PGC 1α-Mediates Mitochondrial Damage in the Liver by Inhibiting the Mitochondrial Respiratory Chain as a Non-cholinergic Mechanism of Repeated Low-Level Soman Exposure

Abstract

This work aimed to assess whether mitochondrial damage in the liver induced by subacute soman exposure is caused by peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) and whether PGC-1α regulates mitochondrial respiratory chain damage. Toxicity mechanism research may provide theoretical support for developing anti-toxic drugs in the future. First, a soman animal model was established in male Sprague–Dawley (SD) rats by subcutaneous soman injection. Then, liver damage was biochemically evaluated, and acetylcholinesterase (AChE) activity was also determined. Transmission electron microscopy (TEM) was performed to examine liver mitochondrial damage, and high-resolution respirometry was carried out for assessing mitochondrial respiration function. In addition, complex I–IV levels were quantitatively evaluated in isolated liver mitochondria by enzyme-linked immunosorbent assay (ELISA). PGC-1α levels were detected with a Jess capillary-based immunoassay device. Finally, oxidative stress was analyzed by quantifying superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG), and reactive oxygen species (ROS) levels. Repeated low-level soman exposure did not alter AChE activity, while increasing morphological damage of liver mitochondria and liver enzyme levels in rat homogenates. Complex I, II and I + II activities were 2.33, 4.95, and 5.22 times lower after treatment compared with the control group, respectively. Among complexes I–IV, I–III decreased significantly (p < 0.05), and PGC-1α levels were 1.82 times lower after soman exposure than in the control group. Subacute soman exposure significantly increased mitochondrial ROS production, which may cause oxidate stress. These findings indicated dysregulated mitochondrial energy metabolism involves PGC-1α protein expression imbalance, revealing non-cholinergic mechanisms for soman toxicity.

INTRODUCTION

Soman (military designation GD) is among the deadliest organicphosphorus (OP) chemical warfare agents, as it rapidly ages, within minutes.¹⁾ Soman’s primary mechanism is the specific, irreversible inhibition of acetylcholinesterase (AChE) in the nervous system, which leads to acetylcholine (ACh) accumulation at the synaptic sites and hyperactivates the cholinergic system.^2–4) The typical toxic symptoms of the cholinergic crisis include salivation, lacrimation, respiratory failure, tremor, and seizures. Besides cholinergic effects, mitochondria, as cell organelles consuming high oxygen amounts and producing energy, may be targeted by OP poisoning as a non-cholinergic mechanism.⁵⁾ Mitochondria, with high abundance in the liver, are important in generating energy for cellular processes in organs and tissues.⁶⁾ The liver is an energy metabolism organ, as well as the site in which toxic molecules are metabolized into less harmful substances for toxicity reduction.⁷⁾ This causes hepatotoxicity and liver damage. Recent evidence confirms the liver is the most important organ targeted by OPs.⁸⁾ Thus, soman toxicity to the hepatic system has recently attracted attention.

The acute effects of soman resulting from AChE suppression have been reported.⁹⁾ However, the adverse impacts and the underlying molecular mechanism at the level of mitochondria following subacute exposure to soman have not been assessed. Therefore, we conducted an in-depth study to examine whether mitochondrial dysfunction is involved in soman-mediated liver damage.

The mitochondrial inner membrane comprises enzymes involved in the electron transport chain (ETC) and ATP production. The ETC contains multiple enzyme complexes such as reduced nicotinamide adenine dinucleotide (NADH)-CoQ reductase, succinate dehydrogenase, ubiquinone–cytochrome c reductase, cytochrome c oxidase and ATP synthase, also termed complexes I to V, respectively.¹⁰⁾ Oxidative phosphorylation (OXPHOS) is a process involving electrons flowing through respiratory chain enzymes, which generates an electrochemical proton gradient that is utilized for ATP synthesis.¹¹⁾ In this process, substrates are converted while ATP is produced.¹²⁾ Complexes I–IV may regulate mitochondrial electron/proton transfer, as well as mitochondrial respiration. As a result, an overall reduction of the activities of these complexes may lead to ETC damage and decrease ATP production.¹³⁾ This would lead to disordered mitochondrial energy metabolism, a pathogenic factor in liver damage due to exogenous poisons. In case of low electron transfer rate, electrons are constantly released by the respiratory chain to generate destructive reactive oxygen species (ROS), and mitochondrial ROS production is elevated.¹⁴⁾ Excessive ROS generation induces oxidative stress, which results in altered endogenous antioxidant enzymes, with subsequent free radical-induced lipid peroxidation.^15,16)

