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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2016 Jun 9;97(2):114–124. doi: 10.1111/iep.12183

Acute paraquat exposure determines dose‐dependent oxidative injury of multiple organs and metabolic dysfunction in rats: impact on exercise tolerance

Rômulo D Novaes 1,✉, Reggiani V Gonçalves 2, Marli C Cupertino 3, Eliziária C Santos 3, Solange M Bigonha 4, Geraldo J M Fernandes 5, Izabel R S C Maldonado 3, Antônio J Natali 6
PMCID: PMC4926049  PMID: 27277193

Summary

This study investigated the pathological morphofunctional adaptations related to the imbalance of exercise tolerance triggered by paraquat (PQ) exposure in rats. The rats were randomized into four groups with eight animals each: (a) SAL (control): 0.5 ml of 0.9% NaCl solution; (b) PQ10: PQ 10 mg/kg; (c) PQ20: PQ 20 mg/kg; and (d) PQ30: PQ 30 mg/kg. Each group received a single injection of PQ. After 72 hours, the animals were subjected to an incremental aerobic running test until fatigue in order to determine exercise tolerance, blood glucose and lactate levels. After the next 24 h, lung, liver and skeletal muscle were collected for biometric, biochemical and morphological analyses. The animals exposed to PQ exhibited a significant anticipation of anaerobic metabolism during the incremental aerobic running test, a reduction in exercise tolerance and blood glucose levels as well as increased blood lactate levels during exercise compared to control animals. PQ exposure increased serum transaminase levels and reduced the glycogen contents in liver tissue and skeletal muscles. In the lung, the liver and the skeletal muscle, PQ exposure also increased the contents of malondialdehyde, protein carbonyl, 8‐hydroxy‐2′‐deoxyguanosine, superoxide dismutase and catalase, as well as a structural remodelling compared to the control group. All these changes were dose‐dependent. Reduced exercise tolerance after PQ exposure was potentially influenced by pathological remodelling of multiple organs, in which glycogen depletion in the liver and skeletal muscle and the imbalance of glucose metabolism coexist with the induction of lipid, protein and DNA oxidation, a destructive process not counteracted by the upregulation of endogenous antioxidant enzymes.

Keywords: liver, lung, oxidative stress, skeletal muscle, pathology

Introduction

Paraquat (PQ) (1,1′‐dimethyl‐4,4′‐bipyridinium dichloride) is a quaternary ammonium herbicide that is highly toxic when absorbed through ingestion, skin contact or inhalation (Dasta 1978; Bismuth et al. 1990; Blanco‐Ayala et al. 2014). The systemic effect of PQ is related to the induction of oxidative stress by generating reactive oxygen species (ROS) through redox cycling by microsomal NADPH‐cytochrome P‐450 reductase (Bismuth et al. 1990), xanthine oxidase (Kelner et al. 1988; Bismuth et al. 1990) and mitochondrial NADH‐quinone oxidoreductase (Shimada et al. 1998; Suntres 2002; Xu et al. 2014). The high ROS production induces a non‐selective oxidation of biomolecules such as lipids, proteins and nucleic acids that lead to cell damage and eventually result in death (Fukushima et al. 2002; Suntres 2002; Sittipunt 2005; Dinis‐Oliveira et al. 2008; Blanco‐Ayala et al. 2014).

Population studies have shown that low‐level PQ exposure may result in respiratory symptoms such as chronic bronchitis and dyspnoea with wheezing (Castro‐Gutiérrez et al. 1997; Dalvie et al. 1999; Dinis‐Oliveira et al. 2008; Cha et al. 2012). In survivors of PQ poisoning, oxygen desaturation was observed during exercise tests, suggesting gas‐exchange abnormalities potentially associated with the lung damage (Schenker et al. 2004). Dalvie et al. (1999) showed a significant relation between respiratory health effects long term after PQ exposure and arterial oxygen desaturation during exercise among workers on deciduous fruit farms. These findings indicate that working with PQ under usual field conditions is associated with abnormal exercise physiology, manifested primarily by changes in respiratory dynamics and by a reduced resistance in aerobic exercises.

In an experimental study conducted by our research group, it was observed for the first time that long term after PQ exposure the rats showed an increased energy cost and reduced performance during aerobic exercise (Lacerda et al. 2009). These findings were consistent with alveolar collapse and impaired oxygen uptake induced by PQ toxicity. However, the relationship between organ damage and energy metabolism remains poorly understood. Furthermore, the acute metabolic effect of PQ exposure on exercise tolerance remains unknown. It has been demonstrated that blood lactate level can be increased in patients with respiratory failure due to acute lung injury and this production is proportional to the severity of lung disease (Borges et al. 2009). In view of its reproducibility and simplicity of administration, PQ has been widely used as an experimental model in the studies on tissue damage mediated by ROS (Bus & Gibson 1984; Lacerda et al. 2009; Novaes et al. 2012a,b). Taking into account that PQ triggers systemic oxidative stress and pro‐inflammatory response, this study was designed to investigate the dose‐dependent effects of PQ exposure on the morphology of the lung, the liver and the skeletal muscle and on oxidative status, glucose metabolism and exercise tolerance in rats.

