Aniline Hydroxylation Research Articles

Metabolism of nitrogen and sulfamethoxazole, performance recovery mechanism, and antibiotic resistance genes (ARGs) behaviors in membrane aerated biofilm reactor (MABR)

The limited information on micro-mechanism of sulfamethoxazole (SMX) metabolism and antibiotics resistance genes (ARGs) behaviors within membrane-aerated biofilm reactor (MABR) blocked the application of MABR in treating antibiotics containing wastewater. In this work, SMX metabolism and the changes of ARGs were investigated in MABR during 0.0–0.7 mg/L SMX. The system achieved a 92.3% SMX removal at 0.7 mg/LSMX, leading to the formation of 17 lower toxic metabolites, which indicated the robust SMX biodegradation capability of MABR. The metabolic pathways of SMX in MABR were first elucidated, revealing key processes including isoxazole ring opening (primarily in anaerobic zones), aniline hydroxylation, and cleavage of the S-N bond in the sulfonyl group. Microbial analysis revealed the involvement of multiple microbes in SMX biodegradation, and among these, sulfate-reducing bacteria (SRB; Desulfovibrio, Desulforhopalus, Desulfomicrobium) facilitated the isoxazole ring opening in the anaerobic zone, while Rhizobium and Chryseobacterium contributed to ipso-hydroxylation and cleavage of the -C-S-N bond in SMX, respectively. Furthermore, the slower accumulation of ARGs under SMX conditions was primarily attributed to the enhanced efflux pump activity, antibiotic target replacement mechanisms, and secretion of extracellular polymeric substances (EPS), suggesting the potential of MABR to inhibit ARG proliferation. Our research filled the knowledge gap in the micro-mechanism of SMX metabolism and ARGs fate within MABR processing SMX-containing wastewater, laying a theoretical foundation for the broader application of MABR in practical engineering.

Bio-Inspired Deaminative Hydroxylation of Aminoheterocycles and Electron-Deficient Anilines.

Among the tools available to chemists for drug design of bioactive compounds, the bioisosteric replacement of atoms or groups of atoms is the cornerstone of modern strategies. Despite the undeniable interest in amino-to-hydroxyl interchange, enzymatic deaminative hydroxylation remains unmatched. Herein, we report a user friendly and safe procedure to selectively convert aminoheterocycles to their hydroxylated analogues by means of a simple pyrylium tetrafluoroborate salt. The hydroxylation step relies on a Lossen-type rearrangement under mild conditions thus avoiding the use of strong hydroxide bases. In addition to biorelevant heterocycles, the deaminative hydroxylation of electron-deficient anilines was also demonstrated. Finally, mechanistic experiments allowed the identification of the key intermediates, thus unveiling a rather unusual mechanism for this formal aromatic substitution.

Garcinia mangostana and α-Mangostin Revive Ulcerative Colitis-Modified Hepatic Cytochrome P450 Profiles in Mice.

<b>Background and Objective:</b> Ulcerative colitis (UC) is inflammation of the large intestine with ulceration but can also cause extraintestinal manifestations (EIM) by damaging surrounding organs such as the liver. <i>Garcinia mangostana</i> (GM) pericarp and α-mangostin (MGS) have been reported to have anti-inflammatory activity. This study evaluated the effects of GM pericarp extract and MGS on the expression of hepatic cytochrome P450 (CYP) enzymes as an EIM of UC. <b>Materials and Methods:</b> Male ICR mice were orally administered GM pericarp extract (40, 200 and 1000 mg/kg/day), MGS (30 mg/kg/day) or sulfasalazine (SUL) (100 mg/kg/day) daily for 7 days. On days 4-7, UC was induced by dextran sulfate sodium (DSS 40 kDa, 6 g/kg/day). Profiles of CYP mRNA expression were determined by RT/qPCR. Alkoxyresorufin <i>O</i>-dealkylation (including ethoxy-, methoxy-, pentoxy- and benzyloxy-resorufin), aniline hydroxylation and erythromycin <i>N</i>-demethylation CYP responsive activities were also examined. <b>Results:</b> The DSS-induced UC mice showed suppressed expression<i> </i>of <i>Cyp1a1</i>, <i>Cyp1a2</i>, <i>Cyp2b9/10</i>, <i>Cyp2e1</i>, <i>Cyp2c29</i>, <i>Cyp2d9</i>, <i>Cyp3a11</i> and <i>Cyp3a13</i> mRNAs. The GM pericarp extract and MGS restored expression of all investigated CYPs and their responsive enzyme activities in DSS-induced UC mice to levels comparable to the control and parallel to the effects of the anti-inflammatory control SUL. <b>Conclusion:</b> The GM is a promising therapy to restore UC-modified hepatic CYP profiles.

