Hyperbaric oxygen treatment and N -acetylcysteine ameliorate acetaminophen-induced liver injury in a rat model

Abstract

An overdose of acetaminophen (APAP) produces centrilobular hepatocellular necrosis. We aimed to investigate the hepatoprotective effects of N-acetylcysteine (NAC) only and hyperbaric oxygen (O₂) treatment (HBOT) combined with NAC, and their anti-inflammatory properties in liver tissue. In the current study, a total of 32 male Sprague Dawley rats were divided into 4 groups: sham, APAP, NAC, and NAC + HBOT. In the APAP, NAC, and NAC + HBOT groups, liver injury was induced by oral administration of 1 g/kg APAP. The NAC group received 100 mg/kg NAC per day. NAC + HBOT group received intraperitoneal injection of 100 mg/kg/day NAC and were given HBOT at 2.8 ATA pressure with 100% O₂ inhalation for 90 min every 12 h for 5 days. Rats in the sham group received distilled water only by gastric tube. All animals were killed on day 6 after APAP or distilled water administration. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, hepatic neopterin, tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6) levels were measured. There was a significant increase in serum AST and ALT activities in the APAP group compared with the sham group (in both p = 0.001). NAC and NAC + HBOT groups had significant decreases in hepatic neopterin, TNF-α, and IL-6 levels compared with the APAP group. APAP administration caused extensive hepatic necrosis. NAC and NAC + HBO treatments significantly reduced APAP-induced liver injury. Our results showed that the liver damage in APAP toxicity was attenuated by NAC and NAC + HBO treatments. NAC + HBOT exhibit hepatoprotective activity against APAP-induced liver injury in rats.

Introduction

Acetaminophen (APAP) is a safe analgesic and antipyretic drug and is commonly used in the world. However, overdose of APAP induces massive hepatocyte death with high morbidity and mortality. ¹ APAP-induced acute liver injury is the most frequent cause of liver failure in Western countries and the United States. ¹ APAP intoxication is characterized by centrilobular hepatocellular necrosis. ²

Currently, the underlying mechanisms of APAP toxicity are still not clear. Following exposure to toxic doses of APAP, APAP is converted to N-acetyl-p-benzoquinoneimine (NAPQI) by cytochrome P450. NAPQI cause inflammatory response, the production of cytokines, and hepatocyte death. ^1,2 Recent evidences have suggested that several inflammatory cells and soluble mediators play roles in APAP-induced hepatotoxicity. ³ In the inflammatory response dependent on APAP-induced liver injury, Kupffer cells and hepatic macrophages are essential players. ⁴ Activated macrophages appear to play a key role in the APAP-induced liver injury process and produce pro-inflammatory cytokines, including TNF-α, interleukins (IL-6, IL-1β, and IL-1α), and interferon-γ (IFN-γ). ^{5 –9}

Hyperbaric oxygen (O₂) treatment (HBOT) is defined as the inhalation 100% O₂ at greater than one atmosphere absolute (ATA) in a pressurized chamber. Generally, HBOT is administered at pressures of 2.0 ATA and above in many clinical applications. ¹⁰ Some studies have reported improvements using HBOT in several animal models. ^{11 –16} Recently, several studies have suggested that HBOT also has potent anti-inflammatory effects. ¹⁰

N-Acetylcysteine (NAC) is a precursor of reduced glutathione and also a potent anti-inflammatory agent. ¹⁷ It is an antidote against APAP overdose hepatotoxicity in clinical practice. Various studies have reported the antioxidant and anti-inflammatory roles of NAC in APAP-induced liver injury. ^18,19 NAC also decreases the synthesis and release of pro-inflammatory cytokines. ²⁰

In the present studies, we investigated the hepatoprotective effects of NAC and HBOT in parallel with their anti-inflammatory properties against APAP-induced liver injury in a rat model.

Materials and methods

Animals

Animal care and all experimental procedures were performed in accordance with the European Union Directive 609/86 for care and use of laboratory animals. The animals were housed in an air-conditioned room at 25°C with a 12-h dark/light cycle and were fed with standard rodent chow and tap water ad libitum. A total of 32 male Sprague Dawley rats (230–250 g) were obtained from the Rat Experimental Animals Laboratory, Stock Company, Yenimahalle, Ankara, Turkey. All procedures involving the use of laboratory animals were reviewed and approved by the Animal Ethics Committee of Gulhane Military Medical Academy, Etlik, Ankara, Turkey.