In addition, mitochondrial peroxisome proliferator-activated receptor-γ coactivator 1 alpha (PGC-1α) represents an important modulator of energy metabolism.¹⁷⁾ Under in vitro conditions, PGC-1α drives mitochondrial biogenesis. Mitochondrial OXPHOS is closely associated with ATP biosynthesis.¹⁸⁾ PGC-1α coactivates multiple transcription factors, which robustly upregulate many nuclear genes encoding almost all mitochondrial proteins involved in the ETC.¹⁹⁾ Besides, PGC-1α regulates respiratory genes after post-translational modifications that regulate protein–protein interactions.^20–22) Reduced PGC-1α expression alters energy metabolism in the mitochondria by downregulating the nuclear subunits of complexes I–V, thereby promoting liver injury.^17,23,24) It is unclear whether soman-induced liver injury is associated with mitochondrial energy metabolism impairment caused by liver PGC-1α protein downregulation.

To explore the mechanism underpinning liver damage in Sprague–Dawley (SD) rats exposed to low-level soman, the effects of soman exposure on liver function, mitochondrial OXPHOS, PGC-1α expression levels, and mitochondrial oxidative stress in rat liver were determined. Additionally, liver AChE activity was quantitated to examine whether the observed liver injury was associated with the toxicity mechanism of the cholinergic pathway.

Taken together, this study on the toxicity mechanism of soman provides some theoretical support for developing anti-toxic drugs in the future. These findings suggest an approach that could help elucidate the molecular mechanisms of soman-mediated mitochondrial energy metabolism disorder in liver damage.

MATERIALS AND METHODS

Animals and Treatment

Totally 42 male SD rats (seven-week-old, 190–210 g) were obtained from Huafukang Animal (China). All rats underwent housing under specific pathogen-free (SPF) conditions at 22 ± 1 °C and 55 ± 5% humidity with a 12-h photoperiod. The rats were acclimatized for seven days, with freely available rodent chow and tap water. All animal protocols abided by the guidelines for the use of experimental animals of State Key Laboratory of NBC Protection for Civilians, following the Chinese National standard GB/T 35892-2018 laboratory animal guideline for ethical review of animal welfare. These animal experiments had approval from the State Key Laboratory of NBC Protection for Civilian Committee for Animal Experimentation (No. LAE-2021-10-001).

Acute Subcutaneous Toxicity

The acute subcutaneous toxicity assay was carried out based on a procedure described in the national standard of China (GB/T 21603-2008), with some modifications. Due to slow detoxification rate following subcutaneous (s.c.) treatment, the long-term impacts of nerve agents could be assessed via this route, as a reasonable alternative to intravenous administration for respiratory exposures in studies assessing nerve agents.²⁵⁾ Thirty animals were randomly assigned to five groups of 6 before exposure to soman. Soman (> 97% purity, mol. wt. 182.19; Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, China) stock solution (20 mg/mL) was prepared with isopropanol. This was diluted with normal saline to the appropriate concentration before use. The rats in various groups received a single subcutaneous injection of soman at 60, 80, 100, 120, and 140 µg per kilogram body weight (BW), respectively, in a volume of 0.4 mL/kg BW. Meanwhile, control animals were administered normal saline at 0.4 mL/kg. The mortality and survival status of rats were observed twice a day for seven days after the single subcutaneous injection. The bliss method determined the LD₅₀ and LD₀₁ of soman in rats.

Subacute Subcutaneous Toxicity

The subacute subcutaneous toxicity study was based on a protocol proposed by the national standard of China (GBZ/T 240.15-2011), with some modifications. Before the assay, twelve adult male SD rats were randomized into the control and soman administration groups. Based on LD₀₁ in the acute subcutaneous toxicity study, the soman group was treated by subcutaneous injection of soman at a dose of 38.59 µg (0.6 × LD₀₁) per kg BW, which does not kill any rat. Control animals were administered normal saline at an equal volume. All animals were injected once a day for seven consecutive days. The animals were weighed daily, and the general behavior changes of rats were monitored continuously for the seven days. At study end, anesthesia was performed with carbon dioxide inhalation, before euthanasia. The livers were harvested, flash frozen (liquid nitrogen) and kept at −80 °C.

Transmission Electron Microscopy

The dissected liver sections were cut into cubes of about 1 mm for each side, which were post-fixed with 2.5% glutaraldehyde fixative solution for 3 h at 4 °C. After phosphate-buffered saline (PBS) washes, the specimens were fixed with 0.5% osmium acid for 2 h, followed by dehydration in a graded ethanol. Then, Spurr embedding was carried out, and 60–80 nm ultrathin sections were obtained with an UltraCut R ultramicrotome (Leica, Germany). Upon double-staining with uranyl acetate and lead citrate, a transmission electron microscope (JEM1230, Japan) was utilized to analyse the samples.