Efficient ozone catalysis by manganese iron oxides/activated carbon for sulfamerazine degradation

In this study, the manganese iron oxides/activated carbon (MnxFeyOz/AC) was prepared via the simple co-precipitation method, characterized and used for ozone catalytic degradation of sulfamerazine (SMZ). The best catalytic ozonation activity of MnxFeyOz/AC was obtained at a ratio of Fe/Mn = 1:11.2. The degradation ratio (~90.5 %) was improved by 25.7 % compared to that of ozonation system. The effects of various factors, including SMZ concentrations, ozone dosage, catalyst dosage, and initial pH on the catalytic performance were investigated. The presence of organic matter and ions in rivers, tap-water, and secondary effluent could decrease the catalytic performance. The MnxFeyOz/AC could promote more ozone dissolution in solution, while the surface hydroxyl as active sites could promote the production of hydroxyl radicals. The hydroxyl radical could destroy the SMZ structure by the cleavage of SN and CS bonds, and hydroxylation of aniline. The results of toxicity assessment indicated that catalytic ozonation could effectively control the acute and chronic toxicity of the SMZ water samples. In addition, the results of characterization of MnxFeyOz/AC before and after the ozonation experiments were discussed, and a possible degradation mechanism was proposed. Importantly, the electrical energy per order value was about 1.37 kW·h/(m3 h−1), which was lower than those of other technologies. This work presents an efficient and promising catalytic ozonation technique for the elimination of SMZ from solution, and provides interesting information for the elucidation of the mechanism of action.

Nucleophilic Functionalizations of Aniline Derivatives: Aromatic Pummerer Reaction for Umpolung Halogenation and Hydroxylation on Benzene Ring.

In this paper, a metal-free protocol of nucleophilic ortho-halogenation and hydroxylation of anilines via an aromatic Pummerer process is reported.

Modulating proposed electron transfer pathways in P450BM3 led to improved activity and coupling efficiency

Electrochemical in vitro reduction of P450 enzymes is a promising alternative to in vivo applications. Previously we presented three engineered P450BM3 variants for aniline hydroxylation, equipped with a carbon nanotube binding-peptide (CNT-tag) for self-assembly on CNT electrodes. Compared to wildtype P450BM3 the NADPH-dependent activity was enhanced, but the coupling efficiency remained low. For P450BM3 Verma, Schwaneberg and Roccatano (2014, Biopolymers 101, 197–209) calculated putative electron transfer pathways (eTPs) by MD simulations. We hypothesised that knockouts of these transfer pathways would alter the coupling efficiency of the system. The results revealed no improved system for the electrically-driven P450s. For the NADPH-driven P450s, however, the most active eTP-mutant showed a 13-fold increased activity and a 32-fold elevated coupling efficiency using NADPH as reducing equivalent. This suggests an alternative principle of electron transport for the reduction by NADPH and an electrode, respectively. The work presents moreover a tool to improve the coupling and activity of P450s with non-natural substrates.

Effectiveness of the liver micronucleus assay using juvenile mice.

This study investigated the effectiveness of the liver micronucleus (MN) assay using juvenile mice. Therefore, we analyzed various hepatic cytochrome P450 (CYP)- mediated activities of ethoxyresorufin O-deethylation, pentoxyresorufin O-dealkylation, tolbutamide hydroxylation, bufuralol 1’-hydroxylation, aniline hydroxylation and midazolam 4-hydroxylation by CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A, respectively, in non-treated male ICR mice aged between 3 and 8 weeks. The enzyme efficiency levels in 3- and 4-week-old mice were approximately similar to or higher than those in 8-week-old mice, except for CYP1A and CYP2E in 3- and 4-week-old mice, respectively. Since these results suggest that juvenile mice have sufficient activities for most CYP enzymes, we also conducted a liver MN assay using diethylnitrosamine (DEN), a rodent hepatocarcinogen, on male ICR mice aged between 3 and 6 weeks. A peripheral blood (PB) MN assay was performed simultaneously in 4-week-old mice. Assays incorporating DEN produced positive results in 3- and 4-week-old mice and showed a dose-dependent increase in the micronucleated hepatocyte frequencies at 4 weeks. Both the liver MN assay in 5- and 6-week-old mice and the PB MN assay had negative results when using DEN. These results suggest that 3- and 4-week-old mice have micronuclei-inducing potential in the liver to detect genotoxic compounds using the liver MN assay.

