Liver is frequently exposed to metabolic insults due to its major role in metabolism and detoxification of endogenous and exogenous compounds including drugs and xenobiotics [
Phenacetin was extensively used as an analgesic and fever-reducing agent for many years; however, its use was prohibited in the late 1970s due to its potential to cause renal nephropathy [
N-acetylcysteine (NAC) is a precursor of the amino acid L-cysteine which is a component of the biologic antioxidant glutathione (GSH). In addition to its indirect antioxidant effect (through incorporation in GSH formation), NAC exhibits also direct antioxidant properties through the interaction of its free thiol group with the electrophilic groups of ROS [
N-acetyl-L-methionine (NAM) is capable of replacing the dietary requirements for methionine. Methionine is an essential methyl donor in mammals and acts as an efficient scavenger of several oxidizing molecules. Methionine is also required for synthesis of cysteine which is the limiting amino acid for GSH synthesis [
N-acetylglucosamine, an amide of glucosamine and acetic acid, has a beneficial effect in the treatment of joint disorders, e.g., osteoarthritis and rheumatoid arthritis [
Several studies have investigated the damaging effects of paracetamol, when used in toxic doses, on the liver or the efficacy of various antioxidants to neutralize such effect, but studies regarding phenacetin could seldom be found. This is the first study aiming to carry out biochemical and histopathological assessments of possible hepatic-oxidative stress induced by paracetamol and phenacetin, when used in therapeutic doses, in male albino rats and also to compare and detect which of the three antioxidants (N-acetylcysteine, N-acetylmethionine, and N-acetylglucosamine) has the best antioxidant and hepatoprotective efficacy against the drug-induced liver injury, if any.
In this study, 90 male Wister albino rats, obtained from the Animal House of the Faculty of Medicine, Assiut University, Assiut, Egypt, were used. Their body weights ranged from 120 to 140 gm. Rats were housed in cages, kept at room temperature with normal 12h light/12h dark cycle, and treated according to the guidelines of the Animal House of Assiut University, where standard commercial pellets for feeding, water
The included rats were divided randomly into 9 groups of 10 rats each. All chemicals were dissolved in 1% dimethyl sulfoxide (DMSO) and rats were treated daily for 15 days. The different groups of the rats and their treatment regimens were illustrated in Figure All used chemicals were purchased from Sigma Aldrich Chemical Co. (UK). All used reagents were of analytical grade and highest purity. The CAS numbers for PA, PH, NAC, NAM, and NAG were 103-90-2, 62-44-2, 616-91-1, 65-82-7, and 7512-17-6, respectively. Calculations of NAC, NAM, and NAG doses were based on theoretical chemistry from previously published work [
Study design: rats were divided randomly into 9 groups of 10 rats each.
Rats of the different groups were anesthetized using diethyl ether inhalation and killed by cervical dislocation 24 hours after the last dose. At time of scarifying, the blood samples were collected from the retroorbital veins into plain tubes and were centrifuged at 4,000 r.p.m for 10 min, and the separated sera were used for alanine transaminase (ALT), aspartate transaminase (AST), alfa fetoprotein (AFP), and 8-hydroxy-guanine (8-OH-Gua) measurements.
Rats' livers were quickly removed and washed with isotonic saline solution 0.9%, and each one was divided into two parts.
The first part was fixed immediately in 10% neutral buffered formalin for 48 hours at room temperature and then processed to prepare the paraffin sections. Serial sections of 7
The second part was frozen in ice bath during scarifying and then washed, and 300 mg of the liver tissue was homogenized in 3 ml (0.1M) phosphate buffer (pH 7.4) to prepare 10% W/V homogenates, using homogenizer (Glas-Col, USA). The homogenates were centrifuged at 6,000 r.p.m for 1 hour at 4°C (MIKRO 220R Germany) and the isolated supernatants were preserved at -20°C for the subsequent biochemical measurements in the form of malondialdehyde (MDA), nitric oxide (NO), reduced GSH, total thiols, and AFP.
(1) Serum AST and ALT measurements were done, by colorimetric method (UNICO 1200), using commercially available assay kits supplied by Egyptian Company for Biotechnology (S.A.E) with catalog no. 292002 and 291002, respectively.
