced by acetaminophen, leading to a noteworthy decrease in serum levels of aminotransferases, including AST and ALT.^19,63 Our research similarly indicated that exposure to phosphine significantly elevated the levels of AST and ALT in comparison to the control group. Notably, simultaneous treatment with CoQ₁₀ effectively attenuated the increase in these biomarkers in the serum.

Conclusion

Several studies have underscored the protective properties of CoQ₁₀ in diverse pathological conditions. The present investigation sought to examine the potential protective effects of CoQ₁₀ against liver damage induced by AlP and to elucidate the underlying mechanisms involved in this protective action. According to the study findings, the average survival time of animals poisoned with AlP increased, suggesting that CoQ₁₀ may exert protective effects against various mechanisms associated with AlP toxicity. The examination of liver tissue revealed mitochondrial dysfunction, characterized by oxidative stress, a significant decrease in cellular energy levels, and disruption of mitochondrial complexes resulting from AlP poisoning. CoQ₁₀ intervention demonstrated an amelioration of these changes, exerting therapeutic effects by inhibiting oxidative stress pathways, enhancing the function of mitochondrial complexes, and improving the aerobic energy pathway and apoptosis. These study results prompt further exploration into the potential therapeutic effects of CoQ₁₀ in clinical conditions involving AlP poisoning.

Footnotes

Acknowledgements

This study received support from a grant provided by AJA University of Medical Sciences, Tehran, Iran, with the code IR.AJAUMS.REC.1402.023. I express my heartfelt gratitude to Dr. Maryam Akhgari from the Legal Medicine Research Center of Iran for her assistance in reviewing and editing the manuscript. I also extend my thanks to Dr. Sara Mirhaghi Saatchi, a specialist at the Food and Drug Organization of Iran, for her collaboration in writing the article.

Author contributions

MRH conceptualized the study, conducted the literature review, carried out the research, drafted the manuscript, and performed statistical analysis. ZH provided guidance as an advisor, contributing to manuscript editing. MC and MRP were involved in the animal and biochemical aspects of the study. EN supervised the entire research process. All authors have reviewed and approved the final version of the manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical statement

ORCID iD

Mohammad Reza Hooshangi Shayesteh

References

Gupta

Ahlawat

(1995) Aluminum phosphide poisoning—a review. Journal of Toxicology: Clinical Toxicology 33(1): 19–24.

Haghi Aminjan

Abtahi

Hazrati

, et al. (2019) Targeting of oxidative stress and inflammation through ROS/NF-kappaB pathway in phosphine-induced hepatotoxicity mitigation. Life Sciences 232: 116607.

Anand

Binukumar

Gill

(2011) Aluminum phosphide poisoning: an unsolved riddle. Journal of Applied Toxicology: JAT 31(6): 499–505.

Anand

Kumari

Kaushal

, et al. (2012) Effect of acute aluminum phosphide exposure on rats—a biochemical and histological correlation. Toxicology Letters 215(1): 62–69.

Moghadamnia

(2012) An update on toxicology of aluminum phosphide. Daru: Journal of Faculty of Pharmacy, Tehran University of Medical Sciences 20(1): 25–28.

Hsu

C-H

Chi

B-C

Liu

M-Y

, et al. (2002) Phosphine-induced oxidative damage in rats: role of glutathione. Toxicology 179(1–2): 1–8.

Nath

Bhattacharya

Tuck

, et al. (2011) Mechanisms of phosphine toxicity. Journal of Toxicology 2011: 494168.

Hooshangi Shayesteh

Haghi-Aminjan

Baeeri

, et al. (2022) Modification of the hemodynamic and molecular features of phosphine, a potent mitochondrial toxicant in the heart, by cannabidiol. Toxicology Mechanisms and Methods 32(4): 288–301.

Sarter

(2002) Coenzyme Q10 and cardiovascular disease: a review. The Journal of Cardiovascular Nursing 16(4): 9–20.

10.

Hargreaves

(2014) Coenzyme Q10 as a therapy for mitochondrial disease. The International Journal of Biochemistry & Cell Biology 49: 105–111.

11.