Effects of Soman on Liver Histopathology in Rat

Livers of control and soman-treated groups were fixed with 10% neutral buffered formalin solution for 72 h, then dehydrated and embedded in paraffin to make conventional paraffin sections. After paraffin embedding, the sections were cut into 4 µm thickness and stained with hematoxylin–eosin (H&E). The histopathological changes of liver tissues were visualized using light microscopy (Nikon, Japan).

Biochemical Tests

Aspartate (AST) and Alanine (ALT) Aminotransferase Quantitation

Serum enzymes were carried out on a biochemistry autoanalyzer (AU480, Olympus, Tokyo, Japan) following standard procedures of biochemistry by colorimetric methods using commercial kits (Nanjing Jiancheng Biological Engineering Institute, China). Data were given as units per gram of protein (U/g protein).

AChE Activity Assessment

AChE activity in the rat liver tissue was tested using a colorimetric assay kit (Elabscience Biotechnology Co., Ltd., E-BC-K174-M, China) as previously described.²⁶⁾ AChE activity was expressed as units per milligrams of protein (U/mg protein). After the reaction, a multimode microplate reader (Spark^®, TECAN, Switzerland) was utilized for absorbance reading at 412 nm.

Mitochondrial Respiratory Function Analyses

High-Resolution Respirometry Analysis of Mitochondrial Respiration Capacity

A Oxygraph-2k high-resolution respirometer (Oroboros Instruments, Austria) was used to determine mitochondrial respiratory function. Mitochondrial complex-associated respiratory rates were examined as shown in Fig. 1. Substrates and inhibitors for determining the activities of mitochondrial complexes were added according to standard protocols.^27,28) A Clark electrode is typically used for the measurement of oxygen concentration. The oxygen consumption rate (OCR) was obtained with DatLab 7.4 (Oroboros Instruments), as pmol/(s*mg). Briefly, the whole liver was quickly extracted, and 3 mg of wet hepatic tissue was placed into 2 mL ice-cold fetal bovine serum (FBS; Gibco, Nebraska, U.S.A.) solution (mitochondrial respiration medium), and the mixture was homogenized at 4 °C for 1 min. Several indexes were assessed by adding multiple substrates and inhibitors, including 2 M glutamate (G, 10 µL; Sigma-Aldrich, U.S.A.) and 400 mM malate (M, 10 µL; Sigma-Aldrich), 300 mM ADP (10 µL; 117105; Merck KGaA, Germany), 4 mM cytochrome c (Cyc c, 5 µL; Sigma-Aldrich), 1 M succinate (S, 20 µL; Sigma-Aldrich), 1 mM rotenone (Rot, 1 µL; Sigma-Aldrich), 5 mM oligomycin (Omy, 2 µL; Sigma-Aldrich), 1 mM trifluorocarbonylcyanide phenylhydrazone (FCCP, 1 µL; Sigma-Aldrich) and 5 mM antimycin A (Ama, 1 µL; Sigma-Aldrich).

Fig. 1. Workflow for the Analysis of Oxidative Phosphorylation

Addition of G and M to provoke the activation of complex I. D and C to active complex I-dependent respiration. S to active complex I + II-dependent respiration. Inhibition of complex I-dependent respiration by adding Rot. Omy (turn off ATPase activity) to measure ATP production. FCCP to measure maximal respiration, and Ama for the final measurement of non-mitochondrial oxygen consumption.

Without adding exogenous substrates and ADP, basal mitochondrial respiration was measured. Respiratory oxygen consumption for ATP production was measured under 5 mM Omy. Finally, sequential injections of 1 mM FCCP and 5 mM Ama were utilized to measure maximal respiration and non-mitochondrial respiration. Complex-I and II-associated respiratory levels were measured by supplementing 300 mM ADP and 1 mM Rot, respectively. Addition of 1M S was used for the assessment of complex I + II-associated respiration.

Enzyme-Linked Immunosorbent Assay (ELISA) Analyses of Complexes I–IV

Frozen liver tissues (approximately 50 mg) were quickly thawed on ice and homogenized in 500 µL ice-cold PBS. After a 10-min centrifugation (10000 × g) at 4 °C, supernatants were assessed for protein amounts with a bicinchoninic acid (BCA) protein quantitative assay kit per manufacturer’s instructions. These supernatants were employed to assess the concentrations of complexes I–IV.

