Identification of UDP-glucuronosyltransferase 1A10 in non-malignant and malignant human breast tissues

UGT1A10 was recently identified as the major isoform that conjugates estrogens. In this study, real-time PCR revealed high levels of UGT1A10 and UGT2B7 mRNA in human breast tissues. The expression of UGT1A10 in breast was a novel finding. UGT1A10 and UGT2B7 mRNAs were differentially expressed among normal and malignant specimens. Their overall expression was significantly decreased in breast carcinomas as compared to normal breast specimens (UGT1A10: 68 ± 26 vs. 252 ± 86, respectively; p < 0.05) and (UGT2B7: 1.4 ± 0.7 vs. 12 ± 4, respectively; p < 0.05). Interestingly, in African American women, UGT1A10 expression was significantly decreased in breast carcinomas in comparison to normals (57 ± 35 vs. 397 ± 152, respectively; p < 0.05). Among Caucasian women, UGT2B7 was significantly decreased in breast carcinomas in comparison to normals (1.1 ± 0.5 vs. 13.5 ± 6, respectively; p < 0.05). Glucuronidation of 4-hydroxylated estrone (4-OHE₁) was significantly reduced in breast carcinomas compared to normals (30 ± 15 vs. 106 ± 31, respectively; p < 0.05). Differential down-regulation of UGT1A10 and UGT2B7 mRNAs, protein, and activity in breast carcinomas compared to the adjacent normal breast specimens from the same donor were also found. These data illustrate the novel finding of UGT1A10 in human breast and confirm the expression of UGT2B7. Significant individual variation and down-regulation of expression in breast carcinomas of both isoforms were also demonstrated. These findings provide evidence that decreased UGT expression and activity could result in the promotion of carcinogenesis.     

Protective effect of diallyl sulfone against acetaminophen-induced hepatotoxicity in mice

Diallyl sulfone (DASO₂) is a metabolite of diallyl sulfide, a compound derived from garlic. The present study investigated the effect of DASO₂ as a protective agent against acetaminophen (APAP)-induced hepatotoxicity in mice. Oral administration of DASO₂ protected mice against the APAP-induced hepatotoxicity in a dose- and time-dependent manner. When administrated 1 hour prior to, immediately after, or 20 minutes after a toxic dose of APAP, DASO₂ at a dose of 25 mg/kg completely protected mice from development of hepatotoxicity, as indicated by liver histopathology and serum lactate dehydrogenase levels. Protective effect was observed when DASO₂ at a dose as low as 5 mg/kg was given to mice 1 hour prior to APAP administration. Oral administration of DASO₂ to mice 1 hour prior to a toxic dose of APAP significantly inhibited the APAP-induced glutathione depletion in the liver. DASO₂ treatment also decreased the levels of oxidative APAP metabolites in the plasma without affecting the concentrations of nonoxidative APAP metabolites. In liver microsomes, 0.1 mM of DASO₂ caused a 60% decrease in the rate of APAP oxidation to N-acetyl-p-benzoquinone imine, which was determined as glutathione conjugate. This inhibitory effect is mainly due to its inhibition of cytochrome P450 2E1 activity; with an IC₅₀ value equal to 0.11 mM. DASO₂ also slightly inhibited the activities of P450s 3A and 1A, with IC₅₀ values >5 mM. Furthermore, a single oral dose of DASO₂ inactivated P450 2E1- and P450 1A-dependent activities in liver microsomes. The results suggest that the protective effect of DASO₂ against APAP-induced hepatotoxicity is due to its ability to block acetaminophen bioactivation mainly by the inactivation and inhibition of P450 2E1. © 1996 John Wiley & Sons, Inc.     