Hidalgo-Gutiérrez

González-García

Díaz-Casado

, et al. (2021) Metabolic targets of coenzyme Q10 in mitochondria. Antioxidants 10(4): 520.

12.

Mirmalek

Gholamrezaei Boushehrinejad

Yavari

, et al. (2016) Antioxidant and anti-inflammatory effects of coenzyme Q10 on L-arginine-induced acute pancreatitis in rat. Oxidative Medicine and Cellular Longevity 2016: 5818479.

13.

Kwong

Kamzalov

Rebrin

, et al. (2002) Effects of coenzyme Q10 administration on its tissue concentrations, mitochondrial oxidant generation, and oxidative stress in the rat. Free Radical Biology & Medicine 33(5): 627–638.

14.

Jafari

Baghaei

Solgi

, et al. (2015) An electrocardiographic, molecular and biochemical approach to explore the cardioprotective effect of vasopressin and milrinone against phosphide toxicity in rats. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association 80: 182–192.

15.

Kariman

Heydari

Fakhri

, et al. (2012) Aluminium phosphide poisoning and oxidative stress: serum biomarker assessment. Journal of Medical Toxicology: Official Journal of the American College of Medical Toxicology 8: 281–284.

16.

Mostafazadeh

Pajoumand

Farzaneh

, et al. (2011) Blood levels of methemoglobin in patients with aluminum phosphide poisoning and its correlation with patient’s outcome. Journal of Medical Toxicology: Official Journal of the American College of Medical Toxicology 7(1): 40–43.

17.

Ali

Faddah

Abdel-Baky

, et al. (2010) Protective effect of L-carnitine and coenzyme Q10 on CCl₄-induced liver injury in rats. Scientia Pharmaceutica 78(4): 881–896.

18.

El-Sheikh

Morsy

Mahmoud

, et al. (2012) Effect of coenzyme-Q10 on doxorubicin-induced nephrotoxicity in rats. Advances in Pharmacological Sciences 2012: 1–8.

19.

Fouad

Jresat

(2012) Hepatoprotective effect of coenzyme Q10 in rats with acetaminophen toxicity. Environmental Toxicology and Pharmacology 33(2): 158–167.

20.

Rauscher

Sanders

Watkins

(2001) Effects of coenzyme Q10 treatment on antioxidant pathways in normal and streptozotocin‐induced diabetic rats. Journal of Biochemical and Molecular Toxicology 15(1): 41–46.

21.

Henry

Chiamori

Golub

, et al. (1960) Revised spectrophotometric methods for the determination of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, and lactic acid dehydrogenase. American Journal of Clinical Pathology 34(4_ts): 381–398.

22.

Young

Tang

Chao

, et al. (2008) Quantitative rat liver function test by galactose single point method. Laboratory Animals 42(4): 495–504.

23.

Aebi

(1974) Catalase. Methods in Enzymatic Analysis 2: 671–678.

24.

Ranjbar

Ghahremani

Sharifzadeh

, et al. (2010) Protection by pentoxifylline of malathion-induced toxic stress and mitochondrial damage in rat brain. Human & Experimental Toxicology 29(10): 851–864.

25.

Chen

Zhong

, et al. (2010) 2′, 7′-Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: forty years of application and controversy. Free Radical Research 44(6): 587–604.

26.

Abdolghaffari

Baghaei

Solgi

, et al. (2015) Molecular and biochemical evidences on the protective effects of triiodothyronine against phosphine-induced cardiac and mitochondrial toxicity. Life sciences 139: 30–39.

27.

Mohammadi

Karimi

Mahdi Rezayat

, et al. (2011) Benefit of nanocarrier of magnetic magnesium in rat malathion-induced toxicity and cardiac failure using non-invasive monitoring of electrocardiogram and blood pressure. Toxicology and Industrial Health 27(5): 417–429.

28.

Sherwood

Hirst

(2006) Investigation of the mechanism of proton translocation by NADH: ubiquinone oxidoreductase (complex I) from bovine heart mitochondria: does the enzyme operate by a Q-cycle mechanism? The Biochemical Journal 400(3): 541–550.