ELISA is a method used to detect an antigen within a sample quantitatively. Complex I–IV levels were quantitated in isolated liver mitochondria with an ELISA kit (Jiangsu Jingmei Biotechnology, China). The Spark^® microplate reader was utilized for absorbance reading at 450 nm. Complex I–IV concentrations were expressed as pg/mL.

Oxidative Stress Analysis

Frozen liver tissues (approximately 50 mg) were thawed on ice and homogenized in 500 µL ice-cold PBS. The samples underwent a 10-min centrifugation at 10000 × g and 4 °C. The resulting supernatants were assessed for protein amounts with the BCA protein quantitative assay kit. The liver tissue supernatants were tested for ROS, superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), and oxidized glutathione (GSSG) amounts.

Liver mitochondrial ROS generation was assessed using an ELISA kit following the manufacturer’s protocol (Jiangsu Jingmei Biotechnology Co., Ltd.). ROS concentration was expressed in IU/mL. The Spark^® microplate reader was utilized for absorbance reading.

SOD, MDA, GSH, and GSSG activities were assessed with colorimetric assay kits (Nanjing Jiancheng Biological Engineering Institute, China), as specified by the manufacturer. Total GSH and GSSG levels were determined by the 5,5′-dithiobis (2-nitrobenzoic acid)-GSSG recycling assay.²⁹⁾ GSH and GSSG concentrations were calculated and expressed in µmol per liter of supernatant (µmol/L). SOD activity was expressed in U/mg protein. MDA level was expressed in nmol/mg protein.

Capillary Western Blot Analysis

Tissue samples underwent lysis with CelLytic MT (Sigma-Aldrich) reagent supplemented with protease and phosphatase inhibitor cocktail (100 : 1 : 1) (EpiZyme Biotech Corp., Ltd., China). The lysates underwent a 10-min centrifugation at 10000 × g and 4 °C. A BCA protein assay kit (ZJ102, EpiZyme Biotech Corp., Ltd.) was utilized for protein quantitation.

The PGC-1α and β-actin (Abcam, ab72230, MA, U.S.A.; Cell Signaling Technology, 4970S, MA, U.S.A.) proteins were detected on a Jess capillary-based immunoassay device (Jess, ProteinSimple, San Jose, CA, U.S.A.) as directed by the manufacturer. Briefly, tissue lysates (0.4 µg/µL in sample buffer) were added to the fluorescent master mix and incubated for 5 min at 95 °C. The latter reactions, as well as the blocking reagent, primary antibodies targeting PGC-1α and β-actin (1 : 50), HRP-linked secondary antibodies, and the chemiluminescent substrate were added to appropriate of the assay plate. Chemiluminescent signals were detected by a charge-coupled device (CCD) camera; the resulting image was analyzed with the Compass software (Version 6.0.0, ProteinSimple) and data were expressed as peak intensity.

Statistical analysis

GraphPad Prism 8.3 (GraphPad Software Inc., CA, U.S.A.) was employed to analyze data (mean ± standard error of the mean (S.E.M.)). The student’s t-test was performed for between-group comparisons. p < 0.05 indicated statistical significance.

RESULTS

Acute Subcutaneous Toxicity

First, soman doses and mortality rates are shown in Table 1. In the acute subcutaneous toxicity study, the dead rats had severe poisoning symptoms before death, including seizures, muscular twitching (fasciculations), floppy (flaccid) paralysis, and cessation of breathing (apnea). The high-dose soman-treated rats died within 10–30 min, and low-dose treated animals died within about one day. They could survive continuously in case of no death for more than one day. Compared with the control group, the weight change decreased slightly within one week, with no significant difference. The LD₅₀ of soman was 98.22 (95% confidence interval [CI] 81.34–118.62) µg/kg. The LD₀₁ of soman was 64.31 (95%CI 39.60–104.44) µg/kg.

Table 1. Acute Toxicity of Soman in SD Rats

Dose (µg/kg)	Soman			LD50 (µg/kg) (95% confidence interval)	LD01 (µg/kg) (95% confidence interval)
	N	Mortality	Ratio
60	6	0	0/6	98.22 (81.34–118.62)	64.31 (39.60–104.44)
80	6	1	1/6
100	6	3	3/6
120	6	5	5/6
140	6	6	6/6

Subacute Subcutaneous Toxicity

Next, soman was subcutaneously injected at 38.59 µg/kg (0.6 × LD₀₁) in the subacute subcutaneous toxicity study based on the above LD₀₁. This administered dose was lower than LD₀₁ and did not cause death for seven days. During the seven days observation period, the animals administered soman showed fatigue at 1 h after exposure, but returned to normal about 2–3 h later. There were no significant changes in behavior parameters, including apathy, hyperactivity, and morbidity, in any of the animals.