Melatonin Protects against Apoptosis-Inducing Factor (AIF)-Dependent Cell Death during Acetaminophen-Induced Acute Liver Failure

Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure and is primarily caused by cytochrome P450 (CYP) 2E1-driven conversion of APAP into hepatotoxic metabolites. Several reports showed that melatonin attenuated APAP-induced acute liver failure. Nevertheless, the exact mechanism remains obscure. In the present study, we investigated the effects of melatonin on apoptosis-inducing factor (AIF)-dependent cell death in APAP-induced acute liver failure. Mice were intraperitoneally (i.p.) injected with different doses of melatonin (1.25, 5, 20 mg/kg) 30 min before APAP (300 mg/kg, i.p.). As expected, melatonin significantly alleviated APAP-induced cell death, as determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Further analysis showed that melatonin significantly attenuated APAP-induced activation of the serine/threonine kinase receptor interacting protein 1 (RIP1). In addition, melatonin inhibited APAP-induced hepatic c-Jun N-terminal kinase (JNK) phosphorylation and mitochondrial Bax translocation. Correspondingly, melatonin inhibited APAP-induced translocation of AIF from mitochondria to nuclei. Interestingly, no changes were induced by melatonin on hepatic CYP2E1 expression. In addition, melatonin had little effect on APAP-induced hepatic glutathione (GSH) depletion. In conclusion, melatonin protects against AIF-dependent cell death during APAP-induced acute liver failure through its direct inhibition of hepatic RIP1 and subsequent JNK phosphorylation and mitochondrial Bax translocation.     

Mice deficient in Cu,Zn-superoxide dismutase are resistant to acetaminophen toxicity

Although antioxidants are used to treat an overdose of the analgaesic/antipyretic drug APAP (acetaminophen), roles of antioxidant enzymes in APAP-induced hepatotoxicity remain controversial. Our objective was to determine impacts of knockout of SOD1 (superoxide dismutase; Cu,Zn-SOD) alone or in combination with selenium-dependent GPX1 (glutathione peroxidase-1) on APAP-induced hepatotoxicity. All SOD1-null (SOD1-/-) and SOD1- and GPX1-double-knockout mice survived an intraperitoneal injection of 600 mg of APAP per kg of body mass, whereas 75% of WT (wild-type) and GPX1-null mice died within 20 h. Survival time of SOD1-/- mice injected with 1200 mg of APAP per kg of body mass was longer than that of the WT mice (934 compared with 315 min, P<0.05). The APAP-treated SOD1-/- mice had less (P<0.05) plasma ALT (alanine aminotransferase) activity increase and attenuated (P<0.05) hepatic glutathione depletion than the WT mice. The protection conferred by SOD1 deletion was associated with a block of the APAP-mediated hepatic protein nitration and a 50% reduction (P<0.05) in activity of a key APAP metabolism enzyme CYP2E1 (cytochrome P450 2E1) in liver. The SOD1 deletion also caused moderate shifts in the APAP metabolism profiles. In conclusion, deletion of SOD1 alone or in combination with GPX1 greatly enhanced mouse resistance to APAP overdose. Our results suggest a possible pro-oxidant role for the physiological level of SOD1 activity in APAP-mediated hepatotoxicity.     

New Therapeutic Approach: Diphenyl Diselenide Reduces Mitochondrial Dysfunction in Acetaminophen-Induced Acute Liver Failure

The acute liver failure (ALF) induced by acetaminophen (APAP) is closely related to oxidative damage and depletion of hepatic glutathione, consequently changes in cell energy metabolism and mitochondrial dysfunction have been observed after APAP overdose. Diphenyl diselenide [(PhSe)₂], a simple organoselenium compound with antioxidant properties, previously demonstrated to confer hepatoprotection. However, little is known about the protective mechanism on mitochondria. The main objective of this study was to investigate the effects (PhSe)₂ to reduce mitochondrial dysfunction and, secondly, compare in the liver homogenate the hepatoprotective effects of the (PhSe)₂ to the N-acetylcysteine (NAC) during APAP-induced ALF to validate our model. Mice were injected intraperitoneal with APAP (600 mg/kg), (PhSe)₂ (15.6 mg/kg), NAC (1200 mg/kg), APAP+(PhSe)₂ or APAP+NAC, where the (PhSe)₂ or NAC treatment were given 1 h following APAP. The liver was collected 4 h after overdose. The plasma alanine and aspartate aminotransferase activities increased after APAP administration. APAP caused a remarkable increase of oxidative stress markers (lipid peroxidation, reactive species and protein carbonylation) and decrease of the antioxidant defense in the liver homogenate and mitochondria. APAP caused a marked loss in the mitochondrial membrane potential, the mitochondrial ATPase activity, and the rate of mitochondrial oxygen consumption and increased the mitochondrial swelling. All these effects were significantly prevented by (PhSe)₂. The effectiveness of (PhSe)₂ was similar at a lower dose than NAC. In summary, (PhSe)₂ provided a significant improvement to the mitochondrial redox homeostasis and the mitochondrial bioenergetics dysfunction caused by membrane permeability transition in the hepatotoxicity APAP-induced.     