CYP2E1 hydroxylation of aniline involves negative cooperativity

CYP2E1 plays a role in the metabolic activation and elimination of aniline, yet there are conflicting reports on its mechanism of action, and hence relevance, in aniline metabolism. Based on our work with similar compounds, we hypothesized that aniline binds two CYP2E1 sites during metabolism resulting in cooperative reaction kinetics and tested this hypothesis through rigorous in vitro studies. The kinetic profile for recombinant CYP2E1 demonstrated significant negative cooperativity based on a fit of data to the Hill equation (n=0.56). Mechanistically, the data were best explained through a two-binding site cooperative model in which aniline binds with high affinity (Ks=30μM) followed by a second weaker binding event (Kss=1100uM) resulting in a threefold increase in the oxidation rate. Binding sites for aniline were confirmed by inhibition studies with 4-methylpyrazole. Inhibitor phenotyping experiments with human liver microsomes validated the central role for CYP2E1 in aniline hydroxylation and indicated minor roles for CYP2A6 and CYP2C9. Importantly, inhibition of minor metabolic pathways resulted in a kinetic profile for microsomal CYP2E1 that replicated the preferred mechanism and parameters observed with the recombinant enzyme. Scaled modeling of in vitro CYP2E1 metabolism of aniline to in vivo clearance, especially at low aniline levels, led to significant deviations from the traditional model based on non-cooperative, Michaelis–Menten kinetics. These findings provide a critical mechanistic perspective on the potential importance of CYP2E1 in the metabolic activation and elimination of aniline as well as the first experimental evidence of a negatively cooperative metabolic reaction catalyzed by CYP2E1.

Effect of N-acetyl cysteine (NAC), an organosulfur compound from Allium plants, on experimentally induced hepatic prefibrogenic events in wistar rat

Aim of present study was to investigate the effect of NAC on experimental chronic hepatotoxicity models induced by carbon tetrachloride (CCl4) and thioacetamide (TAA). CCl4 toxicity was induced by administering 200μl CCl4 (diluted 2:3 in coconut oil)/100g body weight, p.o., twice weekly for 8 weeks. TAA toxicity was induced by administering 150mg/kg b. wt. of TAA i.p., twice weekly for 8 weeks. NAC treatment was started along with toxicants (CCl4 and TAA) for 8 weeks and continued for further 4 weeks. Self reversal group was kept without any treatment for 4 weeks after completion of toxicant treatments. Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Bilirubin were measured in serum. Hydroxyproline (HP), lipid peroxidation (LPO), catalase (CAT), Glutathione peroxidase (GPx) and Glutathione (GSH) were determined in liver samples by colorimetric methods. Cytochrome P450 2E1 (CYP 450 2E1), activity was determined as hydroxylation of aniline in liver microsomes. General examination and histological analysis were also performed. Serum markers of liver damage (AST, ALT, ALP and Bilirubin) were increased by CCl4 and TAA intoxication (p<0.001), whereas co-treatment with NAC reversed such changes (p<0.001). HP was enhanced in toxicant groups (p<0.001 in CCl4 and TAA), but inhibited by NAC (p<0.001). LPO was increased while as GSH, CAT and GPx decreased by the administration of CCl4 and TAA (p<0.001); co-administration of NAC restored these liver markers to normal levels (p<0.001). Biochemical determinations were corroborated by general and histological findings. Keeping in view the biochemical and histopathological studies, it was concluded that CCl4 and TAA are strong hepatotoxic agents that produce liver fibrosis with close proximity to human etiology (micronodular cirrhosis) and NAC has a significant protective activity against CCl4 and TAA. NAC has also been validated as a model against oxidative burden in chronic liver pathology.