In comparison with control animals, body weight gain in soman-treated rats showed a decreasing trend during the six days of observation. There was a significant difference on day 7 between the two groups. The mean body weight of animals in the control group was 232.27 ± 3.47 g, versus 218.67 ± 5.89 g in soman-treated animals (Fig. 2A). The average weight gain was significantly lower in the soman group compared with the control group (p < 0.01) (Fig. 2B). This was an obvious sign of poisoning after soman administration. Loss of body weight is commonly observed after organophosphate intoxication in vertebrates.³⁰⁾

Fig. 2. Effect of Soman on Rat Weight

(A) Rat weight after continuous soman administration for seven days. (B) Weight gains in the soman and control groups on day 7. Data are mean ± S.E.M. (n = 6). ** p < 0.01 versus control group.

Biochemical Evidence of Liver Dysfunction

Morphological Damage of Liver Mitochondria

Next, all mitochondria in control samples maintained a normal structure as illustrated in Fig. 3, including rod-like, dense, and organized cristae (Fig. 3A). Meanwhile, mitochondria in soman-treated animals exhibited overtly abnormal morphology (Fig. 3B). Some mitochondria were swollen, with significantly increased volume; the matrix in the membrane was reduced, and the cristae were broken and disappeared, with vacuoles. In addition, glycogen was aggregated around the nucleus.

Fig. 3. Electron Micrographs of Liver Mitochondria in Rats after Subacute Soman Exposure

Liver sections were fixed, and electron microscopy was performed. (A) In the control group, many mitochondria and the endoplasmic reticulum can be seen in the cytoplasm. The mitochondrial membrane structure is complete, and cristae are visible. (B) In the soman group, mitochondria were significantly aggregated and fused, with irregular morphology.

Histopathological Analysis of Liver Injury

As shown in Fig. 4, The liver cells are relatively abundant, the hepatic lobules were clear, and the hepatic cords and hepatocytes were regularly arranged in the control group (Fig. 4A). In the soman group, it was found that some hepatocytes were swollen and deformed, inflammatory infiltration in the portal area, and the hepatic cords were not regularly arranged, and vascular congestion (Fig. 4B).

Fig. 4. Effects of Soman on Liver Histopathology in Rat (100×)

The histopathological changes of liver tissues were observed under the optical microscope by H&E staining. (A) In the control group, the liver tissues were intact, the hepatic lobules were clear, and the hepatocytes were regularly arranged. (B) In the soman group, some hepatocytes edema (blue arrows), inflammatory infiltration (black arrows) and vascular congestion (red arrows).

Soman-Induced Liver Injury

Liver damage was assessed by determining serum ALT and AST amounts. Table 2 depicts ALT and AST amounts in control- and soman-treated animals. ALT and AST activities were markedly elevated in soman-treated rats in comparison with control animals (p < 0.05). These data indicated soman induced liver injury.

Table 2. Effect of Subacute Soman Exposure on the AST and ALT Amounts in the Serum of Rat

	Control group	Soman group
AST activity (U/g protein)	106.79 ± 15.64	168.62 ± 19.31*
ALT activity (U/g protein)	54.32 ± 2.31	67.97 ± 2.12**

* p < 0.05, ** p < 0.01 versus control group. Data are mean ± S.E.M., n = 6.

Soman Does Not Inhibit AChE

Additionally, AChE activity was assessed, and no marked difference was found between the two groups (Fig. 5). The above finding suggested soman's effect on liver damage less likely resulted from AChE inhibition.

Fig. 5. Effect of Soman on AChE in the Liver Tissue

There was no significant difference between the soman and control groups in AChE activity. Data are mean ± S.E.M., n = 6.

Mitochondrial Energy Metabolism Is Impaired by Soman

Soman Inhibits Mitochondrial Respiration Capacity in the Liver

As illustrated in Fig. 6A, soman-treated rats exhibited markedly reduced compared with the control group in mitochondrial respiratory capacity. The toxic effects of soman were reflected by decreased non-mitochondrial respiration and ATP-associated respiration, with significant differences (all p < 0.05). Data confirmed that basal respiratory activity was remarkably reduced in soman-treated rats compared with control animals (Fig. 6B). Furthermore, ATP production declined to 35% of the control level (Fig. 6C), while maximal respiration significantly decreased by 36% (Fig. 6D). Meanwhile, non-mitochondrial oxygen consumption also significantly decreased in the soman group compared with control animals (p < 0.05; Fig. 6E). These results indicated impaired mitochondrial respiratory function following subacute soman administration.