Materials and methods

Animals and ethics

Adult male 16‐week‐old Wistar rats weighing 314.69 ± 5.85 g were kept under a controlled temperature (21 ± 2°C), a relative air humidity from 60 to 70% and a photoperiod (12‐/12‐h light/darkness). The animals had free access to rat chow and water.

Treatments

PQ was dissolved in 0.5 ml of 0.9% NaCl saline solution and injected intraperitoneally (i.p.). Control rats (SAL) were concurrently treated with 0.5 ml saline solution (0.9% NaCl) i.p. According to the treatment administered, 32 rats were randomized into four groups with eight animals each: Group 1 (SAL): 0.9% NaCl solution; Group 2 (PQ10): 10 mg/kg PQ solution (Fukushima et al. 1994); Group 3 (PQ20): 20 mg/kg PQ solution (Borges et al. 2009); and Group 4 (PQ30): 30 mg/kg PQ solution (Novaes et al. 2012a,b).

Exercise tolerance and metabolic parameters

Seventy‐two hours after PQ administration, each rat was subjected to an incremental aerobic running test until fatigue, according to the incremental running protocol described by Novaes et al. (2011). Briefly, the test was performed on the motor‐driven treadmill (Insight Instruments®, Ribeirão Preto, Brazil) at a constant slope of 5% with the starting speed at 10 m/min−1. Treadmill velocity was increased 1 m/min−1 every 3 min, and each rat ran until fatigue. Fatigue was defined as the point at which the animals were no longer able to keep pace with the treadmill and interrupted the race by over 10 s. Time until the fatigue (min), distance travelled (m), maximal speed (m/min) and workload were used as indexes of exercise tolerance. Workload (kgm) was calculated using the equation W = body mass (kg) × TTF (min) × treadmill speed (m/min) × sine θ (treadmill inclination), where TTF is time until the fatigue (Brooks et al. 1984).

Immediately before and every three minutes during the running test blood lactate and blood glucose levels were measured by Accutrend Lactate® (Roche, Basel, Switzerland) and OneTouch Ultra® (Jonson & Jonson, CA, USA), respectively. The transition point between the aerobic metabolism and anaerobic metabolism was determined by assessing the lactate threshold, which is represented by the inflection point at which the blood lactate levels exhibited an exponential growth and lost the linearity with the increases in exercise intensity (time and velocity) (Skinner & Mclellan 1980; APS 2006).

Euthanasia and organ oedema

Twenty‐four hours following the end of aerobic exercise protocol, the animals were euthanised with ketamine (10 mg/kg wt., i.p.) and xylazine (2 mg/kg wt., i.p.). The lungs, the liver and the gastrocnemius skeletal muscle of each animal were removed in totum and weighed. Fragments of the lung and liver tissues and the entire left gastrocnemius skeletal muscle were weighed and used to measure water content per tissue weight unity (ml/g). This parameter was determined after drying the fragments at 60°C for 96 h using the relation (wet weight – dry weight) × 103/wet weight (Novaes et al. 2012a).

Glycogen analysis

Glycogen was extracted according to the method described by Hassid and Abraham (1957). Briefly, the fresh liver and muscle samples (50 mg) were digested by heating (100°C) in 0.5 ml of 5 N KOH for 60 min. Glycogen was purified and precipitated by 99% ethanol in boiling water and then centrifuged at 8000 g for 20 min. The pellets obtained were resuspended in distilled water (1 ml) and 3 ml of anthrone solution (50 mg diluted in 50 ml of 84% H2SO4) and incubated for 10 min at 100°C. The absorbance was measured at 620 nm (Power Wave X, Winooski, VT, USA).

Hepatic function markers

After euthanasia, a total blood sample (2 ml) was collected by cardiac puncture and centrifuged at 3000 g for 20 min. The serum thus obtained was used for biochemical determination of total bilirubin and the hepatic enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) according to the instructions of commercial kits (Human in Vitro Diagnostics, Minas Gerais, Brazil).