Peroxide-Dependent Analyte Conversion by the Heme Prosthetic Group, the Heme Peptide “Microperoxidase-11” and Cytochrome c on Chitosan Capped Gold Nanoparticles Modified Electrodes

In view of the role ascribed to the peroxidatic activity of degradation products of cytochrome c (cyt c) in the processes of apoptosis, we investigate the catalytic potential of heme and of the cyt c derived heme peptide MP-11 to catalyse the cathodic reduction of hydrogen peroxide and to oxidize aromatic compounds. In order to check whether cyt c has an enzymatic activity in the native state where the protein matrix should suppress the inherent peroxidatic activity of its heme prosthetic group, we applied a biocompatible immobilization matrix and very low concentrations of the co-substrate H2O2. The biocatalysts were entrapped on the surface of a glassy carbon electrode in a biocompatible chitosan layer which contained gold nanoparticles. The electrochemical signal for the peroxide reduction is generated by the redox conversion of the heme group, whilst a reaction product of the substrate oxidation is cathodically reduced in the substrate indication. The catalytic efficiency of microperoxidase-11 is sufficient for sensors indicating HRP substrates, e.g., p-aminophenol, paracetamol and catechol, but also the hydroxylation of aniline and dehalogenation of 4-fluoroaniline. The lower limit of detection for p-aminophenol is comparable to previously published papers with different enzyme systems. The peroxidatic activity of cyt c immobilized in the chitosan layer for catechol was found to be below 1 per mill and for p-aminophenol about 3% as compared with that of heme or MP-11.

Protective effects of thiopronin against isoniazid-induced hepatotoxicity in rats

Isoniazid is a widely used drug for the treatment of tuberculosis, but hepatotoxicity is a major concern during treatment. Thiopronin contains an SH-group and is generally considered an antioxidant. The aim of the present study was to investigate the effects of thiopronin during liver injury and DNA damage induced by isoniazid. Rats were injected daily with isoniazid (100 mg/kg, i.p.) for 21 days with or without thiopronin co-administration (60 mg/kg, i.p.) from day 11 to day 21. The influence of thiopronin on isoniazid-induced DNA oxidative damage was analyzed in precision-cut rat liver slices by HPLC–MS/MS. Thiopronin prevented isoniazid-induced hepatotoxicity, indicated by both diagnostic indicators of liver damage (alanine aminotransferase and aspartate aminotransferase) and histopathological analysis. In vivo, thiopronin significantly inhibited isoniazid-induced CYP2E1 activity as assessed by both chlorzoxazone hydroxylase and aniline hydroxylase ( p < 0.001). Thiopronin concentration-dependently inhibited CYP2E1-dependent aniline hydroxylation, and the Dixon plots suggest that thiopronin is a competitive inhibitor of CYP2E1. Thiopronin markedly attenuated isoniazid-induced inhibition of the detoxification system through cytosolic glutathione S-transferases (GSTs), including mu GST and alpha GST. In precision-cut liver slices, the free radical scavenging activity of thiopronin reduced the generation of DNA adducts induced by isoniazid ( p < 0.05). Altogether, these results suggest that thiopronin exerts its hepatoprotective activity against isoniazid-induced hepatotoxicity by inhibiting the production of free radicals in addition to its role as a scavenger. Thiopronin may reduce free radical generation via inhibition of hepatic CYP2E1 and increase the removal of free radicals directly or through the induction of cytosolic GSTs.

Hepatoprotective Effects of an Anthocyanin Fraction from Purple-Fleshed Sweet Potato Against Acetaminophen-Induced Liver Damage in Mice