Fig. 6. Effect of Soman on Mitochondrial Respiration Function in the Rat Liver

(A) Representative recordings of mitochondrial respiration (Soman, blue line; Control, red line). (B) Quantification of basal respiration. (C) ATP production. (D) Maximal respiration. (E) Non-mitochondrial oxygen consumption. Data are mean ± S.E.M. (n = 3–4 per group). * p < 0.05, ** p < 0.01 versus control group.

Soman Downregulates Mitochondrial Complex I–II Activities in the Liver

Mitochondrial complex I, II and I + II activities in the liver of soman-treated rats are depicted in Fig. 7. Mitochondrial complex I and II activities were starkly reduced after soman treatment in comparison with control values. Complex I activity was reduced by 57% (Fig. 7A), and that of complex II by 68% (Fig. 7B). Of note, succinate (inducing parallel electron input from complexes I + II) markedly decreased the O₂ flux in soman-treated rats, by 81% (Fig. 7C). The above data indicated soman alters mitochondrial energy metabolism by suppressing the activities of complexes I, II, and I + II.

Fig. 7. Effects of Soman on Mitochondrial Complex Activities in the Rat Liver

(A) Complex I activity. (B) Complex II activity. (C) Complex I + II activity. Data are mean ± S.E.M., n = 3–4, * p < 0.05, ** p < 0. 01 and *** p < 0.001 versus control group.

Soman Downregulates Complexes I–III

Then, we used specific ELISA kits to evaluate the enzymatic activities of individual OXPHOS complexes (I–IV) in liver tissue homogenates. Complex I, II, and III activities were remarkably lowered following soman treatment. As evident from Fig. 8, complex I activity decreased from 72.82 ± 3.22 pg/mL in the control group to 60.61 ± 3.30 pg/mL in soman-treated animals, corresponding to a 25% reduction. Similarly, complex II activity decreased from 151.85 ± 6.56 pg/mL in control rats to 135.06 ± 2.31 pg/mL after soman treatment, reflecting a 16% reduction. Significantly, the activity of complex III also decreased from 27.44 ± 0.62 pg/mL in the control group to 22.97 ± 0.89 pg/mL in soman-treated animals, corresponding to a 21% reduction. However, these two groups had no significant difference in complex IV activity. This indicated that soman-associated inhibition of respiratory function was less likely to involve complex IV. The above findings indicated besides decreasing mitochondrial complex I and II activities, soman may also decrease mitochondrial complex III activity. Therefore, overall reduced complex I, II, and III activities could impair the mitochondrial respiration chain and dysregulate mitochondrial energy metabolism.

Fig. 8. Effects of Soman Exposure on the Contents of Mitochondrial Respiratory Chain Complexes in the Liver Tissue

Data are mean ± S.E.M., n = 6, * p < 0.05, ** p < 0.01 versus control group.

Soman-Induced Oxidative Stress

ROS Generation

As shown in Fig. 9A, liver mitochondria had overtly increased ROS amounts in soman-exposed rats in comparison with control animals (p < 0.05). This finding suggested enhanced mitochondrial ROS production in the liver after soman treatment.

Fig. 9. Effects of Soman on the Levels of Oxidative Stress Products in the Rat Liver

(A) ROS levels. (B) SOD activities. (C) GSH levels. (D) MDA levels. (E) GSH/GSSG ratios. Data are mean ± S.E.M., n = 6, * p < 0.05, ** p < 0.01 and *** p < 0.001 versus control group.

Liver SOD, MDA, GSH, and GSH/GSSG Changes

SOD, MDA, GSH, and GSSG are major molecular markers of oxidative stress. Therefore, we studied changes in these four substances after soman exposure. The results revealed markedly reduced mitochondrial SOD activity (Fig. 9B) and GSH amounts (Fig. 9C) in soman-exposed rats in comparison with control animals (both p < 0.05). In addition, rats administered soman showed increased liver mitochondrial MDA in comparison with control rats (p < 0.01; Fig. 9D). These data suggested the liver of soman-intoxicated rats was not protected against oxidative damage. Moreover, GSH/GSSG ratios were lower in liver mitochondria from the soman group compared with the control group (p < 0.001; Fig. 9E). Thus, these data suggested that subacute soman exposure might lead to oxidative stress, with decreased amounts of antioxidant biomarkers (SOD, GSH, and GSH/GSSG) and elevated prooxidant (MDA) levels.