Lipid, protein and DNA oxidation

For the analysis of tissue malondialdehyde (MDA), an end product of lipid peroxidation, aliquots of the frozen liver, lung and gastrocnemius muscle (100 mg) were homogenized in PBS, the homogenate was centrifuged, the supernatant was reacted with thiobarbituric acid and the formation of thiobarbituric acid‐reactive substance was monitored at 535 nm as described previously (Gutteridge & Halliwel 1990). Protein carbonyl (CnP) content was measured in the tissue pellets by adding 0.5 ml of 10 mM 2,4‐dinitrophenylhydrazine (DNPH). The reaction involved the derivatization of the carbonyl group with DNPH, leading to the formation of a stable 2,4‐dinitrophenyl (DNP) hydrazone product. The absorbance was measured spectrophotometrically at 370 nm (Levine et al. 1990). Total protein levels in the lung, the liver and the muscle homogenates were measured using the Bradford method (Bradford 1976).

DNA oxidative damage was investigated after nucleic acid extraction and purification according to Coombs et al. (1999). Briefly, the fragments of the lung, the liver and the skeletal muscle (20 mg) were incubated with proteinase K (10 mg/ml) at 55°C. Genomic DNA was extracted by the phenol–chloroform (1:1) method, and 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) content was determined according to Kakimoto et al. (2002). Briefly, DNA was resuspended in 10 mM/l Tris–HCl and 0.1 mM/l EDTA. Five microlitres of 200 mM/l sodium acetate buffer and 5 μg nuclease P1 (Aldrich Chemical Co., Milwaukee, WI, USA) were added to 45 μl DNA samples. The mixtures were incubated at 37°C for 1 h to digest the DNA to nucleotides. Then, 5 μl of 500 mM/l Tris–HCl, 10 mM/l MgCl2 and 0.6 units of alkaline phosphatase (Aldrich Chemical Co.) were added to the samples. The mixtures were incubated at 37°C for 1 h to hydrolyse the nucleotides to nucleosides. The nucleoside samples were used for the determination of 8‐OHdG by competitive enzyme‐linked immunosorbent assay (Cell Biolabs Inc., San Diego, CA, USA).

Antioxidant enzymes

The lung, the liver, and the muscle fragments (500 mg) were homogenized in 50 mM phosphate buffer, and the resulting suspension was centrifuged at 3000 g (4°C for 10 min). The supernatant was used to measure the enzyme activity. Catalase (CAT) was evaluated according to the method described by Aebi (1984), by measuring the rate of decomposition of hydrogen peroxide (H2O2). Catalase activity was measured in a reaction mixture (3 ml) containing 100 mM Na2HPO4 buffer pH 6.8 (2 ml), 30 mM H2O2 (0.5 ml) and 0.5 ml enzyme extract. The decrease in absorbance due to H2O2 depletion was monitored spectrophotometrically at 240 nm for 3 min (PAUV.1600, Pro‐analysis, São Paulo, SP, Brazil). Superoxide dismutase (SOD) activity was based on the generation of superoxide radicals produced by xanthine and xanthine oxidase, which react with 2‐(4‐iodophenyl)‐3‐(4‐nitrophenol)‐5‐phenyltetrazolium chloride (INT) to form a red formazan dye (Sarban et al. 2005). The standard assay was performed in 3 ml of 0.05 M potassium phosphate buffer at pH 7.8 containing 10−4 M EDTA at 25°C. The reaction mixture contained 1 × 10−5 M ferricytochrome c, 5 × 10−5 M xanthine and xanthine oxidase to produce a rate of reduction of ferricytochrome c at 550 nm. One unit of SOD was defined as the amount of enzyme necessary to produce 50% inhibition in the INT reduction rate. The reaction was read at 550 nm by spectrophotometry (PAUV.1600, Pro‐analysis, São Paulo, SP, Brazil).

Morphological analysis

Tissue fragments were placed in Karnovsky's solution for 24 h and embedded in glycol–methacrylate (for the lung and the gastrocnemius skeletal muscle [right side]) or paraffin (for the liver). Sections (3 μm thick) were cut with a Multicut 2045® rotary microtome (Reichert‐Jung, Jena, Germany) and stained with haematoxylin and eosin (for the lung), carmine method for glycogen (for the liver) or toluidine blue and basic fuchsine (for the skeletal muscle).