The present study was undertaken to examine the protective effects of an anthocyanin fraction (AF) obtained from purple-fleshed sweet potato on acetaminophen (paraceptamol [APAP])-induced hepatotoxicity in mice and to determine the mechanism involved. Mice pretreated with AF prior to APAP administration showed significantly lower increases in serum alanine aminotransferase and aspartate aminotransferase activities and hepatic malondialdehyde formation than APAP-treated animals without AF. In addition, AF prevented hepatic glutathione (GSH) depletion by APAP, and hepatic GSH levels and GSH S-transferase activities were up-regulated by AF. APAP-induced hepatotoxicity was also prevented by AF, as indicated by liver histopathology findings. In addition, the effects of AF were examined on cytochrome P450 (CYP) 2E1, the major isozyme involved in APAP bioactivation. Treatment of mice with AF significantly and dose-dependently reduced CYP2E1-dependent aniline hydroxylation and CYP2E1 protein levels. Furthermore, AF had an antioxidant effect on FeCl(2)/ascorbate-induced lipid peroxidation in mouse liver homogenates and had superoxide radical scavenging activity. These results suggest that AF protects against APAP-induced hepatotoxicity by blocking CYP2E1-mediated APAP bioactivation, by up-regulating hepatic GSH levels, and by acting as a free radical scavenger.

The Influence of Ethanol Pretreatment on the Effects of Nine Hepatotoxic Agents

The hepatotoxic effects of carbon tetrachloride (0.01 ml/kg i.p.), thioacetamide (50 mg/kg intraperitoneally), paracetamol (0.5 g/kg intraperitoneally), and allyl alcohol (0.05 ml/kg intraperitoneally) as estimated by determination of serum enzyme activities (GOT, GPT, SDH) were enhanced in mice treated with one oral dose of 4.8 g/kg ethanol 16 hrs. previously. Pretreatment of mice with ethanol did not increase the hepatotoxic actions of bromobenzene (0.25 ml/kg intraperitoneally), phalloidin (1.5 mg/kg intraperitoneally), alpha-amanitin (0.75 mg/kg intraperitoneally), and praseodymium (12 mg/kg intravenously) though there was a trend to higher enzyme activities in the case of bromobenzene. In guinea-pigs ethanol also aggravated CCl4-induced liver damage, but only strengthened the hepatotoxic activity of D-galactosamine (150 mg/kg intraperitoneally). Treatment with 4.8 g/kg ethanol did not influence liver glutathione levels in mice but increased aniline hydroxylation in the 9000 x g liver homogenate supernatant of mice and guinea-pigs. A dose of 2.4 g/kg ethanol, on the other hand, neither increased aniline hydroxylase activity nor enhanced carbon tetrachloride-induced hepatotoxicity in mice. It is assumed that the enhanced sensitivity to hepatotoxic agents after treatment with ethanol may be due to an enhanced microsomal activation of these substances.

Saponins isolated from the root of Platycodon grandiflorum protect against acute ethanol-induced hepatotoxicity in mice

The protective effects of saponins isolated from the root of Platycodon grandiflorum (Changkil saponins: CKS) against alcoholic steatosis in liver injury induced by acute ethanol administration were investigated. Pretreatment with CKS prior to ethanol administration significantly prevented the increases in serum alanine aminotransferase activity, hepatic TNF-α level, hepatic lipid peroxidation and hepatic triglyceride level. CKS prevented ethanol-induced steatosis and necrosis, as indicated by liver histopathological studies. Additionally, CKS protected against ethanol-induced depletion of hepatic glutathione levels. CYP2E1 has been suggested as a major contributor to ethanol-induced oxidative stress and liver injury. The concurrent administration of CKS efficaciously abrogated the CYP2E1 induction and CYP2E1-dependents hydroxylation of aniline as compared to the individual treatment at higher doses. These findings suggest that CKS may prevent ethanol-induced acute liver injury, possibly through its ability to block CYP2El-mediated ethanol bioactivation and its free radical scavenging effects.

Purification of CYP2B‐like protein from feral leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular, and immunological characterization

In this study, CYP2B-immunoreactive protein was purified to electrophoretic homogeneity from the liver microsomes of leaping mullet. The purified cytochrome P450 (CYP) gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a M(r) of 49,300 Da. Absolute absorption spectrum of the purified CYP showed a maximum at 417 nm and CO-difference spectrum of dithionite-reduced cytochrome P450 gave a peak at 450 nm. The purified CYP was found to be active in N-demethylation of benzphetamine, erythromycin, and ethylmorphine, and O-dealkylation of pentoxyresorufin in the reconstituted system. However, it was unable to catalyze O-dealkylation of ethoxyresorufin, methoxyresorufin, benzyloxyresorufin, and hydroxylation of lauric acid and aniline. The purified CYP showed strong cross-reactivity with anti-sheep lung CYP2B, a homologue of CYP2B4. N-terminal amino acid sequence of the mullet P450 had the highest degree of homology with CYP2Bs among the known CYPs. Spectral, electrophoretic, immunochemical, N-terminal amino acid sequence, and biocatalytic properties of the purified CYP are most similar to those of mammalian cytochrome P4502B. All these data indicate that the purified CYP is certainly 2B-like. In this study, we not only purified biocatalytically active CYP2B-like protein from fish, but also demonstrated detailed functional properties of CYP2B-like protein for the first time.

