Efficacy of free glutathione and niosomal glutathione in the treatment of acetaminophen-induced hepatotoxicity in cats

Acetaminophen (APAP) administration results in hepatotoxicity and hematotoxicity in cats. The response to three different treatments against APAP poisoning was evaluated. Free glutathione (GSH) (200mg/kg), niosomal GSH (14 mg/kg) and free amino acids (180 mg/kg of N-acetylcysteine and 280 mg/kg of methionine) were administered to cats that were intoxicated with APAP (a single dose of 150 mg/kg, p.o.). Serum concentration of alanine aminotransferase (ALT) along with serum, liver and erythrocyte concentration of GSH and methemoglobin percentage were measured before and 4, 24 and 72 hours after APAP administration. Free GSH (200 mg/kg) and niosomal GSH (14 mg/kg) were effective in reducing hepatotoxicity and hematotoxicity in cats intoxicated with a dose of 150 mg/kg APAP. We conclude that both types of treatments can protect the liver and haemoglobin against oxidative stress in APAP intoxicated cats. Furthermore, our results showed that treatment with niosomal GSH represents an effective therapeutic approach for APAP poisoning.Fil: Denzoin Vulcano, L. A.. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigacion Veterinaria de Tandil; ArgentinaFil: Confalonieri, O.. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Clinicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigacion Veterinaria de Tandil; ArgentinaFil: Franci, R.. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Clinicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigacion Veterinaria de Tandil; ArgentinaFil: Tapia, Maria Ofelia. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigacion Veterinaria de Tandil; ArgentinaFil: Soraci, Alejandro Luis. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigacion Veterinaria de Tandil; Argentin

Hepatoprotective effect of obeticholic acid on acetaminophen induced hepatotoxicity in mice

Acetaminophen (APAP) is commonly used as analgesic and antipyretic drug for relieving mild and moderate pain, but at high doses produces hepatic necrosis. Though, Obeticholic acid (OCA) has been tested in range of diseases, its therapeutic potential against APAP-induced hepatic injury remains to be elucidated. Thus, in this study, we investigated the preventive effect of OCA along with N-acetylcysteine (NAC) and Silymarin (SIL) against acetaminophen-induced hepatotoxicity in mice. SIL (100 mg/kg, po) and OCA (30 mg/kg, po) were administered continuously for six days prior to APAP administration. After sixth dose, animas were fasted for 12 h and treated with 300 mg/kg APAP and then received SIL (100 mg/kg, po), NAC (500 mg/kg, ip) and OCA (30 mg/kg, po) at 1 h after APAP. Mice were sacrificed 6 h after APAP injection. Analysis of serum Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), liver glutathione (GSH) and histopathology were employed for assessment of hepatotoxicity. APAP group showed a significant increase in ALT, AST, ALP and centriolobular hepatic necrosis with a significant decrease in glutathione in comparison to control group. All these parameters were significantly improved in all the three treated groups when compared to APAP group. In conclusion, Obeticholic acid (OCA), Silymarin (SIL) and N-acetylcysteine (NAC) are suggested to protect against APAP-induced hepatotoxicity in mice by ameliorating liver enzymes, antioxidant effect and decreasing liver necrosis

Flavonoids in natural products for the therapy of liver diseases: progress and future opportunities

The liver is the largest, important organ and the site for essential biochemical reactions in the human body. It has the function to detoxify toxic substances and synthesize useful biomolecules. Liver diseases related complications represent a significant source of morbidity and mortality worldwide, creating a substantial economic burden. Oxidative stress, excessive inflammation, and dysregulated energy metabolism significantly contributed to liver diseases. Therefore, discovery of novel therapeutic drugs for the treatment of liver diseases are urgently required. For centuries, flavonoids and their preparations which have the beneficial health effects in chronic diseases have been used to treat various human illnesses. Flavonoids mainly include flavones, isoflavones, flavanols, dihydroflavones, dihydroflavonols, anthocyanins and chalcones. The primary objective of this review is to assess the efficacy and safety of flavonoids, mainly from a clinical point of view and considering clinically relevant end-points. We summarized the recent progress in the research of hepatoprotective and molecular mechanisms of different flavonoids bioactive ingredients and also outlined the networks of underlying molecular signaling pathways. Further pharmacology and toxicology research will contribute to the development of natural products in flavonoids and their derivatives as medicines with alluring prospect in the clinical application

Evaluation of Anti-Inflammatory and Hepatoprotective Potency of a Selected Medicinal Plant

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Efeito hepatoprotetor da silimarina (Silybum marianum) sobre a hepatotoxicidade induzida por paracetamol (APAP) em ratos espontaneamente hipertensos (SHR)