Soman Inhibits PGC-1α Protein Expression

Capillary immunoblot was applied for the detection of PGC-1α, which is related to mitochondrial metabolism function. In comparison with control rats, liver PGC-1α protein amounts were markedly downregulated in soman-treated rats (p < 0.05, Fig. 10), which indicated that soman downregulated PGC-1α protein. These results suggested soman potentially disturbed mitochondrial energy metabolism, which might involve PGC-1α.

Fig. 10. Effect of Soman on PGC-1α Expression in the Rat Liver

(A) Representative immunoblots illustrating the effect of soman on PGC-1α expression in the liver. (B) Quantification of PGC-1α expression in the liver. Data are mean ± S.E.M., n = 3, * p < 0.05 versus control group.

DISCUSSION

The current study treated SD rats with soman, observed mitochondrial damage in the liver, and assessed mitochondrial respiration function. Mitochondrial damage in the liver was increased after soman administration, reflected by decreased complex I–III activity and PGC-1α levels in comparison with control animals. These findings suggest dysregulated mitochondrial energy metabolism may involve PGC-1α protein expression imbalance.

This study revealed enhanced morphological damage in liver mitochondria as well as increased AST and ALT activities in liver homogenate of rats after soman treatment. Besides, previous reports have established that organophosphorus flame retardants (OPFRs) reduced the numbers of mitochondrial. The reduced content of mitochondrial networks was confirmed to be one of the pathological phenomena caused by the mitochondrial dysfunction of OPFRs.³¹⁾ In addition, a recent study by Zhang et al. showed that the disorder of mitochondrial fusion and fission results in further reduction of the number of mitochondria so that it is not enough to clear excessive ROS, and mitochondrial structure changes to form mitochondrial membrane permeable transport pores (mPTPs), which leads to cell necrosis and apoptosis, organ failure, and metabolic dysfunction, increasing morbidity and mortality.³²⁾ These findings support our data suggesting that soman toxicity will cause a decrease in the number of mitochondria, which is due to morphological damage caused by mitochondrial fusion and fission. An oxidative state for the liver in rats with adjuvant-induced arthritis was also reported based on liver homogenates.³³⁾ Besides, complex I, II, III, and I + II activities were inhibited. Studies also revealed complex II suppression is physiologically important in aging, diseases, and the mKATP channel.³⁴⁾ Suppressed complex I and III activities result in increased cytoplasmic hydrogen peroxide amounts with no oxidative stress induction.³⁵⁾ Soman exposure downregulated the PGC-1α protein. Previous studies also demonstrated PGC-1α downregulation and its consequences.^36,37) These results implied that subacute low-level soman exposure causes liver damage and mitochondrial energy metabolism impairment, which are closely associated with PGC-1α downregulation.

Correspondingly, liver histopathology showed that the soman administration resulted in a large number of hepatocytes with hepatocyte edema, inflammatory infiltration, and vascular congestion in the liver of rats. liver injury increases the amounts of liver AST and ALT, which enter the circulation. Therefore, AST and ALT are considered major indicators of liver damage or dysfunction.³⁸⁾ In the present work, elevated AST and ALT amounts may indicate increased liver damage in soman-treated rats. In agreement, multiple reports^39–41) demonstrated significantly elevated amounts of rat and human liver enzymes after exposure to OP pesticides. Increased liver transaminase activity indicates liver damage results from oxidative stress after OP treatment.⁴²⁾ In addition, transmission electron microscopy results showed damaged morphology for liver mitochondria. Furthermore, AChE data showed that soman likely causes liver damage without AChE inhibition. However, the exact molecular mechanism of soman-induced liver damage deserves further investigation.

Moreover, this study revealed that subacute soman exposure reduced mitochondrial ETC complex I, II, I + II, and III activities, thereby adversely affecting ATP-linked respiration. ATP amounts in mitochondria obtained from the liver of soman-treated rats indicated basal respiration, ATP synthesis, maximal respiration, and inhibited non-mitochondrial oxygen consumption. In fact, similar results have also been shown that soman intoxication entails a reduction in the cerebral level of ATP causing inhibition of cerebral glycolysis by inducing metabolism of glucose and pyruvate.⁴³⁾ Glaros et al. demonstrated that VX also disrupts anaerobic metabolism causing a dysfunction in glycolysis/TCA cycle by directly inhibiting mitochondrial isocitrate dehydrogenase (IDH2).⁴⁴⁾ This eventually leads to disordered mitochondrial energy metabolism (Fig. 11). Previous studies indicated that OPs could impair mitochondrial energy metabolism by altering mitochondrial respiration and respiratory chain enzyme activity^45,46) as well as energy production.^47–50) Recent studies have also demonstrated mitochondrial enzyme alterations are most likely the secondary effects of poisoning.⁵¹⁾ Additionally, suppressed complexes I, II, and IV and enhanced ROS production indicate an overall alteration of the electron transfer pattern, which results in absolute mitochondrial impairment associated with dichlorvos toxicity.⁵²⁾ This evidence supported our current results.