Protective effect of caffeic acid phenethyl ester against carbon tetrachloride-induced hepatotoxicity in mice

This study was designed to investigate the protective effects of the phenethyl ester of caffeic acid (CAPE) against carbon tetrachoride (CCl 4)-induced hepatotoxicities in mice. Pretreatment with CAPE prior to administration of CCl 4 significantly prevented the increases in serum alanine, aspartate aminotransferase and alkaline phosphatase activities, hepatic lipid peroxidation formation, and depletion of glutathione content. In addition, CAPE prevented CCl 4-induced apoptosis and necrosis, as indicated by liver histopathology and DNA laddering studies. To determine whether the Fas/Fas ligand (FasL) pathway is involved in CCl 4-induced acute liver injury, Fas and FasL proteins and caspase-3 and -8 activities were tested by western blotting and ELISA. CAPE markedly decreased CCl 4-induced Fas/FasL protein expression levels and, in turn, attenuated CCl 4-induced caspase-3 and -8 activities in mouse liver. Moreover, the effect of CAPE on CYP2E1, the major isozyme involved in CCl 4 bioactivation, was investigated. Treatment with CAPE significantly decreased the CYP2E1-dependent hydroxylation of aniline. In addition, CAPE attenuated the CCl 4-mediated depletion of antioxidant enzyme (catalase, superoxide dismutase and glutathione- S-transferase) activities. These findings suggest that the protective effects of CAPE against CCl 4-induced acute liver injury may involve its ability to block CYP2El-mediated CCl 4 bioactivation and to protect against Fas/FasL-mediated apoptosis.

EFFECTS OF STRUCTURALLY AND FUNCTIONALLY DIVERSE GROUP OF PHYTOCHEMICALS ON ANILINE HYDROXYLATION (CYP2E1) OF CONTROL AND ACETONE‐INDUCED RAT LIVER MICROSOMES

Although the last decade focused on exploring disease fighting properties of hundreds of phytochemicals, major gaps remain to be filled in understanding their mechanisms of actions in vivo. Our laboratory pioneered investigating anti‐toxic properties of several phytochemicals, however, information on their bioavailability, impact on drug metabolism, precise role during pathogenesis of a number of diseases, and drug‐drug interactions remains unclear. Interestingly, CYP2E1 has evolved as a major player in this field. This study investigated the effects of structurally &amp; functionally diverse group of phytochemicals on aniline hydroxylation pattern of control and acetone induced (1% in drinking water for 6 days) rat liver microsomes in vitro. Trans cinnamic acid (TCA), Resorcinol (RSL), Rutin (RUT), Quercetin (QTN), Hesperidin (HES) and Curcumin (CUR) were used at 25, 50, 100μM, 1 and 5mM concentrations. Data show that TCA and HES did not impact aniline hydroxylation at all, whereas RSL turned out to be a potent inhibitor. RUT showed 22% inhibition at 25–100 μM, 33% and 53% inhibition at 1mM &amp; 5mM respectively. CUR and QTN showed slight inhibition, 9% and 33% respectively but only at highest dose. This study suggests that different phytochemicals may have different mechanisms of interfering with drug metabolism; therefore, products containing these phytochemicals must be used with caution during drug therapy.