(2) AFP assays in serum and hepatic tissue homogenates assays were done, using commercially available ELISA assay kits supplied by CHECK, INC (USA) with catalog no. 40-052-115007.
(3) Serum 8-OH-Gua determination was done, using high performance liquid chromatography (HPLC; Agilent Technologies 1200 Series, G1315D DAD): a 20
Calibration curves for HPLC assay of 8-OH-Gua at concentrations 1, 5, 15, and 25 mg/L.
(4) MDA, NO, reduced GSH, total thiols, and total proteins measurements in hepatic tissue homogenates were done using chemical methods (SpectraMax Plus, Molecular Devices, USA) according to Wills [
Statistical Package for Social Science (SPSS) for Windows, version 5.0 (Graph Pad software, Inc., San Diego, CA, USA) was used. Experimental data were expressed as mean ± standard deviation (SD). The results were analyzed using one way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison test as a posttest to determine significant differences between means. The level of significance was considered when p<0.05.
The mean ± SD values of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels, AFP, and 8-OH-Gua among the studied groups were presented in (Table
Mean ± SD of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels, AFP, and 8-OH-Gua among the studied groups.
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| 10 | 81.45 ± 37.8 | 100.2 ± 27.84 | 12.23 ± 0.4265 | 2.240 ± 1.072 |
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| 10 | 124.9 ± 38.2 | 150.5 ± 50.7 | 12.58 ± 0.617 | 3.643 ± 1.01 |
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| 10 | 73.11 ± 17.0 | 66.93 ± 32.6 | 12.45 ± 0.271 | 0.8158 ± 0.360 |
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| 10 | 63.34 ± 10.0 | 53.34 ± 14.2 | 12.43 ± 0.428 | 1.981 ± 0.305 |
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| 10 | 60.58 ± 12.2 | 55.38 ± 15.2 | 12.50 ± 0.298 | 1.097 ± 0.244 |
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| 10 | 127.1 ± 2 | 150.7 ± 57.5 | 12.74 ± 0.622 | 4.643 ± 1.03 |
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| 10 | 55.46 ± 17.7 | 76.82 ± 26.6 | 12.22 ± 0.399 | 1.535 ± 0.121 |
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| 10 | 52.85 ± 13.2 | 60.57 ± 7.7 | 12.03 ± 0.609 | 1.892 ± 0.772 |
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| 10 | 54.46 ± 14.3 | 59.00 ± 14.2 | 12.28 ± 0.277 | 1.026 ± 0.432 |
Cotreatment of the rats with NAC, NAM, or NAG was associated with a significant decrease in serum levels of ALT, AST, and 8-OH-Gua in the paracetamol (73.11 U/l ± 17.08, 63.34 U/l ± 10.03, and 60.58 U/l ± 12.23; 66.93 U/l ± 32.64, 53.34 U/l ± 14.24, and 55.38 U/l ± 15.22; 0.8158 mg/l ± 0.3600, 1.981 mg/l ± 0.3059, and 1.097 mg/l ± 0.2448) and phenacetin groups (55.46 ± 17.76 U/l, 52.85 U/l ± 13.29, and 54.46 U/l ± 14.37; 76.82 U/l ± 26.64, 60.57 U/l ± 7.75, and 59.00 U/l ± 14.23; 1.535 mg/l ± 0.1212, 1.892 mg/l ± 0.7727, and 1.026 mg/l ± 0.4326), respectively, when compared with drug-only groups. The levels of significance were similar (p<0.001) among paracetamol and phenacetin groups treated with either NAC or NAG but phenacetin-NAM group showed higher significant decrease in the serum 8-OH-Gua levels (p<0.001) than in paracetamol-NAM group (p<0.01).