The aim of this work was to investigate the effect of silymarin on a hypertensive state and the hepatic function alterations in an cetaminophen induced model (APAP) of hepatotoxicity in Spontaneously Hypertensive Rats (SHR). The animals were divided into 6 experimental groups (n=12 each group): (I), normotensive Wistar rats (N) were used as a control group, received a saline solution (NaCl 0,9% , orally); (II), SHR that received a saline solution (NaCl 0,9% , orally); (III) and (IV) , N rats and SHR treated with APAP (3g/kg; orally), respectively; (V) and (VI), N and SHR, pretreated with silymarin (SLM) (200mg/kg, orally) respectively during 7 days before the APAP administration. Twelve hours after APAP administration, all animals were euthanized and the hepatic function was determined by plasmatic biomarkers: alanine (ALT) and aspartato aminotransferase (AST), alkaline phosphatase (ALP), glucose (GLU) and gamma glutamyl transferase (Y-GT). Samples of hepatic tissue were used to determine: the activity of the myeloperoxidase enzyme (MPO), the nitric oxide (NO) production. Indeed, tissue samples were selected for histological examination. The results showed that hepatotoxicity was increased in SHR and N animals (groups III and IV). Thus, SLM treatment reversed all changes observed (groups V and VI). Data were expressed as means ± SEM for each group. The results were statistically analysed by ANOVA (One-way), followed by Tukey's test (p < 0, 05). The results were compared with normotensive animals. None significant differences were observed between the animals SHR and N related to ALT levels, indicating that hypertensive state did not interfere in basal hepatic functions. Although there was a significant increase in the ALT levels for APAP treated animals in both groups, the SLM pre-treatment restored these alterations, ALT: (I) 51.6 ± 1.9 U/L; (II) 57.3 ± 3 U/L; (III) 181.5 ± 18.3* U/L (72%); (IV) 196 ± 23.7* U/L (71%); (V) 65 ± 4.7** U/L (64%); (VI) 83.1 ± 7.9** U/L (58%), p<0.0001; a significant difference in the AST levels was observed between N and SHR groups, increased after APAP treatment. The pre-treatment with SLM reduced the AST levels in N and SHR animals, AST: (I) 74.3 ± 4.2 U/L; (II) 103.5 ± 5 U/L; (III) 189.5 ± 16.2* U/L (61%); (IV) 224.7 ± 23.8* U/L (54%); (V) 113.6 ± 11.9** U/L (40%); (VI) 131 ± 7.9** U/L (42%), p<0.0001. After APAP treatment a significant difference in the ALP levels was observed between the SHR and N, which was nor restored after SLM treatment, ALP: (I) 198 ± 14.9 U/L; (II) 270.4 ± 4.7# U/L; (III) 178.2 ± 6.5 U/L (11%); (IV) 245.3 ± 17.62## U/L (10%); (V) 204.3 ± 33.9 U/L (13%); (VI) 195.3 ± 7.05 U/L (20%), p=0.0015; there was no significant difference on GLU levels between SHR and N groups, in both treatments with APAP or SLM, GLU: (I) 150.4 ± 7.9 mg/dl; (II) 150.4 ± 6.7 mg/dl; (III) 156.4 ± 5.2 mg/dl (4%); (IV) 143.4 ± 10.3 mg/dl (5%); (V) 167.2 ± 3.4 mg/dl (7%); (VI) 167.8 ± 4.8 mg/dl (16%), p=0.17. In the ?-GT levels between SHR and N, none significant difference was verified. However it was observed only for SHR animals when compared to that treated with APAP or SLM Y-GT: (I) 1.94 ± 0.2 U/L; (II)1.85 ± 0.2 U/L; (III)1.55 ± 0.4 U/L (25%); (IV) 4.12 ± 1 U/L* (55%); (V) 1.85 ± 0.2 U/L (19%); (VI) 1.66 ± 0.1**U/L (61%), p=0.01; the inflammatory response was evaluated by the activity of MPO, by the production of NO on the hepatic tissue, by histological analysis and by leukocyte infiltration into hepatic tissue. The leukocyte infiltration and MPO activity were increased after APAP treatment whereas SLM reduced the leukocyte migration in both groups. The MPO activity was similar when compared SHR and N groups, MPO: (I) 0.11 ± 0.02U/L; (II) 0.19 ± 0.03U/L; (III) 0.36 ± 0.03*U/L (69%); (IV) 0.54 ± 0.05*U/L (65%); (V) 0.13 ± 0.04**U/L (64%); (VI) 0.23 ± 0.04**U/L (57%), p<0.0001; the NO production, a free radical involved in the inflammatory process, was similar between SHR and N groups. A significant increase in NO production was observed in these groups after APAP treatment. The treatment with SLM decreased NO contents, suggesting anti-free radical activities and anti-inflammatory of the SLM, NO: (I) 34.9 ± 4.7; (II) 38.5 ± 3.8; (III) 68 ± 9.6* (49%); (IV) 84.5 ± 4.8* (54%); (V) 38.9 ± 2** (43%); (VI) 45.9 ± 3.9** (46%), p<0.0001. Liver injury was assessed using histological studies by hematoxylin and eosin staining. Together, our data indicated that hypertensive state affects significantly in the acetaminophen-induced hepatotoxicity. In additions, SLM by acts reducing the functional and histopathological alterations reduces the APAP-hepatotoxicity; probably due to their effects in the free radical production inducing hepatic injury.Este estudo teve como objetivo investigar o efeito da silimarina sobre o estado hipertensivo e as alterações das funções hepáticas