Protective Effect of Eugenol against Acetaminophen-Induced Hepatotoxicity in Human Hepatocellular Carcinoma Cells via Antioxidant, Anti-Inflammatory, and Anti-Necrotic Potency

BACKGROUND: Overdoses acetaminophen (APAP) could cause acute liver failure, even though it used is for analgesics. APAP could cause hepatotoxicity due to multiple mediators of inflammation and oxidative stress. Eugenol has been reported to have anti-inflammatory and antioxidant activity but its hepatoprotective effect has not been widely reported. AIM: The purpose of this research is to know if eugenol could protect HepG2 cells from APAP. METHODS: HepG2 that induced by APAP as hepatotoxicity cells model was treated by using eugenol at 6.25 and 25 μg/mL. The protective effects of eugenol toward hepatotoxicity were evaluated by determine tumor necrosis factor-α (TNF-α) concentration, apoptotic activity, reactive oxygen species (ROS) level, also cytochrome (CYP)2E1 and GPX gene expression. RESULTS: Eugenol at 6.25 and 25 μg/mL concentration can reduce TNF-α concentration, the apoptotic, necrotic, dead cells, and ROS level. Besides it can increase the gene expression (GPX and CYP2E1). The best hepatoprotective effect was found when using the eugenol at 25 μg/mL. CONCLUSION: Therefore, eugenol can be used to protect HepG2 cells against APAP.

Ethnic differences in excretion of butadiene–DNA adducts by current smokers

1,3-Butadiene (BD) is a known human carcinogen used in the synthetic polymer industry and also found in cigarette smoke, automobile exhaust and wood burning smoke. BD is metabolically activated by cytochrome P450 monooxygenases (CYP) 2E1 and 2A6 to 3,4-epoxy-1-butene (EB), which can be detoxified by GST-catalyzed glutathione conjugation or hydrolysis. We have previously observed ethnic differences in urinary levels of EB–mercapturic acids in white, Japanese American and Native Hawaiian smokers. In the present study, similar analyses were extended to urinary BD–DNA adducts. BD-induced N7-(1-hydroxy-3-buten-2-yl) guanine (EB–GII) adducts were quantified in urine samples obtained from smokers and non-smokers belonging to three racial/ethnic groups: white, Japanese American and Native Hawaiian. After adjusting for sex, age, nicotine equivalents, body mass index and batch, we found that Japanese American smokers excreted significantly higher amounts of urinary EB–GII than whites [1.45 (95% confidence interval: 1.12–1.87) versus 0.68 (95% confidence interval: 0.52–0.85) fmol/ml urine, P = 4 × 10−5]. Levels of urinary EB–GII in Native Hawaiian smokers were not different from those in whites [0.67 (95% confidence interval: 0.51–0.84) fmol/ml urine, P = 0.938]. There were no racial/ethnic differences in urinary EB–GII adduct levels in non-smokers. Racial/ethnic differences in urinary EB–GII adduct levels in smokers could not be explained by GSTT1 gene deletion or CYP2A6 enzymatic activity. Urinary EB–GII adduct levels in smokers were significantly associated with concentrations of BD metabolite dihyroxybutyl mercapturic acid. Overall, our results reveal that urinary EB–GII adducts in smokers differ across racial/ethnic groups. Future studies are required to understand genetic and epigenetic factors that may be responsible for these differences.

A PRELIMINARY STUDY ON CYTOCHROME 2E1 GENE SINGLE NUCLEOTIDE POLYMORPHISMS AMONG ETHNIC FULANI IN NORTH WESTERN NIGERIA

Single nucleotide polymorphisms of xenobiotics metabolizing enzymes are critical in inter-individual variability in drug response. Cytochrome P450 2E1, which is highly polymorphic belongs to these enzymes and ethnicity signicantly determine their expressions. Volunteers from Fulani extractions were recruited through their expressed consents. Five mls of blood sample was collected in EDTA container used for DNA extraction, PCR and sequencing. The consensus sequence generated for individual participants were located to the Cyp 2E1 gene and spanned both target rs 2031920 to rs 3813867 polymorphism region. Chromatograms were validated with Bioedit software. Three (3) volunteers (15%) revealed new rs 147346389 A>C polymorphism at nucleotide position 4029. Also, three (3) other volunteers (15%) revealed rs 35806299 A>G polymorphism at position 3661. One (5%) new (unlabelled) A>C polymorphism was revealed at position 3906.

Weaning Mice and Adult Mice Exhibit Differential Carbon Tetrachloride-Induced Acute Hepatotoxicity

Age is a risk factor for drug-induced liver injury (DILI). However, there is a limited understanding of pediatric DILI. Here, 2-week-old weaning and 8-week-old adult male ICR mice were intraperitoneally injected with CCl4 (0.1 mmol/kg equal to 15.4 mg/kg) to comparatively evaluate the time-dependent liver damage and cellular events. CCl4 significantly enhanced the serum alanine aminotransferase/aspartate aminotransferase levels and hepatic centrilobular necrosis in the weaning mice, whereas it induced mild liver injury in the adult mice. CCl4-treated weaning mice exhibited higher hepatic levels of pro-apoptotic proteins (Bax, cleaved caspase-3, -7, and -9), activated MAPKs (p-JNK and p-Erk), and endoplasmic reticulum stress indicators (ATF6 and CHOP) and lower hepatic anti-apoptotic Bcl-2 levels than the adult mice. The weaning mice exhibited enhanced basal hepatic glutathione (GSH) levels due to high glutamate cysteine ligase (GCL) and low anti-cysteine dioxygenase (CDO) enzyme levels. However, CCl4 markedly reduced the hepatic GSH levels only in the weaning mice. Furthermore, higher hepatic levels of oxidative stress-induced malondialdehyde, 4-hydroxynonenal, nitrotyrosine-protein adducts, and oxidized proteins were observed in CCl4-treated weaning mice than in CCl4-treated adult mice. The enhanced levels of hepatic cytochrome P450 (CYP) 2E1 and CYP3A, and decreased hepatic GSH S-transferase (GST)-π and GSH reductase (GR) levels in the weaning mice may contribute to their enhanced susceptibility to liver damage.