Fig. 11. Graphical Abstract

Furthermore, it was suggested that mitochondrial dysfunction may impair mitochondrial energy metabolism by inhibiting the ETC. This may promote oxidative damage because electrons constantly leak from the respiratory chain to generate deleterious ROS, resulting in liver injury.^53–55) The above data indicated subacute soman exposure markedly increased mitochondrial ROS production. Excessive ROS production further disrupts mitochondrial antioxidant defence and causes oxidate stress.⁵⁶⁾ It is widely accepted that oxidative stress results from increased ROS amounts, constituting a key contributor to liver damage. Next, we evaluated oxidative stress biomarkers in liver mitochondria to determine the involvement of oxidative stress in liver damage. Our data suggested repeated exposure to soman decreased SOD activity, GSH levels, and GSH/GSSG ratio while increasing MDA levels in liver mitochondria. The physiological activities of essential endogenous antioxidants, such as including GSH and SOD, are critical in preventing oxidant-associated damage to macromolecules. It has been reported MDA is the end-product of lipid peroxidation, which is formed by ROS degradation of polyunsaturated lipids.⁵⁷⁾ Previously, individual GSH and GSSG concentrations and GSSG/GSH ratio were assessed to quantitate oxidative stress in tissues.⁵⁸⁾ The above results showed that exposure to soman promotes mitochondrial oxidative stress through ROS production by mitochondria.

On the other hand, PGC-1α is important in modulating energy metabolism and mitochondrial function.⁵⁹⁾ In recent years, mitochondrial impairment involving PGC-1α dysregulation has been reported in liver metabolic diseases.^60,61) PGC-1α is involved in energy metabolic processes by regulating the expression of nuclear and mitochondrial genes, including electron transport system effectors and OXPHOS constituents, including ETC respiration submits (complexes I–IV).²³⁾ Therefore, our results indicated decreased mitochondrial electron transfer abilities for complexes I, II, I + II, and III, along with altered ATP-related respiration. This might result from inhibited mitochondrial PGC-1α protein. Earlier reports have confirmed that monocrotophos (MCP), a widely used neurotoxic organophosphate, induced mitochondrial dysfunctions in human mesenchymal stem cells and SHSY-5Y cells by AMPK/SIRT1/PGC-1α signaling cascade.⁶²⁾ Abd-Elhakim et al. demonstrated chlorpyrifos (CPF) induced reprotoxic effect could be related to the modulation of NR5A1, HSD17B3, and SIRT1/TERT/PGC-1α pathway in the testicular tissue.⁶³⁾ However, the molecular mechanism of soman induced PGC-1α has not been reported. In the present study, we observed a remarkable reduction of PGC-1α protein. This may be one of the reasons why soman damaged mitochondrial function. Therefore, future investigation using omics techniques would be useful for elucidating the molecular mechanisms of downregulation PGC-1α expression by soman treatment in the rat’s liver.

This study had limitations. Only 42 male SD rats were used in this study to establish a soman animal model to observe mitochondrial damage in the liver, which is a relatively small size. However, this is a preliminary study on this topic. Therefore, we plan to carry out future research with an extended experimental plan with a large sample size.

CONCLUSION

Subacute soman exposure causes liver damage by altering mitochondrial energy metabolism, resulting from suppressed ETC enzyme activities and mitochondrial respiration. Results showed soman could inhibit mitochondrial activity, disrupt energy metabolism, and increase ROS production to promote oxidative stress-induced toxicity by downregulating the protein level of PGC-1α. The present study unveils a mechanism for soman toxicity other than AChE suppression in the rat liver, suggesting a non-cholinergic mechanism for organophosphate toxicity. This novel mechanism involves mitochondrial energy metabolism impairment and oxidative stress. Future research on PGC-1 protein intervention may help unveil the exact molecular mechanism of soman-mediated mitochondrial energy metabolism disorder in liver injury.

Acknowledgments

We thank the reviewers for helpful comments on this article.

Conflict of Interest

The authors declare no conflict of interest.

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