Sulfation and glucuronidation of acetaminophen by cultured hepatocytes reproducing in vivo sex-differences in conjugation on matrigel and type 1 collagen

Summary The sulfate and glucuronide conjugation of acetaminophen (APAP) by hepatocytes cultured on Matrigel or type 1 collagen was compared to APAP metabolism in vivo. The metabolic fate of low (15 mg/kg), medium (125 mg/kg), and high (300 mg/kg) doses of APAP injected intraperitoneally were determined in male and female rats. Males excreted more APAP as the sulfate conjugate than females, which correlated with the twofold greater APAP sulfotransferase activity in the male vs. females (301±24 vs. 156±18 pmol · mg⁻¹ protein · min⁻¹). Also, as sulfate conjugation became saturated, there was a dose-related shift in APAP metabolism from sulfate to glucuronide conjugation in both sexes. After death, the livers of the same animals were perfused with collagenase and the hepatocytes cultured in modified Waymouth’s medium on either Matrigel or rat-tail collagen, with various doses of APAP (0, 0.125, 0.25, 0.5, and 1.0 mM). Sex differences in APAP sulfation and glucuronidation persisted in culture for up to 4 days, with sulfation predominating in the male similar to in vivo. With increasing APAP concentration (dose), there was a saturation of sulfate conjugation and a shift to glucuronidation as observed in vivo. Sex differences in APAP sulfation and glucuronidation were no longer significant by Day 4 in culture. Sulfation, and to a lesser extent, glucuronidation, were more stable on Matrigel than collagen. We concluded that APAP metabolism of freshly isolated hepatocytes could replicate in vivo sex differences in conjugation, and that Matrigel was superior to collagen as substrate.     

Kava extract, an herbal alternative for anxiety relief, potentiates acetaminophen-induced cytotoxicity in rat hepatic cells

The widely used over-the-counter analgesic acetaminophen (APAP) is the leading cause of acute liver failure in the United States and due to this high incidence, a recent FDA Advisory Board recommended lowering the maximum dose of APAP. Kava herbal dietary supplements have been implicated in several human liver failure cases leading to the ban of kava-containing products in several Western countries. In the US, the FDA has issued warnings about the potential adverse effects of kava, but kava dietary supplements are still available to consumers. In this study, we tested the potential of kava extract to potentiate APAP-induced hepatocyte cytotoxicity. In rat primary hepatocytes, co-treatment with kava and APAP caused 100% loss of cell viability, while the treatment of kava or APAP alone caused ∼50% and ∼30% loss of cell viability, respectively. APAP-induced glutathione (GSH) depletion was also potentiated by kava. Co-exposure to kava decreased cellular ATP concentrations, increased the formation of reactive oxygen species, and caused mitochondrial damage as indicated by a decrease in mitochondrial membrane potential. In addition, similar findings were obtained from a cultured rat liver cell line, clone-9. These observations indicate that kava potentiates APAP-induced cytotoxicity by increasing the magnitude of GSH depletion, resulting in oxidative stress and mitochondrial dysfunction, ultimately leading to cell death. These results highlight the potential for drug-dietary supplement interactions even with widely used over-the-counter drugs.     

Short term effects of dexamethasone on hyaluronidase activity and sperm characteristics in rams

The aim of this study was to determine the effects of dexamethasone on sperm characteristics and hyaluronidase activity of serum and semen. In this investigation, 14 healthy Akkaraman rams, at the age of 2 years and weighing between 50–60 kg, were used. The rams were randomly divided into two groups. After the last administration of dexamethasone intramuscularly at a dose of 0.25 mg/kg, semen and blood samples were taken at different times. The results showed that the serum hyaluronidase activity was increased significantly (p < 0.001) in the treatment group when compared with the control group except for the 1st hour. There was a significant difference (p < 0.001, 0.01, 0.05) in the hyaluronidase activity of semen between the treatment group and the control group. Furthermore, there was a significant difference (p < 0.01) in sperm concentration between both groups at all the times except the 96th hour. There were statistically significant (p < 0.05) differences in semen volume between the treatment and control groups. There were also significant differences (p < 0.05) in sperm motility between the treatment and control groups except for the 72 and 96th hours.