The presence and the intensity of the inflammatory process in the lung tissue and skeletal muscle were evaluated by the correlation index between the total number of inflammatory cells observed in control and intoxicated animals. These cells were evaluated in a test area of 3.4 × 103 μm2 at the magnification of ×1000 across 10 random, non‐coincident microscopic fields of each animal (Lacerda et al. 2009). Forty random fields from each staining method and group were analysed with ×20 objective lens. For the stereological analysis, a test system with 300 points was used in a standard test area of 73 × 103 μm2 (Mandarim‐de‐Lacerda 2003). The stereological parameter of volume density (Vv) was estimated by point counting for alveolar septum [septum] and alveolar space [space] in the lung tissue, hepatocytes [h] and glycogen cytoplasm inclusions [glyc] in the liver tissue and muscle fibres in the skeletal muscle using the following formula: Vv = P P [structure]/P T, where P P is the number of points that hit the structure and P T is the total test points (Mandarim‐de‐Lacerda 2003).

The slides were visualized and the images were captured using a light microscope (Olympus BX‐60®, Tokyo, Japan) connected to a digital camera (Olympus QColor‐3®, Tokyo, Japan). All morphological analyses were performed using the image analysis software Image Pro‐Plus 4.5® (Media Cybernetics, Silver Spring, MD, USA).

Statistical analysis

Data were expressed as mean ± standard deviation (mean ± SD). The normality of the data distribution was verified using the D'Agostino–Pearson normality test. Biochemical and biometric data were subjected to the one‐way anova test, followed by Tukey's test for multiple comparisons. Kruskal–Wallis test was used to compare the stereological data. A statistical significance was established at P < 0.05.

Ethical approval

The protocols were approved by the internationally accepted laboratory animal use and care stated in the guidelines and rules of the institutional Ethics Committee (UFV approval protocol 064/2010).

Results

Exercise tolerance and serum biochemical analysis

Animals treated with PQ showed an anticipation of the lactate threshold and a significant reduction in all indexes of exercise tolerance (Figures 1 and 2). In these groups, the animals showed a short time period before the onset of the fatigue, reduced distance travelled and maximal speed workload compared to SAL animals. All parameters were changed in a dose‐dependent way (Figure 2).

Figure 1.

Figure 1

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Temporal evolution of blood lactate levels in control and paraquat (PQ)‐exposed rats during the progressive running test. Lactate threshold is represented by the inflection point at which the blood lactate levels exhibited an exponential growth and lost a linearity with the increases in exercise intensity. The animals were exposed to different doses of PQ (10, 20 or 30 mg/kg). Control animals (SAL) received 0.9% NaCl saline solution.

Figure 2.

Figure 2

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Parameters of exercise tolerance in control and paraquat (PQ)‐exposed rats. The animals were exposed to different doses of PQ (10, 20 or 30 mg/kg). Control animals (SAL) received 0.9% NaCl saline solution. TTF, time to fatigue; W, workload. Data are expressed as mean ± SD. a,b,c Different letters in columns indicate a statistical difference between the groups (P < 0.05), and groups that have some common letter do not differ statistically, one‐way anova followed by Tukey's test post hoc test.

There was a significant difference in the blood glucose levels before and after exercise in the groups PQ20 and PQ30 compared to groups SAL and PQ10, indicating a different glucose consumption during rest and exercise. In the groups PQ20 and PQ30, there was a significant reduction in blood glucose levels during exercise. Lactate levels at rest were similar between the groups. At the fatigue point, the lactate levels were significantly higher in the animal groups treated with PQ compared to the animals treated with SAL, with the worst results being observed in the group PQ30, which indicates a dose‐dependent effect (Table 1).

Table 1.

Blood glucose and lactate levels in rest and at the fatigue point in the running test in control and paraquat‐exposed rats

Groups Glucose (mg/dl) Lactate (mM/l)
Rest Fatigue Rest Fatigue
SAL 115.13 ± 3.03a 101.38 ± 3.08a 1.65 ± 0.08a 4.04 ± 0.07a
PQ10 100.63 ± 2.73a 87.63 ± 2.56b 1.68 ± 0.09a 4.41 ± 0.09b
PQ20 83.38 ± 4.89b 57.75 ± 3.67c 1.71 ± 0.10a 4.96 ± 0.10c
PQ30 80.51 ± 6.07b 53.88 ± 3.66c 1.69 ± 0.08a 5.08 ± 0.09c
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Animals were exposed to different doses of paraquat (PQ 10, 20 or 30 mg/kg). Control animals received saline (SAL) alone. Data are expressed as mean ± SD. a,b,c Different letters in columns indicate a statistical difference between the groups (P < 0.05), and groups that have some common letter do not differ statistically, one‐way anova followed by Tukey's test post hoc test.

Oxidative damage and antioxidant enzymes

The oxidative damage to lipid, proteins and DNA triggered by PQ showed a dose‐dependent effect in all the tissues investigated. MDA, CnP and 8‐OHdG levels were higher in the liver and the lung from the animals in the PQ20 and PQ30 groups compared to SAL (P < 0.05). In the liver and the skeletal muscle, these parameters were similar in groups PQ20 and PQ30. In the group PQ30, CnP and 8‐OHdG levels were higher in the skeletal muscle compared to the other groups (Figures 3 and 4).