Experimental protocol

Rats were randomly divided into four groups with eight animals in each group. The groups include sham, control (only APAP treated), NAC (APAP + NAC therapy), and NAC + HBOT (APAP + NAC therapy + HBOT). The APAP, NAC, and NAC + HBOT groups of animals were given a single dose of 1 g/kg body weight of APAP (MSB Drug Factory, Ankara, Turkey; suspended in 3 mL hot distilled water at 50°C) by gastric tube. Rats in the sham group received distilled water by gastric tube. NAC and HBO treatments were performed 24 h after the administration of APAP. The rats in the NAC and NAC + HBOT groups were administrated with NAC at a dose of 100 mg/kg/day via intraperitoneal route for five consecutive days. The NAC + HBOT group underwent HBOT at 2.8 ATA pressure with 100% O₂ inhalation for 90 min every 12 h for 5 days. Animals were killed on day 6 after APAP administration. Whole blood was drawn from the heart. The abdomen was opened, and livers were removed and cleaned. Liver tissue samples were stored in 10% formalin solution for histological analysis. The remaining liver tissues were immediately frozen in liquid nitrogen and stored in a deep freezer at −80°C until all assays.

Tissue preparations

The frozen liver tissues were homogenized in phosphate buffer solution (pH 7.4) by means of a homogenizator (Heidolph Diax 900, Heidolph Elektro GmbH, Kelhaim, Germany) on an ice cube. Homogenates were centrifuged at 14,000 r/min in 4°C for 10 min. The supernatants were used for entire assays. The protein content of liver homogenates was measured by the method described by Lowry et al. using bovine serum albumin as the standard. ²¹

Biochemical parameters

Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured with a spectrophotometric technique by the Olympus AU-2700 autoanalyzer using commercial kits (Olympus, Hamburg, Germany) and are expressed in units per liter. Liver neopterin levels were measured with a high-performance liquid chromatography device (1200 Series System, Agilent Technologies, Santa Clara, CA, USA), using the method defined by Agilli et al. ²²

Liver cytokine assays

Levels of tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) were measured by enzyme-linked immunosorbent assay kits (Bender MedSystems GmbH, Vienna, Austria).

Liver histopathology

For histopathological analysis, liver sections were cut into equal parts of 5 μm thickness, fixed in 4% buffered neutral formalin solution for 24 h, and embedded in paraffin wax. Then, all the sliced sections were stained with hematoxylin and eosin (H&E). Slides were examined under a light microscope. Each slide was independently evaluated and scored, while the examiner was unaware of the group to which the specimen belonged. Liver injury was scored using the parameters such as sinusoidal dilatation (0–4), inflammatory cell infiltration (0–6), hemorrhage (0–6), and necrosis (0–8) defined by Gul et al. ²³

Statistical analysis

All the statistical analyses were performed using Statistical Package for Social Sciences 15.0 software (SPSS Inc., Chicago, Illinois, USA). For the overall comparisons, we used Kruskal–Wallis test. For the pairwise comparisons, we used Bonferroni-corrected Mann–Whitney U test with respect to distribution. χ ² test was used for comparing the grades of liver injury. The results were expressed as median (minimum–maximum) and frequencies (%). The values of p < 0.05 and p < 0.0083 were considered statistically significant for overall comparisons and for Bonferroni-corrected pairwise comparisons, respectively.

Results

No deaths were observed in all the groups of rats that were given APAP or distilled water during 24-h period. All biochemical parameters belonging to the four experimental groups are shown in Table 1. The serum activities of AST and ALT enzymes are widely used as biochemical markers for evaluation of liver injury. As expected, serum AST and ALT activities were different among all groups (in both p = 0.001), after treatment with APAP. Administration of the toxic dose of APAP to NAC + HBOT group after 24 h showed significant decrease in the serum ALT and AST activities compared with the APAP-treated (control) group (Table 1).

Table 1.