These findings indicate that dexamethasone increases hyaluronidase activity of serum and semen, but it decreases sperm concentration, semen volume and sperm motility in rams. Therefore the use of these drugs in breeding rams during breeding season is not suitable.     

Enhanced Production of Adenosine Triphosphate by Pharmacological Activation of Adenosine Monophosphate-Activated Protein Kinase Ameliorates Acetaminophen-Induced Liver Injury

The hepatic cell death induced by acetaminophen (APAP) is closely related to cellular adenosine triphosphate (ATP) depletion, which is mainly caused by mitochondrial dysfunction. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a key sensor of low energy status. AMPK regulates metabolic homeostasis by stimulating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. We found that the decrease in active phosphorylation of AMPK in response to APAP correlates with decreased ATP levels, in vivo. Therefore, we hypothesized that the enhanced production of ATP via AMPK stimulation can lead to amelioration of APAP-induced liver failure. A769662, an allosteric activator of AMPK, produced a strong synergistic effect on AMPK Thr172 phosphorylation with APAP in primary hepatocytes and liver tissue. Interestingly, activation of AMPK by A769662 ameliorated the APAP-induced hepatotoxicity in C57BL/6N mice treated with APAP at a dose of 400 mg/kg intraperitoneally. However, mice treated with APAP alone developed massive centrilobular necrosis, and APAP increased their serum alanine aminotransferase and aspartate aminotransferase levels. Furthermore, A769662 administration prevented the loss of intracellular ATP without interfering with the APAP-mediated reduction of mitochondrial dysfunction. In contrast, inhibition of glycolysis by 2-deoxy-glucose eliminated the beneficial effects of A769662 on APAP-mediated liver injury. In conclusion, A769662 can effectively protect mice against APAP-induced liver injury through ATP synthesis by anaerobic glycolysis. Furthermore, stimulation of AMPK may have potential therapeutic application for APAP overdose.     

Arjunolic acid,a triterpenoid saponin,prevents acetaminophen (APAP)-induced liver and hepatocyte injury via the inhibition of APAP bioactivation and JNK-mediated mitochondrial protection

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug and is safe at therapeutic doses but its overdose frequently causes liver injury. In earlier studies, we demonstrated that arjunolic acid (AA) has a protective effect against chemically induced hepatotoxicity. The purpose of this study was to explore whether AA plays any protective role against APAP-induced acute hepatotoxicity and, if so, what molecular pathways it utilizes for the mechanism of its protective action. Exposure of rats to a hepatotoxic dose of acetaminophen (700 mg/kg, ip) altered a number of biomarkers (related to hepatic oxidative stress), increased reactive oxygen species production, reduced cellular adenosine triphosphate level, and induced necrotic cell death. Arjunolic acid pretreatment (80 mg/kg, orally), on the other hand, afforded significant protection against liver injury. Arjunolic acid also prevented acetaminophen-induced hepatic glutathione depletion and APAP metabolite formation although arjunolic acid itself did not affect hepatic glutathione levels. The results suggest that this preventive action of arjunolic acid is due to the metabolic inhibition of specific forms of cytochrome P450 that activate acetaminophen to N-acetyl-p-benzoquinone imine. In addition, administration of arjunolic acid 4 h after acetaminophen intoxication reduced acetaminophen-induced JNK and downstream Bcl-2 and Bcl-xL phosphorylation, thus protecting against mitochondrial permeabilization, loss of mitochondrial membrane potential, and cytochrome c release. In conclusion, the data suggest that arjunolic acid affords protection against acetaminophen-induced hepatotoxicity through inhibition of P450-mediated APAP bioactivation and inhibition of JNK-mediated activation of mitochondrial permeabilization.     