Figure 3.

Figure 3

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Malondialdehyde (MDA) and carbonyl protein (CnP) levels in the lung, the liver and the skeletal muscle from control and paraquat (PQ)‐exposed rats. The animals were exposed to different doses of PQ (10, 20 or 30 mg/kg). Control animals (SAL) received 0.9% NaCl saline solution. Data are expressed as means ± SD. a,b,c Different letters indicate a statistical difference between the groups (P < 0.05), one‐way anova followed by Tukey's post hoc test.

Figure 4.

Figure 4

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8‐Hydroxy‐2′‐deoxyguanosine (8‐OHdG) levels in the liver, the lung and the skeletal muscle from control and paraquat (PQ)‐exposed rats. The animals were exposed to different doses of PQ (10, 20 or 30 mg/kg). Control animals (SAL) received 0.9% NaCl saline solution. The box represents the interquartile interval with the median indicated (horizontal line), and whiskers represent the superior and inferior quartiles. a,b,c Different letters indicate a statistical difference between the groups (P < 0.05), one‐way anova followed by Tukey's post hoc test.

Catalase and SOD activities in the liver tissue were significantly higher in group PQ30 compared to those in SAL and PQ10 ones. The animals in group PQ20 also showed high levels of SOD compared to the animals in SAL and PQ10 (P < 0.05). In the lung tissue, the activities of both enzymes were significantly higher in groups PQ10 and PQ20 compared to those in the SAL (P < 0.05). Both enzymes presented an increased activity in all groups exposed to PQ (P < 0.05) (Figure 5).

Figure 5.

Figure 5

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Catalase (CAT) and superoxide dismutase (SOD) activities in the liver, the lung and the skeletal muscle from control and paraquat (PQ)‐exposed rats. The animals were exposed to different doses of PQ (10, 20 or 30 mg/kg). Control animals (SAL) received 0.9% NaCl saline solution. Data are expressed as means ± SD. a,b,c Different letters indicate a statistical difference between the groups (P < 0.05), one‐way anova followed by Tukey's post hoc test.

Serum enzymes

Serum ALT levels were significantly higher in the animals in PQ10, PQ20 and PQ30 group compared to those in the SAL group (P < 0.05). Higher AST levels were found in the group PQ30 compared to the other groups (P < 0.05). The total bilirubin levels were significantly higher in all animals treated with paraquat (P < 0.05) compared to those in SAL. For all parameters, a dose‐dependent effect of PQ exposure was observed (Figure 6).

Figure 6.

Figure 6

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Hepatic function markers in serum from control and paraquat (PQ)‐exposed rats. The animals were exposed to different doses of PQ (10, 20 or 30 mg/kg). Control animals (SAL) received 0.9% NaCl. ALT, alanine aminotransferase; AST, aspartate aminotransferase. Data are expressed as means ± SD. a,b,c Different letters indicate a statistical difference between the groups (p < 0.05), one‐way anova followed by Tukey's post hoc test.

Oedema and organ morphology

The water content in all organs was significantly higher in PQ20 and PQ30 groups compared to PQ10 and SAL groups (P < 0.05) (Table 2). Figure 7 shows that the histological organization of lung tissue samples in PQ10, PQ20 and PQ30 groups demonstrated progressive abnormalities, with thick‐lined alveolar septa, alveolar collapse and increased tissue cellularity. PQ30 group showed vascular congestion, an intense area of haemorrhage and an inflammatory infiltrate. In this group, the alveolar obliteration by the accumulation of fibrinous exudates was evident. The morphological analysis confirmed the histopathological abnormalities, showing that the volume density of alveolar spaces (Vv[space]) was significantly reduced and the number of inflammatory cells was significantly increased in all groups exposed to PQ compared to SAL (P < 0.05) (Table 2). In the liver tissue, the proportion of the histological area occupied by hepatocytes (Vv[h]) was higher and that of glycogen‐containing cytoplasmic inclusions (Vv[glyc]) was reduced in PQ20 and PQ30 groups compared to SAL group (P < 0.05) (Figure 7, Table 2). In the skeletal muscle from PQ20 and PQ30 groups hypertrophied fibres were observed with increased thickness (P < 0.05) and heterogeneous staining pattern (Figure 7). A marked reduction in the hepatic and muscle glycogen contents was confirmed in the same groups by biochemical analysis (P < 0.05) (Table 2).

Table 2.