CCl4-Induced Liver Injury Was Ameliorated by Qi-Ge Decoction through the Antioxidant Pathway

Qi-Ge decoction (QGD), which is derived from the Huangqi Gegen decoction, contains three traditional Chinese herbs: Astragalus membranaceus (Huangqi), Pueraria lobata (Gegen), and Citri Reticulatae Blanco Pericarpium (Chenpi). Gastric mucosal damage caused by ethanol was prevented and alleviated by QGD. However, the role of QGD in protecting the liver from toxins has not been reported. High-performance liquid chromatography with diode-array detection was used to qualitatively analyze QGD. Positive control (silymarin 100 mg/kg/day), QGD (20, 10, or 5 g/kg/day), and Nrf2 inhibitor brusatol (0.4 mg/kg/2 d) were administered to rats for 7 days, and then, liver injury was induced by injecting 2 mL/kg 25% CCl4. After 24 h, blood and liver were collected for analysis and evaluation. QGD was found to contain 12 main components including calycosin, puerarin, and hesperidin. QGD treatment significantly reduced liver damage and decreased serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase activities. QGD increased superoxide dismutase and catalase activities, and glutathione levels, but decreased malondialdehyde levels in livers from CCl4-treated rats. Compared to rats treated with CCl4 alone, after QGD administration, mRNA and protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 were increased, while those of Kelch-like ECH-related protein 1 (Keap1) and cytochrome P450 (CYP)2E1 were decreased. However, these improvements in QGD were reversed by brusatol. In conclusion, QGD can achieve its hepatoprotective effect through an antioxidant mechanism by activating the Nrf2 pathway.

Phenylpropionc acid produced by gut microbiota alleviates acetaminophen-induced hepatotoxicity

ABSTRACTAcetaminophen (APAP) overdose causes hepatic injury and is major contributor to acute liver injury cases. To investigate potential roles of gut microbiota in APAP-induced liver injury, C57BL/6 mice from Jackson (JAX) or Taconic (TAC) were challenged with APAP. TAC mice were more susceptible to APAP toxicity, and this disappeared upon co-housing of JAX and TAC mice. When the cecum contents from JAX and TAC mice were transplanted to germ-free mice, the mice that received TAC gut microbiota exhibited more significant hepatotoxicity after APAP administration. Non-targeted metabolomic analysis using portal vein serum and liver tissue of the mice led to identification of 19 metabolites the levels of which are associated with JAX or TAC gut microbiota. A gut bacteria-derived metabolite phenylpropionic acid (PPA) levels in cecum contents and blood were higher in mice harboring JAX gut microbiota. PPA supplementation in drinking water alleviated APAP-induced hepatotoxicity in TAC mice. This was accompanied by reduced hepatic protein levels of cytochrome P450 (CYP) 2E1, the enzyme responsible for APAP bioactivation to a toxic metabolite. This illustrates a gut microbe-liver interaction mediated by a gut bacteria-derived metabolite in modulating drug-induced liver injury.

Alcoholic Liver Disease: Alcohol Metabolism, Cascade of Molecular Mechanisms, Cellular Targets, and Clinical Aspects

Alcoholic liver disease is the result of cascade events, which clinically first lead to alcoholic fatty liver, and then mostly via alcoholic steatohepatitis or alcoholic hepatitis potentially to cirrhosis and hepatocellular carcinoma. Pathogenetic events are linked to the metabolism of ethanol and acetaldehyde as its first oxidation product generated via hepatic alcohol dehydrogenase (ADH) and the microsomal ethanol-oxidizing system (MEOS), which depends on cytochrome P450 2E1 (CYP 2E1), and is inducible by chronic alcohol use. MEOS induction accelerates the metabolism of ethanol to acetaldehyde that facilitates organ injury including the liver, and it produces via CYP 2E1 many reactive oxygen species (ROS) such as ethoxy radical, hydroxyethyl radical, acetyl radical, singlet radical, superoxide radical, hydrogen peroxide, hydroxyl radical, alkoxyl radical, and peroxyl radical. These attack hepatocytes, Kupffer cells, stellate cells, and liver sinusoidal endothelial cells, and their signaling mediators such as interleukins, interferons, and growth factors, help to initiate liver injury including fibrosis and cirrhosis in susceptible individuals with specific risk factors. Through CYP 2E1-dependent ROS, more evidence is emerging that alcohol generates lipid peroxides and modifies the intestinal microbiome, thereby stimulating actions of endotoxins produced by intestinal bacteria; lipid peroxides and endotoxins are potential causes that are involved in alcoholic liver injury. Alcohol modifies SIRT1 (Sirtuin-1; derived from Silent mating type Information Regulation) and SIRT2, and most importantly, the innate and adapted immune systems, which may explain the individual differences of injury susceptibility. Metabolic pathways are also influenced by circadian rhythms, specific conditions known from living organisms including plants. Open for discussion is a 5-hit working hypothesis, attempting to define key elements involved in injury progression. In essence, although abundant biochemical mechanisms are proposed for the initiation and perpetuation of liver injury, patients with an alcohol problem benefit from permanent alcohol abstinence alone.