Associations between common variation in the aromatase gene promoter region and testosterone concentrations in two young female populations

We recently reported association between a coding-region single nucleotide polymorphism (SNP50) in the aromatase gene that encodes a key enzyme in testosterone metabolism, with risk for the development of precocious pubarche and circulating testosterone concentrations in two independent female populations. We have now explored further association with variation in the promoter-region of the aromatase gene. We genotyped six promoter-region haplotype-tag SNPs in young women from Oxford, UK (n = 109), and in girls with precocious pubarche (n = 186) and controls (n = 71) from Barcelona, Spain. Aromatase distal promoter-region variation was associated with plasma testosterone concentrations in both Oxford (r² = 18.3%, p = 0.01) and Barcelona (r² = 8.5%, p = 0.03) females. These associations were independent of SNP50, but appeared to be dependent on different SNPs in Oxford (r² = 13.7%, p = 0.006 with SNPs 11 (p = 0.009), 28 (p = 0.02) and 39 (p = 0.06)) and Barcelona (r² = 5.9%, p = 0.002 with SNP43 (p = 0.002)) populations. Aromatase distal promoter-region variation was also associated with PCOS symptom score in Oxford women (r² = 14.5%, p = 0.048), but, unlike SNP50, was not associated with precocious pubarche risk in Barcelona girls. In conclusion, aromatase distal promoter-region variation appears to have functional consequences for plasma testosterone concentrations in females. The variable associations with androgen-related clinical features could possibly reflect the tissue-specific promoters of the aromatase gene.     

Protective effects of BML-111 against acetaminophen-induced acute liver injury in mice

The present study was undertaken to investigate the effect of the new formyl peptide receptor 2/lipoxin A₄ receptor agonist BML-111 on acetaminophen (APAP)-induced liver injury in mice and explore its possible mechanism(s). Male Swiss albino mice were intraperitoneally injected with BML-111 (1 mg/kg) twice daily for five consecutive days prior to a single intraperitoneal injection of APAP (500 mg/kg). Results have shown that APAP injection caused liver damage as indicated by significant increase in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). Liver histopathological examination revealed marked necrosis and inflammation. Additionally, APAP decreased activities of hepatic glutathione (GSH) and superoxide dismutase (SOD) with significant increase in the hepatic malondialdehyde (MDA) content. Furthermore, APAP increased serum nitrite/nitrate (NO₂ ^?/NO₃ ^? ) level and hepatic tumor necrosis factor alpha (TNF-α). Pretreatment with BML-111 significantly reversed all APAP-induced pathological changes. BML-111 prevented the increase of AST, ALT, and ALP. Also, BML-111 markedly attenuated APAP-induced necrosis and inflammation. It decreased MDA with increase in SOD and GSH. Importantly, BML-111 decreased NO₂ ^?/NO₃ ^? level and TNF-α. These findings suggest that BML-111 has hepatoprotective effects against APAP-induced liver injury in mice. Its protective effect may be attributed to its ability to counteract the inflammatory ROS generation and regulate cytokine effects.     

Serum pancreatic lipase [EC 3.1.1.3] activity, serum lipid profile and peripheral blood dendritic cell populations in normolipidemic males with psoriasis

The purpose of the study was to explore serum pancreatic lipase activity and the serum lipid profile in relation to peripheral blood dendritic cell subsets and disease severity in males with psoriasis.

Material and methods

The study population consisted of 22 normolipidemic males with psoriasis and 12 aged-matched and body mass index (BMI)-matched healthy males. The percentages of peripheral blood dendritic cell (DC) subsets were evaluated using appropriate monoclonal antibodies and flow cytometry. The serum pancreatic lipase activity and the lipid profile were determined using standard enzymatic and colorimetric techniques.

Results

Pancreatic lipase activity was increased (p = 0.56421), high-density lipoprotein (HDL)-cholesterol concentration (p = 0.00584) was significantly decreased, triglyceride (p = 0.00766) and VLDL-cholesterol (p = 0.00765) levels were significantly increased in serum of psoriatic patients compared to controls. The serum pancreatic lipase activity showed significant correlation with serum triglyceride (r = 0.42; p = 0.04721) and serum VLDL-cholesterol levels (r = 0.42; p = 0.04721) in psoriatic individuals. In psoriatic patients the percentage of myeloid DCs was increased (p = 0.54932), the percentage of lymphoid DCs was decreased (p = 0.14210) and myeloid DC/lymphoid DC ratio was significantly increased (p = 0.03569) compared to healthy individuals.

Conclusion

The direct cause of the abnormal lipid profile in psoriasis and its relationship with the immune system disturbances remains unclear. The reciprocal relationship between serum pancreatic activity and serum triglyceride level appears to confirm the hypothesis about abnormal lipid metabolism in psoriasis.     