Biochemical findings in all the experimental groups.^a

Parameters	Groups				p b (overall)	p c (I vs. II)	p c (I vs. III)	p c (I vs. IV)	p c (II vs. III)	p c (II vs. IV)	p c (III vs. IV)
	Sham (I)	APAP (II)	NAC (III)	NAC + HBOT (IV)
Serum AST (U/L)	67 (56–72)	132 (109–211)	92 (74–105)	97 (84–115)	0.001	0.001	0.001	0.001	0.001	0.001	0.293
Serum ALT (U/L)	31 (17–53)	104 (67–183)	59 (20–74)	53 (30–65)	0.001	0.001	0.013	0.007	0.002	0.001	0.370
Liver neopterin (pmol/mg protein)	41 (29–61)	198 (179–234)	127 (109–175)	95 (77–111)	0.001	0.001	0.001	0.001	0.001	0.001	0.001
Liver TNF-α (pg/mg protein)	130 (108–172)	254 (224–516)	209 (114–243)	163 (120–188)	0.001	0.001	0.009	0.058	0.014	0.001	0.035
Liver IL-6 (pg/mg protein)	1000 (688–1128)	1583 (1314–2368)	1152 (1061–1268)	1008 (747–1322)	0.001	0.001	0.009	0.600	0.001	0.001	0.093

AST: aspartate aminotransferase; ALT: alanine aminotransferase; TNF-α: tumor necrosis factor α; IL-6: interleukin 6; APAP: acetaminophen; NAC: N-acetylcysteine; HBOT: hyperbaric oxygen treatment.

^aAll data were expressed as median (minimum-maximum).

^bKruskal–Wallis Test.

^cBonferroni corrected Mann Whitney U Test [statistically significant level (p < 0.0083)].

We measured the concentrations of liver TNF-α, IL-6, and neopterin levels in rats after APAP exposure. We found that administration of APAP markedly increased liver TNF-α, IL-6, and neopterin levels. APAP induced an inflammatory response in the liver tissue. In contrast, treatments of rats with NAC and NAC + HBOT significantly reduced liver TNF-α, IL-6, and neopterin levels (Table 1 and Figure 1).

Figure 1.

(a) Liver TNF-α, (b) IL-6, and (c) neopterin levels after APAP administration in all groups. Sham (n = 8); APAP: only paracetamol treated (n = 8); NAC: NAC treatment followed by APAP administration (n = 8); and NAC + HBOT: NAC and HBO treatment group followed by APAP administration (n = 8). NAC: N-acetylcysteine; HBOT: hyperbaric oxygen treatment; TNF-α; tumor necrosis factor α; IL-6: interleukin 6; APAP: acetaminophen.

In the APAP group, liver neopterin concentrations (198 (179–234) pmol/mg protein) were significantly increased compared to the other groups (p = 0.001; Figure 1). NAC and NAC + HBO treatments reduced liver neopterin levels significantly (127 (109–175) pmol/mg protein; 95 (77–111) pmol/mg protein; in both, p = 0.001), respectively. However, these values were still higher than the sham group (41 (29–61) pmol/mg protein; in both, p = 0.001; Figure 1).

The lowest liver TNF-α levels (130 (108–172) pg/mg protein) were also reported in the sham group. These concentrations were significantly different than the APAP (254 (224–516) pg/mg protein), NAC (209 (114–243) pg/mg protein), and the NAC + HBOT (163 (120–188) pg/mg protein) groups (p = 0.001, p = 0.009, and p = 0.058, respectively). In the APAP group, TNF-α levels were significantly higher than in the NAC and the NAC + HBOT groups (p = 0.014 and p = 0.001, respectively). In the NAC + HBOT group, liver TNF-α concentration was lower than the NAC group.

The concentrations of liver IL-6 in the sham group (1000 (688–1128) pg/mg protein) were significantly different compared with the APAP (1583 (1314–2368) pg/mg protein) and NAC (1152 (1061–1268) pg/mg protein) groups (p = 0.001 and p = 0.009, respectively). But there was no difference between the sham and the NAC + HBOT (1008 (747–1322) pg/mg protein) group (p = 0.600). In addition, there was no difference between the NAC and the NAC + HBOT (1008 (747–1322) pg/mg protein) group (p = 0.093).

The histological scores of liver injury in all rat groups are shown in Table 2. H&E staining revealed normal hepatic parenchyma in sham rats, whereas the APAP group exhibited extensive hepatic necrosis, as well as infiltration of inflammatory cells, sinusoidal dilatation and ballooning in hepatic parenchyma. In the NAC and NAC + HBOT groups, hepatocytes were relatively well preserved, compared with the APAP group. Sample views of the histological examination of sham, APAP, NAC, and NAC + HBOT group livers are shown in Figure 2.