induzidas por paracetamol (APAP) em ratos espontaneamente hipertensos (SHR). Os animais foram divididos em 6 grupos experimentais (n=12 por grupo): (I), ratos normotensos da linhagem Wistar (N) utilizados como grupo controle, receberam solução salina (NaCl 0,9% , via oral); (II), SHR que receberam salina (NaCl 0,9 %, via oral ); (III) e (IV) , ratos N e SHR tratados com APAP (3g/kg; via oral), respectivamente; (V) e (VI), N e SHR, respectivamente pré-tratados com silimarina (SLM) (200mg/kg , via oral), durante 7 dias antes da administração de APAP. Após doze horas da administração de APAP, todos os animais foram eutanaziados e a função hepática foi determinada pelos marcadores plasmáticos: alanina (ALT) e aspartato aminotransferase (AST), fosfatase alcalina (ALP), glicose (GLU) e gama-glutamil transferase (Y-GT). Amostras de tecido hepático foram utilizadas para determinar: a atividade da enzima mieloperoxidase (MPO), a produção de óxido nítrico (NO) e, posteriormente amostras foram seccionadas para análise histológica. Os resultados demonstram que a hepatotoxicidade está aumentada em animais SHR e N (grupos III e IV) e, que o tratamento com a SLM (grupos V e VI) reverteu as alterações observadas. Os dados foram expressos como a média ± SEM para cada grupo. Os resultados foram analisados estatisticamente por meio de análise de variância (ANOVA One-way) seguida pelo teste de Tukey (p < 0,05). Os resultados foram comparados com o grupo de animais N. Não houve diferença significativa entre animais SHR e N nos níveis de ALT indicando que o estado hipertensivo não interfere na função hepática basal. Porém, houve aumento significativo nos níveis de ALT pelo tratamento com APAP em ambos os grupos, o pré-tratamento com SLM reverteu esta alteração, ALT: (I) 51,6 ± 1,9 U/L; (II) 57,3 ± 3 U/L; (III) 181,5 ± 18,3* U/L (72%); (IV) 196 ± 23,7* U/L (71%); (V) 65 ± 4,7** U/L (64%); (VI) 83,1 ± 7,9** U/L (58%), p<0,0001; houve diferença significativa nos níveis de AST entre os grupos de animais N e SHR, com o aumento significativo pelo tratamento com APAP. O pré-tratamento com SLM promoveu uma redução nos níveis de AST em animais N e SHR, AST: (I) 74,3 ± 4,2 U/L; (II) 103,5 ± 5 U/L; (III) 189,5 ± 16,2* U/L (61%); (IV) 224,7 ± 23,8* U/L (54%); (V) 113,6 ± 11,9** U/L (40%); (VI) 131 ± 7,9** U/L (42%), p<0,0001; houve diferença significativa entre animais SHR e N e também, após o tratamento com APAP nos níveis de ALP. Entretanto, não houve diferença significativa pelo tratamento com SLM, ALP: (I) 198 ± 14,87 U/L; (II) 270,4 ± 4,71# U/L; (III) 178,2 ± 6,49 U/L (11%); (IV) 245,3 ± 17,62## U/L (10%); (V) 204,3 ± 33,92 U/L (13%); (VI) 195,3 ± 7,05 U/L (20%), p=0,0015; não houve diferença significativa nos níveis de GLU entre os animais SHR e N, e nos tratamentos com o APAP e SLM, GLU: (I) 150,4 ± 7,9 mg/dl; (II) 150,4 ± 6,7 mg/dl; (III) 156,4 ± 5,2 mg/dl (4%); (IV) 143,4 ± 10,3 mg/dl (5%); (V) 167,2 ± 3,4 mg/dl (7%); (VI) 167,8 ± 4,84 mg/dl (16%), p=0,1735. Não houve diferença significativa nos níveis de ?-GT entre os animais SHR e N. Apenas em SHR quando comparados estes tratados com APAP e SLM, Y-GT: (I) 1,94 ± 0,2 U/L; (II)1,85 ± 0,2 U/L; (III)1,55 ± 0,4 U/L (25%); (IV) 4,12 ± 1 U/L* (55%); (V) 1,85 ± 0,2 U/L (19%); (VI) 1,66 ± 0,1**U/L (61%), p=0,0102; a resposta inflamatória foi avaliada pela atividade da MPO e pela produção de NO no tecido hepático, e também por meio da histologia e infiltração leucocitária no tecido. A infiltração leucocitária pela atividade de MPO foi de maior intensidade após o tratamento com APAP e o tratamento com SLM diminuiu a migração de leucócitos, em ambos os grupos. Não houve diferença significativa para a atividade da MPO entre os animais SHR e N, MPO: (I) 0,11 ± 0,02U/L; (II) 0,19 ± 0,03U/L; (III) 0,36 ± 0,03*U/L (69%); (IV) 0,54 ± 0,05*U/L (65%); (V) 0,13 ± 0,04**U/L (64%); (VI) 0,23 ± 0,04**U/L (57%), p<0,0001; a produção de NO, um radical livre presente no processo inflamatório não foi diferente entre os grupos de animais SHR e N. Após o tratamento com APAP, houve aumento significativo na produção de NO para ambos os grupos. O tratamento com SLM diminuiu a produção deste radical livre, sugerindo atividades anti-radicais livres e anti-inflamatórias da SLM, NO: (I) 34,9 ± 4,7; (II) 38,5 ± 3,8; (III) 68 ± 9,6* (49%); (IV) 84,5 ± 4,8* (54%); (V) 38,9 ± 2** (43%); (VI) 45,9 ± 3,9** (46%), p<0,0001. A lesão hepática foi avaliada através de estudos histológicos corados por hematoxilina e eosina. Nossos dados, em conjunto, indicam que o estado hipertensivo tem influência significativa na hepatotoxicidade induzida pelo APAP. Além disso, que a SLM por reduzir as alterações funcionais e histopatológicas induzidas pelo APAP reduz esta toxicidade, provavelmente devido aos efeitos desta substância sobre a produção de radicais livres pelo APAP, que poderiam induzir a lesão hepática .46