Abrogation by Trifolium alexandrinum root extract on hepatotoxicity induced by acetaminophen in rats

Abstract

Objective

Acetaminophen (APAP) is a substance that harms human health by stimulating free radical production. This study investigated the ability of Trifolium alexandrinum root (TAR) extract to reduce the hepatotoxicity induced by APAP in rats.

Methods

Animals were classified into four groups and treated for 6 weeks. Group 1: normal control-treated (saline); Group 2: TAR extract-treated (100 mg/kg); Group 3: APAP-treated; Group 4: APAP plus TAR extract.

Results

APAP significantly elevated AST (aspartate amino transferase), ALT (amino alanine transferase), ALP (alkaline phosphatase), GGTP (gamma glutamyl transpeptidase), bilirubin, and malondialdehyde with a significant decrease in glutathione, superoxide dismutase, glutathione peroxidase, catalase, and glutathione S-transferase compared with the control group. Administration of TAR extract combined with APAP improved the liver damage induced by APAP. Histopathological evidence, together with observed DNA fragmentation, supported the detrimental effect of APAP and the ameliorating effect of TAR extract on liver toxicity.

Conclusion

TAR extract has beneficial properties and can reduce the liver damage and toxicity induced by APAP.

Discussion

Free radical mediated processes have been implicated in the pathogenesis of many diseases. The protective effect of TAR root extract on APAP-induced hepatotoxicity in rats appears to be related to inhibition of lipid peroxidation and enhancement of antioxidant enzyme levels, in addition to a free radical scavenging action.     

Comparative evaluation of N-acetylcysteine and N-acetylcysteineamide in acetaminophen-induced hepatotoxicity in human hepatoma HepaRG cells

Acetaminophen (N-acetyl-p-aminophenol, APAP) is one of the most widely used over-the-counter antipyretic analgesic medications. Despite being safe at therapeutic doses, an accidental or intentional overdose can result in severe hepatotoxicity; a leading cause of drug-induced liver failure in the U.S. Depletion of glutathione (GSH) is implicated as an initiating event in APAP-induced toxicity. N-acetylcysteine (NAC), a GSH precursor, is the only currently approved antidote for an APAP overdose. Unfortunately, fairly high doses and longer treatment times are required due to its poor bioavailability. In addition, oral and intravenous administration of NAC in a hospital setting are laborious and costly. Therefore, we studied the protective effects of N-acetylcysteineamide (NACA), a novel antioxidant, with higher bioavailability and compared it with NAC in APAP-induced hepatotoxicity in a human-relevant in vitro system, HepaRG. Our results indicated that exposure of HepaRG cells to APAP resulted in GSH depletion, reactive oxygen species (ROS) formation, increased lipid peroxidation, mitochondrial dysfunction (assessed by JC-1 fluorescence), and lactate dehydrogenase release. Both NAC and NACA protected against APAP-induced hepatotoxicity by restoring GSH levels, scavenging ROS, inhibiting lipid peroxidation, and preserving mitochondrial membrane potential. However, NACA was better than NAC at combating oxidative stress and protecting against APAP-induced damage. The higher efficiency of NACA in protecting cells against APAP-induced toxicity suggests that NACA can be developed into a promising therapeutic option for treatment of an APAP overdose.     

A Txnrd1-dependent metabolic switch alters hepatic lipogenesis,glycogen storage,and detoxification

Besides helping to maintain a reducing intracellular environment, the thioredoxin (Trx) system impacts bioenergetics and drug metabolism. We show that hepatocyte-specific disruption of Txnrd1, encoding Trx reductase-1 (TrxR1), causes a metabolic switch in which lipogenic genes are repressed and periportal hepatocytes become engorged with glycogen. These livers also overexpress machinery for biosynthesis of glutathione and conversion of glycogen into UDP-glucuronate; they stockpile glutathione-S-transferases and UDP-glucuronyl-transferases; and they overexpress xenobiotic exporters. This realigned metabolic profile suggested that the mutant hepatocytes might be preconditioned to more effectively detoxify certain xenobiotic challenges. Hepatocytes convert the pro-toxin acetaminophen (APAP, paracetamol) into cytotoxic N-acetyl-p-benzoquinone imine (NAPQI). APAP defenses include glucuronidation of APAP or glutathionylation of NAPQI, allowing removal by xenobiotic exporters. We found that NAPQI directly inactivates TrxR1, yet Txnrd1-null livers were resistant to APAP-induced hepatotoxicity. Txnrd1-null livers did not have more effective gene expression responses to APAP challenge; however, their constitutive metabolic state supported more robust GSH biosynthesis, glutathionylation, and glucuronidation systems. Following APAP challenge, this effectively sustained the GSH system and attenuated damage.     