Morphological and biochemical parameters of the lung, the liver and the skeletal muscle from control and paraquat‐exposed rats

Parameters SAL PQ10 PQ20 PQ30
Lung
Water content [ml/g] 0.55 ± 0.02a 0.59 ± 0.02a 0.68 ± 0.02b 0.77 ± 0.01c
 Vv[space] (%) 61.32 ± 1.78a 52.79 ± 2.4b 42.88 ± 3.15c 32.02 ± 3.79d
IC (N/170 × 103 μm2) 43.13 ± 1.95a 70.25 ± 3.53b 90.00 ± 3.00c 99.63 ± 4.54d
Liver
Water content [ml/g] 0.51 ± 0.04a 0.53 ± 0.05a 0.62 ± 0.02b 0.70 ± 0.04c
 Vv[h] (%) 74.77 ± 2.31a 77.21 ± 1.53a 82.15 ± 2.06b 84.14 ± 2.45b
 Vv[glycogen] (%) 44.03 ± 2.15a 38.19 ± 2.48b 27.61 ± 3.43c 15.96 ± 4.34d
Glycogen (μM/g) 90.21 ± 12.67a 82.11 ± 10.48a 40.29 ± 9.50b 18.13 ± 6.00c
Skeletal muscle
Water content [ml/g] 0.49 ± 0.05a 0.50 ± 0.06a 0.63 ± 0.04b 0.65 ± 0.04b
Fibre thickness (μm) 18.59 ± 9.83a 21.15 ± 7.44a , b 36.45 ± 11.17b , c 42.37 ± 10.09c
 Vv[fibres] (%) 94.03 ± 3.29a 92.60 ± 5.23a 93.14 ± 4.37a 92.22 ± 4.25a
IC (N/170 × 103 μm2) 47.21 ± 10.15a 44.61 ± 9.63a 49.91 ± 12.94a 46.50 ± 11.33a
Glycogen (μM/g) 78.15 ± 10.31a 75.10 ± 11.59a 48.22 ± 12.75b 20.04 ± 8.51c
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Animals were exposed to different doses of paraquat (PQ 10, 20 or 30 mg/kg). Control animals received saline (SAL) alone. Vv, volume density; space, alveolar space; IC, inflammatory cells. Data are expressed as mean ± SD. a,b,c,d Different letters in rows indicate a statistical difference between the groups (P < 0.05), and groups that have some common letter do not differ statistically, Kruskal–Wallis test.

Figure 7.

Figure 7

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Representative photomicrographs of the lung, the liver and the skeletal muscle from control and paraquat (PQ)‐exposed rats obtained by bright‐field microscopy. Lung: left column (haematoxylin and eosin staining, bar = 50 μm); Liver: middle column (carmine method for glycogen [red spots], bar = 80 μm); Soleus skeletal muscle: right column (toluidine blue and basic fuchsine, bar = 40 μm). The animals were exposed to different doses of PQ 10, 20 or 30 mg/kg). Control animals (SAL) received 0.9% NaCl saline solution. In PQ30, the animals presented an alveolar accumulation of fibrinous exudate, glycogen depletion and muscle fibre hypertrophy.

Discussion

Several studies have investigated the respiratory repercussions after PQ exposure; however, these publications were restricted to evaluating spirometric variables in basal conditions (Castro‐Gutiérrez et al. 1997; Cha et al. 2012) or during physical efforts (Dalvie et al. 1999; Schenker et al. 2004). Considering the possible effects of PQ on energy metabolism, this study investigated the impact of this herbicide on blood glucose and lactate levels, two important molecules with a direct influence on exercise tolerance (Wasserman et al. 1990; De Backer et al. 1997). In fact, the anticipation of the lactate threshold, a premature reduction in blood glucose level and an elevated lactate production were evident during exercise in animals exposed to PQ. Taken together with the reduction in all parameters of exercise tolerance, these findings suggested that intoxicated animals had higher energy expenditure and more active anaerobic metabolism compared to control animals. It has been demonstrated that blood lactate level can be increased in patients with respiratory failure and this production is proportional to the severity of lung disease (Weibel 1973; Routsi et al. 1999). These characteristics are similar to those found in this work, where at the fatigue point the blood lactate levels were significantly higher in the groups treated with PQ, which induced the multiple organ damage in a dose‐dependent way. Although O2 consumption was not investigated, the marked lung injury (especially lung oedema, alveolar inflammatory infiltration and collapse) is coherent with an impaired gas exchange (Weibel 1973; Piantadosi & Schwartz 2004; Lacerda et al. 2009). Thus, under the conditions of limited O2 rate, metabolic deviation towards a more active anaerobic component also represents a typical physiological strategy to ensure an adequate energy production (Wasserman et al. 1990; De Backer et al. 1997). As observed in the present study, in basal conditions (rest), this mechanism can be enough to determine the adequate energy availability and at the same time to maintain an adjusted lactate clearance, restricting its blood accumulation even in PQ‐intoxicated animals. Under resting conditions, this proposition is reinforced by the findings reported by Routsi et al. (1999), who asserted that lactate production does not seem to be attributable to lung tissue hypoxia. However, the high energy requirements during exercise and a potential deficiency in O2 uptake in these animals implies abnormally increased activation of the anaerobic pathway and lactate production, which is not compensated by counter‐regulatory enzymatic clearance and leads to lactate accumulation, a molecule recognizably involved in metabolic acidosis and fatigue (Wasserman et al. 1990; De Backer et al. 1997).