Figure 2.

Liver tissue sections demonstrate histological changes in the study groups. (a) Sham group: normal central vein and hepatic parenchyma. (b) APAP group: massive and severe hepatic necrosis, mild sinusoidal dilatation, ballooning in hepatic parenchyma, and infiltration of inflammatory cells. (c) NAC group: mild necrosis, moderate sinusoidal dilatation, and mild to moderate portal inflammatory cell infiltration. (d) NAC + HBOT group: mild portal inflammatory cell infiltration. (H&E, ×200). H&E: hematoxylin and eosin; APAP: acetaminophen; NAC: N-acetylcysteine; HBOT: hyperbaric oxygen treatment.

Table 2.

The grades of liver injury in all the groups.

Injury parameters	Grade	Groups				p a (overall)	p b (I vs. II)	p b (I vs. III)	p b (I vs. IV)	p b (II vs. III)	p b (II vs. IV)	p b (III vs. IV)
		Sham (I)	APAP (II)	NAC (III)	NAC + HBOT (IV)
		n (%)	n (%)	n (%)	n (%)
Sinusoidal dilatation	0	6 (75)	0 (0)	0 (0)	0 (0)	0.001	<0.001	<0.001	<0.001	0.050	0.024	0.735
	1	2 (25)	0 (0)	3 (37.5)	3 (37.5)
	2	0 (0)	2 (25)	3 (37.5)	4 (50)
	3	0 (0)	4 (50)	1 (12.5)	1 (12.5)
	4	0 (0)	2 (25)	1 (12.5)	0 (0)
Inflammatory cell infiltration	0	7 (87.5)	0 (0)	0 (0)	0 (0)	<0.001	<0.001	<0.001	<0.001	0.001	<0.001	0.203
	1	1 (12.5)	0 (0)	3 (37.5)	5 (62.5)
	2	0 (0)	0 (0)	3 (37.5)	3 (37.5)
	3	0 (0)	2 (25)	2 (25)	0 (0)
	4	0 (0)	3 (37.5)	0 (0)	0 (0)
	5	0 (0)	1 (12.5)	0 (0)	0 (0)
	6	0 (0)	2 (25)	0 (0)	0 (0)
Hemorrhage	0	6 (75)	0 (0)	0 (0)	0 (0)	<0.001	<0.001	<0.001	<0.001	0.007	0.001	0.152
	1	2 (25)	0 (0)	3 (37.5)	5 (62.5)
	2	0 (0)	1 (12.5)	2 (25)	3 (37.5)
	3	0 (0)	2 (25)	3 (37.5)	0 (0)
	4	0 (0)	4 (50)	0 (0)	0 (0)
	5	0 (0)	0 (0)	0 (0)	0 (0)
	6	0 (0)	1 (12.5)	0 (0)	0 (0)
Necrosis	0	8 (100)	0 (0)	0 (0)	0 (0)	0.001	<0.001	<0.001	<0.001	0.109	0.010	0.069
	1	0 (0)	0 (0)	0 (0)	2 (25)
	2	0 (0)	1 (12.5)	3 (37.5)	4 (50)
	3	0 (0)	1 (12.5)	2 (25)	1 (12.5)
	4	0 (0)	1 (12.5)	1 (12.5)	1 (12.5)
	5	0 (0)	2 (25)	1 (12.5)	0 (0)
	6	0 (0)	2 (25)	1 (12.5)	0 (0)
	7	0 (0)	1 (12.5)	0 (0)	0 (0)
	8	0 (0)	0 (0)	0 (0)	0 (0)

APAP: acetaminophen; NAC: N-acetylcysteine; HBOT: hyperbaric oxygen treatment.

^a p Value corresponding to χ ² test.

^b p Value corresponding to corrected, pairwise χ ² test.

Discussion

Rats in the NAC and NAC + HBOT groups had decrement serum AST and ALT activities and reduced liver necrosis. This protective effect was associated with significantly decreased hepatic TNF-α, IL-6, and neopterin levels and reduced infiltrating inflammatory cells in histopathological liver sections compared with the APAP group. In our present study, H&E staining also showed that NAC and NAC + HBO treatments preserved hepatic parenchyma and prevented severe hepatic necrosis.