Oxidative Stress in Drug-Induced Liver Injury (DILI): From Mechanisms to Biomarkers for Use in Clinical Practice

Idiosyncratic drug-induced liver injury (DILI) is a type of hepatic injury caused by an uncommon drug adverse reaction that can develop to conditions spanning from asymptomatic liver laboratoryabnormalitiestoacuteliverfailure(ALF)anddeath.Thecellularandmolecularmecha- nisms involved in DILI are poorly understood. Hepatocyte damage can be caused by the metabolic activation of chemically active intermediate metabolites that covalently bind to macromolecules (e.g., proteins, DNA), forming protein adducts—neoantigens—that lead to the generation of oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress, which can eventually lead to cell death. In parallel, damage-associated molecular patterns (DAMPs) stimulate the immune response, whereby inflammasomes play a pivotal role, and neoantigen presentation on specific human leukocyte antigen (HLA) molecules trigger the adaptive immune response. A wide array of antioxidant mechanisms exists to counterbalance the effect of oxidants, including glutathione (GSH), superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), which are pivotal in detoxification. These get compromised during DILI, triggering an imbalance between oxidants and antioxidants defense systems, generating oxidative stress. As a result of exacerbated oxidative stress, several danger signals, including mitochondrial damage, cell death, and inflammatory markers, and microRNAs (miRNAs) related to extracellular vesicles (EVs) have already been reported as mechanis- tic biomarkers. Here, the status quo and the future directions in DILI are thoroughly discussed, with a special focus on the role of oxidative stress and the development of new biomarkers.This work was supported by the MINECO Retos SAF2016-78711, EXOHEP-CM S2017/BMD- 3727, NanoLiver-CM Y2018/NMT-4949, ERAB Ref. EA 18/14, AMMF 2018/117, FIS-FEDER PI16_01748, PI19-00883, UMA18-FEDERJA-194, PY18-3364_PY19 and UCM-25-2019. FJC is a Ramón y Cajal Researcher RYC-2014-15242 and a Gilead Liver Research 2018. The research group belongs to the validated Research Groups Ref. 970935 “Liver Pathophysiology” and 920631 “Lymphocyte immunobiology” and IBL-6 (imas12-associated). This article/publication is based upon work from COST Action “CA17112—Prospective European Drug-Induced Liver Injury Network” supported by COST (European Cooperation in Science and Technology); www.cost.eu; accessed 4 March 2021. CIBERehd is funded by ISCiii