Double null of selenium-glutathione peroxidase-1 and copper, zinc-superoxide dismutase enhances resistance of mouse primary hepatocytes to acetaminophen toxicity

This study was conducted to determine the impact of knockout of selenium (Se)-dependent glutathione peroxidase-1 (GPX1-/-) or double knockout of GPX1 and copper, zinc (Cu,Zn)-super-oxide dismutase (SOD1) on cell death induced by acetaminophen (APAP) and its major toxic metabolite N-acetyl-P-benzoquinoneimine (NAPQI). Primary hepatocytes were isolated from GPX1-/-, double knockout of GPX1 and SOD1 (DKO), and their wild-type (WT) mice and were treated with 5 mM APAP or 100 microM NAPQI for 0, 6, and 12 hrs. Compared with the WT cells, the GPX1-/- and DKO hepatocytes were more resistant (P < 0.05) to the APAP-induced cell death but less resistant to the NAPQI-induced cell death. The APAP-mediated glutathione (GSH) depletion was greater (P < 0.05) at 6 hrs in the WT cells than in the GPX1-/- and DKO cells, whereas there was no genotype effect on the NAPQI-mediated GSH depletion. The DKO cells had lower (P < 0.05) microsomal cytochrome P450 2E1 activities, but higher (P < 0.05) glutathione reductase and thioredoxin reductase activities than the WT cells at 0 hrs, and they responded differently to the APAP and NAPQI treatments. Glutathione-S-transferase activity was not affected by genotypes or treatments. Neither APAP nor NAPQI induced nitric oxide production or protein nitration in cells of any genotype. However, the GPX1-/- and DKO cells were more resistant to peroxynitrite-mediated protein nitration than were the WT cells. In conclusion, double null of GPX1 and SOD1 enhanced the resistance of mouse primary hepatocytes to APAP toxicity by affecting events prior to or at NAPQI formation. While the double knockout attenuated the peroxynitrite-mediated protein nitration in hepatocytes, no protein nitration was detected in these cells treated with APAP or NAPQI.     

Consummatory behavior and metabolic indicators after central ghrelin injections in rats

Ghrelin, an endogenous ligand for the growth-hormone-secretagogue receptor, is a 28-amino acid peptide with a post-translational acyl modification necessary for its activity. It has central nervous system actions that affect appetite, body mass and energy balance. An intracerebroventricular (ICV) injection protocol of sub-nanomolar doses of ghrelin, known to alter the morphology of ACTH and GH producing pituicytes and plasma levels of these hormones, was used to provide an overview of metabolic changes linked to energy metabolism. Variables measured were: food intake (FI), water intake (WI), fecal mass, urine volume, body weight (BW), retroperitoneal (RP) and epididymal (EPI) white adipose tissue (WAT), and changes in serum leptin, insulin, triglycerides, cholesterol, and glucose. Five injections of rat ghrelin or PBS (n = 8 per group) were given ICV every 24 h (1 μg/5 μL PBS) to adult male rats. Ghrelin had a positive and cumulative effect on FI, WI and BW (p < 0.05), but not feces mass or urine volume (p > 0.05). Centrally applied ghrelin clearly increased RP WAT (by 235%, p < 0.001), EPI WAT (by 85%, p < 0.05) and serum insulin levels (by 43%, p < 0.05), and decreased serum leptin levels (by 77%, p < 0.05) without (p > 0.05) evoking changes in blood triglyceride cholesterol, or glucose levels.

These data and the available literature clearly document that exposure of the brain of normal rats, over time, to sub-nanomolar doses of ghrelin results in metabolic dysregulation culminating in increased body mass, consummatory behavior, and lipid stores as well as changes in blood leptin/insulin levels. Thus, modulation of central ghrelin receptors may represent a pharmacological approach for controlling multiple factors involved in energy balance and obesity.