The mean ± SD values of hepatic homogenate levels of oxidants (MDA and NO), antioxidants (GSH and total thiols), and AFP among the studied groups are shown in (Table
Mean ± SD of liver homogenate levels of oxidants (MDA and NO), antioxidants (GSH and total thiols), and AFP among the studied groups.
| | | | | | |
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| 10 | 55.78 ± 16.08 | 549.0 ± 101.1 | 493.9 ± 98.85 | 2062 ± 667.5 | 312.2 ± 38.44 |
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| 10 | 76.20 ± 10.5 | 621.7 ± 51.8 | 354.0 ± 61.6 | 1299 ± 173. | 375.9 ± 33.6 |
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| 10 | 63.56 ± 9.8 | 566.0 ± 94.1 | 494.4 ± 94.3 | 1542 ± 376. | 282.9 ± 26.3 |
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| 10 | 54.13 ± 13. | 520.0 ± 124. | 449.3 ± 111. | 1640 ± 465. | 258.1 ± 65.4 |
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| 10 | 55.50 ± 14.4 | 449.4 ± 68.6 | 508.8 ± 87. | 1536 ± 369. | 223.9 ± 65.4 |
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| 10 | 80.25 ± 10. | 776.5 ± 154. | 331.3 ± 60.8 | 1215 ± 209. | 433.2 ± 20.0 |
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| 10 | 58.11 ± 20.1 | 630.1 ± 154. | 531.3 ± 179. | 1401 ± 167. | 300.1 ± 66.7 |
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| 10 | 66.33 ± 21.1 | 581.5 ± 161. | 450.0 ± 101. | 1615 ± 361. | 277.4 ± 75.5 |
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| 10 | 53.63 ± 14.9 | 495.4 ± 111. | 507.5 ± 77.9 | 1504 ± 428. | 234.7 ± 55.3 |
Regarding the cotreated NAC, NAM, and NAG-paracetamol groups, there was significant reduction in the MDA (63.56 ± 9.84, 54.13 ± 13.9, and 55.50 ± 14.48), NO (566.0 ± 94.18, 520.0 ± 124.6, and 449.4 ± 68.64), and AFP (282.9 ± 26.32, 258.1 ± 65.42, and 223.9 ± 65.42), with significant increase in GSH levels (494.4 ± 94.35, 449.3 ± 111.9, and 508.8 ± 87.90), respectively, when compared with the PA-only group, except for total thiols which showed nonsignificant changes (p>0.05). These antioxidant effects were more obvious in NAG-treated group (p<0.01 for MDA, NO, and GSH; p<0.001 for AFP) than in NAC-PA and NAM-PA groups.
Regarding the cotreated phenacetin groups, the least antioxidant effect was for those received NAM as there were nonsignificant effects on homogenate levels of MDA (66.33 ± 21.16), GSH (450.0 ± 101.8), or total thiols (1615 ± 361.3), but there was significant decrease in both NO (581.5 ± 161.3, p<0.05) and AFP (277.4 ± 75.51, p<0.01), in comparison to the PH-only group. The best antioxidant effects were for those receiving NAC which cause significant decrease in MDA (58.11 ± 20.13) and NO (630.1 ± 154.5) with p<0.05 for both, AFP (300.1 ± 66.74, p<0.01), with significant increase in GSH (531.3 ± 179.5) and total thiols (1401 ± 167.4) with p<0.01 for both, when compared with the PH-only group. Those treated with NAG revealed significant decrease in MDA (53.63 ± 14.98, p<0.05), NO (495.4 ± 111.2, p<0.01), and AFP (234.7 ± 55.39, p<0.001), with significant increase in GSH (507.5 ± 77.97, p<0.05), but nonsignificant effect has been noticed on the total thiols, in comparison to the PH-only group.
Histopathological changes in the liver architecture of various study groups have been described in Figures
Drug-induced hepatotoxicity is a frequent event, which is difficult to determine, due to underreporting, incomplete observation of exposure, and difficulties in diagnosis or detection [
Although serum AFP is primarily used as a marker for hepatocellular carcinoma, it can be also regularly elevated in a range of nonneoplastic liver diseases such as acute liver injury with extensive necrosis [
In our experiment, the PA- and PH-induced hepatic oxidative damage was evidenced by the liver histopathological findings and the significant increased hepatic levels of MDA and NO and serum levels of 8-OH-Gua, with significant decreased hepatic GSH and total thiols among PA-only and PH-only treated groups when compared with the controls. These findings were in agreement with many studies [
Although there is considerable evidence for the safety and efficacy of NAC in management of PA-induced liver injury [
The data used to support the findings of this study are included within the article.
The authors declare that they do not have any conflicts of interest
All authors contributed equally to this work and approved the final version of the manuscript.