TNF-α and IL-6 are the prototypic pro-inflammatory cytokines produced by macrophages/monocytes. ²⁴ In our study, NAC and NAC + HBO treatments significantly reduced hepatic TNF-α and IL-6 levels. Our results are in accordance with previous reports that hepatic TNF-α and IL-6 levels increase in APAP-induced liver injury. ^25,26 TNF-α and IL-1α were released in APAP-induced liver injury and responsible for pathological manifestations in liver. ²⁷ NAC treatment reduces APAP-induced expression of TNF-α. ²⁸ Thus, NAC may contribute to hepatoprotection in APAP-induced liver injury. On the other hand, increased levels of pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6 may reflect the severity of the inflammatory response in liver injury. ²⁹ In lipopolysaccharide-activated The human monocytic leukemia cell line (THP-1) macrophages, Palacio et al. have reported that NAC downregulates the production of TNF-α and IL-6. ³⁰ The pro-inflammatory transcription factors including nuclear factor- κB (NF-κB) and activator protein-1 have a central role in inflammatory process. ³¹ Activated NF-κB pathway stimulates the gene expressions of cytokines such as TNF-α, IL-1, and IL-6. ³² It has demonstrated that NAC inhibits activation of the NF-κB pathway and improves circulating levels of TNF-α, IL-6, and plasminogen activator inhibitor-1. ³³ NAC also reduces the expression of intercellular adhesion molecule-1. ³⁴

Our results have shown that hepatic neopterin levels in the APAP group were significantly increased compared to other groups. But after NAC and NAC + HBO treatments, these levels decreased until those in the sham group. In addition, our previous studies showed a marked increase in serum neopterin levels in APAP-induced liver injury rat models. ^23,35 Neopterin (6-d-erythro-trihydroxypropylpteridine) is a pteridine derivative and is a marker of inflammation. ³⁶ It is synthesized from guanosine triphosphate and secreted by macrophages/monocytes activated IFN-γ and other cytokines in various infectious diseases and injuries. ^{13,23,36 –38} Evidence suggests that activated macrophages contribute to the pathogenic response to APAP. ³⁹ An elevated neopterin level is a marker of monocyte/macrophage activities and also inflammation in liver injury. ^23,35 Previous studies have reported that neutrophils may have an important role in the pathogenesis of APAP-induced liver injury. Additionally, they have shown the inflammatory response in liver injury. Following APAP overdose, less liver injury was shown in IFN-γ gene knockout mice. ⁴⁰ These observations suggest that IFN-γ may be directly involved in APAP-induced liver injury and subsequent inflammatory cell infiltration. Hepatic IFN-γ levels in APAP-induced liver injury were correlated with severity. ²⁷ Our hepatic neopterin levels were compatible with the severity of liver injury.

HBOT modulates the immune system, stimulating phagocytic and neutrophilic activity in several tissues. And, it also stimulates angiogenesis. ⁴¹ It has shown that HBOT has anti-inflammatory properties and also decreases the production of pro-inflammatory cytokines such as TNF-α, IL-1, IL-6, and IFN-γ in several animal studies. ^{42 –44} In addition, HBOT decreased inflammation as measured by reduced tissue edema and decreased infiltration of the inflammatory cells. ^13,45 Our results have shown that NAC + HBOT obviously reduced hepatic TNF-α and IL-6 levels compared with only NAC treatment. HBOT can control acute inflammation following injury in animals. ^13,45,46 NAC + HBOT may reduce early APAP-induced hepatocellular injury and decrease infiltration of inflammatory cells in liver.

We therefore investigated whether NAC and HBOT would have anti-inflammatory effects on APAP-induced liver injury. As expected, NAC + HBOT decreased the levels of neopterin and the pro-inflammatory cytokines such as TNF-α and IL-6 in rats with APAP-induced liver injury. NAC + HBOT possess anti-inflammatory activities. On the other hand, oxidative stress induces inflammation by upregulating the expression of inflammatory mediators. Pro-inflammatory cytokines such as TNF-α and IFN-γ may mediate inflammatory response in APAP-induced liver injury. Besides, both NAC and HBOT have antioxidant properties. Thus, the combination of NAC and HBOT contributes to decrease of inflammation in hepatic injury. NAC + HBOT exhibit hepatoprotective activity against APAP-induced liver injury in rats.