Admittedly, PQ metabolism can also interfere directly in energy production (Fukushima et al. 1994; Lei et al. 2014; Xu et al. 2014). Thus, a lower energy efficiency due to the increased anaerobic metabolism of glucose is proposed as a mechanism for partially explaining the reduced exercise tolerance in PQ‐exposed animals. A more glycolytic metabolism is consistent with an increased production of lactate and with the anticipation of fatigue point during a progressive exercise protocol, as observed in the present study. This mechanism is not unrealistic considering the negative influence of PQ on the electron transport chain and hence in the aerobic process of energy production. The toxic effect of PQ on mitochondria and disturbances in energy production has been demonstrated clearly in previous studies (Fukushima et al. 1994; Lei et al. 2014). PQ changes energy availability by reducing purine levels (ATP, ADP and AMP) (Fukushima et al. 2002; Lei et al. 2014). Apparently, this effect is mediated by pro‐oxidant events triggered during PQ metabolism. In this process, the high ROS production and the reduction in the activity of antioxidant enzymes cause dysfunction in aerobic metabolism by uncoupling several enzymatic complexes integrated within the electron transport chain (Fukushima et al. 1994; Dinis‐Oliveira et al. 2008; Lei et al. 2014).

Playing an important regulatory role in the metabolism of energy substrates, the liver and skeletal muscles are pivotal organs involved in glucose homeostasis (Ferrer et al. 2003; Emhoff et al. 2013). At rest and during exercise, the mobilization of glucose from hepatic and skeletal muscle glycogen storage is fundamental for the maintenance of basal blood glucose levels and provides energy substrates for muscle contraction (Skinner & Mclellan 1980; Borges et al. 2009; Emhoff et al. 2013). In the present study, even at rest, PQ exposure reduced blood glucose levels in a dose‐dependent manner, a finding related to the depletion of glycogen cytoplasm storages in hepatocytes and skeletal muscle fibres. These results are consistent with lactate levels at the fatigue point, reinforcing the hypothesis that glycogenolysis and glucose anaerobic metabolism are upregulated in PQ‐exposed animals in an attempt to provide the additional energy required during exercise, determining high lactate production. Although the influence of PQ on glucose metabolism has been described (Rose et al. 1974; Giri et al. 1979, 1983), this effect is still not completely understood. It has been reported that high levels of circulating catecholamines and corticosteroids in acute PQ poisoning could be associated with a marked glycogenolysis, gluconeogenesis and the depletion of liver glycogen storages (Rose et al. 1974; Giri et al. 1979; Suntres 2002; Borges et al. 2009). There is previous evidence which indicates that high serum catecholamine and corticosteroid levels in PQ poisoning are primarily related to oxidative insults directed at the adrenal gland cortex and medulla, especially lipid peroxidation, resulting in membrane instability and hormone translocation (Rose et al. 1974; Giri et al. 1979, 1983).