29.

Degli Esposti

(2001) Assessing functional integrity of mitochondria in vitro and in vivo. Methods in Cell Biology 65: 75–96.

30.

Cooperstein

Lazarow

(1951) A microspectrophotometric method for the determination of cytochrome oxidase. The Journal of Biological Chemistry 189(2): 665–670.

31.

Vermes

Haanen

Steffens-Nakken

, et al. (1995) A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. Journal of Immunological Methods 184(1): 39–51.

32.

Krifka

Hiller

K-A

Spagnuolo

, et al. (2012) The influence of glutathione on redox regulation by antioxidant proteins and apoptosis in macrophages exposed to 2-hydroxyethyl methacrylate (HEMA). Biomaterials 33(21): 5177–5186.

33.

Proudfoot

(2009) Aluminium and zinc phosphide poisoning. Clinical Toxicology 47(2): 89–100.

34.

Etemadi-Aleagha

Akhgari

Iravani

(2015) Aluminum phosphide poisoning-related deaths in Tehran, Iran, 2006 to 2013. Medicine 94(38): e1637.

35.

Armandeh

Bameri

Baeeri

, et al. (2021) The role of levosimendan in phosphine-induced cardiotoxicity: evaluation of electrocardiographic, echocardiographic, and biochemical parameters. Toxicology Mechanisms and Methods 31(9): 631–643.

36.

Ahmadi

Shadboorestan

(2016) Oxidative stress and cancer; the role of hesperidin, a citrus natural bioflavonoid, as a cancer chemoprotective agent. Nutrition and Cancer 68(1): 29–39.

37.

Erdemli

Gul

, et al. (2021) Ameliorative effects of crocin on the inflammation and oxidative stress-induced kidney damages by experimental periodontitis in rat. Iranian Journal of Basic Medical Sciences 24(6): 825–832.

38.

Cederbaum

(2009) Role of oxidative stress in alcohol-induced liver injury. Archives of Toxicology 83: 519–548.

39.

Abdollahi

Ranjbar

Shadnia

, et al. (2004) Pesticides and oxidative stress: a review. Medical Science Monitor: International Medical Journal of Experimental and Clinical Research 10(6): 141–147.

40.

Shayesteh

Haghi-Aminjan

Mousavi

, et al. (2019) The protective mechanism of cannabidiol in cardiac injury: a systematic review of non-clinical studies. Current Pharmaceutical Design 25(22): 2499–2507.

41.

Armandeh

Bameri

Haghi-Aminjan

, et al. (2022) A systematic review on the role of melatonin and its mechanisms on diabetes-related reproductive impairment in non-clinical studies. Frontiers in Endocrinology 13: 1022989.

42.

Bameri

Armandeh

Haghi-Aminjan

, et al. (2024) Insights into the role of melatonin on the reproductive system toxicity induced by bisphenol A: a systematic review of preclinical studies. Toxin Reviews 43: 137–156.

43.

Lenaz

Bovina

D'aurelio

, et al. (2002) Role of mitochondria in oxidative stress and aging. Annals of the New York Academy of Sciences 959(1): 199–213.

44.

Rahimi Kakavandi

Asadi

Hooshangi Shayesteh

, et al. (2022) The electrocardiographic, hemodynamic, echocardiographic, and biochemical evaluation of treatment with edaravone on acute cardiac toxicity of aluminum phosphide. Frontiers in Pharmacology 13: 1032941.

45.

Bameri

Armandeh

Baeeri

, et al. (2021) Electrocardiographic, hemodynamic, and biochemical evidence on the protective effects of exenatide against phosphine-induced cardiotoxicity in rat model. Human & Experimental Toxicology 40(12_suppl): S381–S396.

46.

Gebicka

Krych-Madej

(2019) The role of catalases in the prevention/promotion of oxidative stress. Journal of Inorganic Biochemistry 197: 110699.

47.

Salvi

Battaglia

Brunati

, et al. (2007) Catalase takes part in rat liver mitochondria oxidative stress defense. The Journal of Biological Chemistry 282(33): 24407–24415.

48.