Protective effect of saponins derived from the roots of Platycodon grandiflorum against carbon tetrachloride induced hepatotoxicity in mice

The purpose of this study was to investigate the protective effects of the saponins isolated from the root of Platycodi Radix (Changkil saponins: CKS) on carbon tetrachloride (CCl4)-induced hepatotoxicities in mice. Pretreatment with CKS prior to the administration of CCl4 significantly prevented the increase in serum alanine aminotransferase and aspartate aminotransferase activities and hepatic lipid peroxidation formation. In addition, CKS prevented CCl4-induced apoptosis and necrosis, as indicated by a liver histopathologic study and DNA laddering. To determine whether Fas/Fas ligand (FasL) pathway involved in CCl4-induced acute liver injury, Fas and FasL proteins and caspase-3, -8 activities were tested by western blotting and ELISA. CKS markedly decreased CCl4-induced Fas/FasL protein expression levels and in turn attenuated CCl4-induced caspase-3, -8 activities in mouse livers. Additionally, CKS protected the CCl4-induced depletion of hepatic glutathione levels. The effect of CKS on CYP2E1, the major isozyme involved in CCl4 bioactivation, was investigated. Treatment with CKS resulted in a significant decrease in the CYP2E1-dependent hydroxylation of aniline. In addition, CKS exhibited antioxidant effects on FeCl2-ascorbate induced lipid peroxidation in liver homogenates, and on superoxide radical scavenging activity. These findings suggest that the protective effects of CKS against CCl4-induced acute liver injury possibly involve mechanisms related to its ability to block CYP2El-mediated CCl4 bioactivation and its free radical scavenging effects, and that is also protects against Fas/FasL pathway mediated apoptosis.

Protective effects of puerarin on carbon tetrachloride-induced hepatotoxicity

Puerarin, the main isoflavone glycoside found in the root of Pueraria lobata, has been used for various medicinal purposes in traditional Chinese medicine for thousands of years. The purpose of this study was to investigate the protective effects of puerarin against hepatotoxicity induced by carbon tetrachloride (CCl4) and the mechanism of its hepatoprotective effect. In mice, pretreatment with puerarin prior to the administration of CCl4 significantly prevented the increased serum enzymatic activity of alanine aspartate aminotransferase and hepatic malondialdehyde formation in a dose-dependent manner. In addition, pretreatment with puerarin significantly prevented both the depletion of reduced glutathione (GSH) content and the decrease in glutathione S-transferase (GST) activity in the liver of CCl4-intoxicated mice. Hepatic GSH levels and GST activity were increased by treatment with puerarin alone. CCl4-induced hepatotoxicity was also prevented, as indicated by liver histopathology. The effects of puerarin on cytochrome P450 (CYP) 2E1, the major isozyme involved in CCl4 bioactivation, were also investigated. Treatment of the mice with puerarin resulted in a significant decrease in the CYP2E1-dependent aniline hydroxylation in a dose-dependent manner. Consistent with these observations, the CYP2E1 protein levels were also lowered. Puerarin exhibited anti-oxidant effects on FeCl2-ascorbate induced lipid peroxidation in mouse liver homogenates, and on superoxide radical scavenging activity. These results suggest that the protective effects of puerarin against the CCl4-induced hepatotoxicity possibly involve mechanisms related to its ability to block CYP-mediated CCl4 bioactivation, induction of GST activity and free radical scavenging effects.

Hepatoprotective and antioxidant effects of the coffee diterpenes kahweol and cafestol on carbon tetrachloride-induced liver damage in mice

The hepatoprotective effects of kahweol and cafestol, coffee-specific diterpenes, on the carbon tetrachloride (CCl 4)-induced liver damage as well as the possible mechanisms involved in these protections were investigated. Pretreatment with kahweol and cafestol prior to the administration of CCl 4 significantly prevented the increase in the serum levels of hepatic enzyme markers (alanine aminotransferase and aspartate aminotransferase) and reduced oxidative stress, such as reduced glutathione content and lipid peroxidation, in the liver in a dose-dependent manner. The histopathological evaluation of the livers also revealed that kahweol and cafestol reduced the incidence of liver lesions induced by CCl 4. Treatment of the mice with kahweol and cafestol also resulted in a significant decrease in the cytochrome P450 2E1 (CYP2E1), the major isozyme involved in CCl 4 bioactivation, specific enzyme activities, such as p-nitrophenol and aniline hydroxylation. Kahweol and cafestol exhibited antioxidant effects on FeCl 2-ascorbate induced lipid peroxidation in a mouse liver homogenate, and on superoxide radical scavenging activity. These results suggest that the protective effects of kahweol and cafestol against the CCl 4-induced hepatotoxicity possibly involve mechanisms related to their ability to block the CYP2E1-mediated CCl 4 bioactivation and free radical scavenging effects.