ROS production is directly implicated in PQ toxicity (Konstantinova & Russanov 1999; Novaes et al. 2012a,b; Lei et al. 2014; Xu et al. 2014). There is abnormally high induction of radical and non‐radical reactive species during PQ metabolism, especially superoxide anion (O2•−), hydroxyl radical (OH−) and hydrogen peroxide (H2O2), and this is responsible for organ damage as a systemic manifestation of oxidative stress (Fukushima et al. 2002; Sittipunt 2005; Dinis‐Oliveira et al. 2008; Blanco‐Ayala et al. 2014). In the present study, oxidation of lipid, protein and DNA was confirmed in all three organs investigated. Lipid peroxidation plays a pivotal role in PQ toxicity because metabolites generated through PQ metabolism oxidize unsaturated fatty acids of the cellular membranes, leading to the production of lipid peroxyl radicals (LOO−), alkoxy radicals (RO−) and MDA, which cause an oxidative damage in a cyclic process of lipid peroxidation (Dasta 1978; Bismuth et al. 1990; Fukushima et al. 2002; Novaes et al. 2012a). In addition, the high 8‐OHdG tissue levels in animals exposed to PQ indicate that the oxidative damage to genomic DNA occurs in a dose‐dependent manner. Although DNA oxidation has been observed in all three organs investigated, the higher levels of 8‐OHdG in the lung and the liver were associated with PQ toxicity, because these organs are the main targets of PQ accumulation and metabolism (Amirshahrokhi & Bohlooli 2013; Blanco‐Ayala et al. 2014). The genotoxic potential of PQ was proven in previous studies (Ali et al. 1996; Tokunaga et al. 1997). Apparently, DNA damage is a secondary effect of this herbicide, which is mediated by the high tissue levels of reactive metabolites produced as by‐products of PQ enzymatic processing (Ali et al. 1996; Tokunaga et al. 1997). This mechanism reinforces the evidence that the oxidative stress triggered by several xenobiotics, including PQ, can act as a danger signal for the induction of systemic effects in the organism, negatively influencing the structure and function of multiple organs and tissues (Tokunaga et al. 1997; Nikitaki et al. 2015).

To counteract the tissue injury, antioxidant mechanisms are activated as a first line of defence against ROS damage. CAT and SOD play a determinant role as endogenous antioxidant enzymes. In the present study, PQ exposure increased CAT and SOD activities in all organs investigated. There is a sufficient evidence that PQ stimulates the endogenous antioxidant enzymatic system (Huang et al. 1997; Novaes et al. 2012a,b; Amirshahrokhi & Bohlooli 2013). However, taking into account that there is active accumulation of PQ pneumocytes, the high concentrations of this herbicide may cause severe lung injury mediated by a metabolic exhaustion process, and characterized by the attenuation of macromolecule biosynthesis and activity, including antioxidant enzymes (Suntres 2002; Novaes et al. 2012a). An in vitro study demonstrated that the low levels of ROS triggered by PQ stimulates cell proliferation (Huang et al. 1997). However, the same authors also showed that SOD is particularly sensitive to high levels of ROS (especially O2•−), which inhibits this enzyme, increasing cell susceptibility to reactive injury and death.

Confirming the findings of this study, lesions in other organs (i.e. the liver and the skeletal muscle) induced by PQ are less evident compared to the manifestations seen in the lungs (Novaes et al. 2012a,b; Amirshahrokhi & Bohlooli 2013). However, despite the differences seen in the organs affected, overall the pathological manifestations are mainly determined by direct pro‐oxidant processes, especially lipid peroxidation (Dasta 1978; Bismuth et al. 1990; Fukushima et al. 2002; Novaes et al. 2012a; Blanco‐Ayala et al. 2014). During PQ exposure, reactive damage to cell macromolecules is potentially associated with functional as well as morphological and biochemical extraction manifestations of organ damage (Novaes et al. 2012b; Amirshahrokhi & Bohlooli 2013). This characteristic was clearly observed in the liver tissue by the increased serum levels of ALT, AST and total bilirubin in PQ groups, indicating an impaired integrity of cell membrane of hepatocytes. There is a close correlation between the markers of oxidative and functional damage and morphological changes in target organs (Novaes et al. 2012a,b). In the present study, it was observed a morphological reorganization of all the investigated organs, especially at the highest doses of PQ. As a typical manifestation of PQ toxicity, there was a severe subversion of lung structure with an evident inflammatory infiltrate and alveoli collapse. Although this is the first study showing a potential role of liver and skeletal muscle in exercise tolerance during PQ intoxication, it is still not understood to what extent the damage in these organs contributes to restricting exercise tolerance, an issue that requires further investigation.

Taken together, the results indicated that PQ exposure induced a dose‐dependent toxic effect in multiple organs with a marked negative influence on exercise tolerance. The ability of PQ to impair the physical performance coexisted with the liver and muscle glycogen depletion, glucose metabolism dysfunction and anticipation of the lactate threshold during the progressive exercise, subversion of the oxidative status and morphofunctional pathological remodelling of the lung, the liver and the skeletal muscle. Considering all the systemic effects of PQ poisoning, this study provides new evidence that impairment of exercise physiology triggered by this herbicide is complex and multifactorial, and that besides a lung involvement, liver and skeletal muscle injury also have a potential role the in reduced exercise tolerance.

Disclosure statement

The authors declare that there is no conflict of interest.

Author contributions

All listed authors met ICMJE authorship criteria, and nobody who had qualified for authorship had been excluded. The authors contributed to research design, acquisition, analysis and interpretation of data; drafted the manuscript or revised it critically; approved the final version of the manuscript.

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