Deneke

(2000) Thiol-based antioxidants. Current Topics in Cellular Regulation 36: 151–180.

49.

Valmas

Zuryn

Ebert

(2008) Mitochondrial uncouplers act synergistically with the fumigant phosphine to disrupt mitochondrial membrane potential and cause cell death. Toxicology 252(1–3): 33–39.

50.

Lakshmi

(2002) Methemoglobinemia with aluminum phosphide poisoning. The American Journal of Emergency Medicine 20(2): 130–132.

51.

Haghi-Aminjan

Baeeri

Rahimifard

, et al. (2018) The role of minocycline in alleviating aluminum phosphide-induced cardiac hemodynamic and renal toxicity. Environmental Toxicology and Pharmacology 64: 26–40.

52.

Lee

Kim

K-Y

Shim

, et al. (2014) Coenzyme Q10 ameliorates oxidative stress and prevents mitochondrial alteration in ischemic retinal injury. Apoptosis: An International Journal on Programmed Cell Death 19: 603–614.

53.

Zaki

(2016) Strategies for oral delivery and mitochondrial targeting of CoQ10. Drug Delivery 23(6): 1868–1881.

54.

Reed

(2000) Mechanisms of apoptosis. The American Journal of Pathology 157(5): 1415–1430.

55.

Hengartner

(2000) The biochemistry of apoptosis. Nature 407(6805): 770–776.

56.

Galati

O'Brien

(2003) Cytoprotective and anticancer properties of coenzyme Q versus capsaicin. Biofactors 18(1–4): 195–205.

57.

Hosseini

Forouzesh

Maleknia

, et al. (2020) The molecular mechanism of aluminum phosphide poisoning in cardiovascular disease: pathophysiology and diagnostic approach. Cardiovascular Toxicology 20: 454–461.

58.

Shabaan

Mostafa

El-Desoky

, et al. (2023) Coenzyme Q10 protects against doxorubicin-induced cardiomyopathy via antioxidant and anti-apoptotic pathway. Tissue Barriers 11(1): 2019504.

59.

Khan

Abid

Ahmad

, et al. (2017) Cardioprotective effect of coenzyme Q10 on apoptotic myocardial cell death by regulation of bcl-2 gene expression. Journal of Pharmacology & Pharmacotherapeutics 8(3): 122–127.

60.

Chen

Hou

Shibu

, et al. (2017) Protective effect of Co‐enzyme Q10 on doxorubicin‐induced cardiomyopathy of rat hearts. Environmental Toxicology 32(2): 679–689.

61.

Huang

X-J

Choi

Y-K

H-S

, et al. (2006) Aspartate aminotransferase (AST/GOT) and alanine aminotransferase (ALT/GPT) detection techniques. Sensors 6(7): 756–782.

62.

Lee

Kim

Poterucha

(2012) Evaluation of elevated liver enzymes. Clinics in Liver Disease 16(2): 183–19998.

63.

Esfahani

Esmaeilzadeh

Bagheri

, et al. (2013) The effect of co-enzyme q10 on acute liver damage in rats, a biochemical and pathological study. Hepatitis Monthly 13(8).

Evaluation of the protective effect of coenzyme Q 10 on hepatotoxicity caused by acute phosphine poisoning

Abstract

Keywords

Introduction

Materials and methods

Ethics

Chemicals

Animals

AlP LD50 calculation

Treatment strategy

Serum and liver tissue sampling

Hepatic serum enzyme evaluation

Measurement of catalase activities

Measurement of ROS

Determination of FRAP

Total thiol measurement

Assessment of liver mitochondrial function

Assessment of mitochondrial complex I activity

Assessment of mitochondrial Complex II activity

Assessment of mitochondrial Complex IV activity

Utilizing flow cytometry to examine necrosis and apoptosis

Statistical analysis

Results

Oxidative stress parameters in liver tissue

Mitochondrial liver activity

Apoptosis and necrosis detection using flow cytometry technique

Levels of AST and ALT in serum

Discussion

Conclusion

Footnotes

Acknowledgements

Author contributions

Declaration of conflicting interests

Funding

Ethical statement

ORCID iD

References