Activation of the adenosine A2B receptor even beyond the therapeutic window of N-acetylcysteine accelerates liver recovery after an acetaminophen overdose.

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the USA. The short therapeutic window of the current antidote, N-acetylcysteine (NAC) highlights the need for novel late acting therapeutics. The neuronal guidance cue netrin-1 provides delayed protection against APAP hepatotoxicity through the adenosine A2B receptor (A2BAR). The clinical relevance of this mechanism was investigated here by administration of the A2BAR agonist BAY 60-6583, after an APAP overdose (300 or 600 mg/kg) in fasted male and female C57BL/6J mice with assessment of liver injury 6 or 24 h after APAP in comparison to NAC. BAY 60-6583 treatment 1.5 h after APAP overdose (600 mg/kg) protected against liver injury at 6 h by preserving mitochondrial function despite JNK activation and its mitochondrial translocation. Gender independent protection was sustained when BAY 60-6583 was given 6 h after APAP overdose (300 mg/kg), when NAC administration did not show benefit. This protection was accompanied by enhanced infiltration of macrophages with the reparative anti-inflammatory phenotype by 24 h, accompanied by a decrease in neutrophil infiltration. Thus, our data emphasize the remarkable therapeutic utility of using an A2BAR agonist, which provides delayed protection long after the standard of care NAC ceased to be effective.

Novel strategies for the treatment of acetaminophen hepatotoxicity.

INTRODUCTION: Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the western world. Despite extensive investigations into the mechanisms of cell death, only a single antidote, N-acetylcysteine, is in clinical use. However, there have recently been more efforts made to translate mechanistic insight into identification of therapeutic targets and potential new drugs for this indication. AREAS COVERED: After a short review of the key events in the pathophysiology of APAP-induced liver injury and recovery, the pros and cons of targeting individual steps in the pathophysiology as therapeutic targets are discussed. While the re-purposed drug fomepizole (4-methylpyrazole) and the new entity calmangafodipir are most advanced based on the understanding of their mechanism of action, several herbal medicine extracts and their individual components are also considered. EXPERT OPINION: Fomepizole (4-methylpyrazole) is safe and has shown efficacy in preclinical models, human hepatocytes and in volunteers against APAP overdose. The safety of calmangafodipir in APAP overdose patients was shown but it lacks solid preclinical efficacy studies. Both drugs require a controlled phase III trial to achieve regulatory approval. All studies of herbal medicine extracts and components suffer from poor experimental design, which questions their clinical utility at this point.

Novel Therapeutic Approaches Against Acetaminophen-induced Liver Injury and Acute Liver Failure.

Liver injury and acute liver failure caused by acetaminophen (APAP, N-acetyl-p-aminophenol, paracetamol) overdose is a significant clinical problem in most western countries. The only clinically approved antidote is N-acetylcysteine (NAC), which promotes the recovery of hepatic GSH. If administered during the metabolism phase, GSH scavenges the reactive metabolite N-acetyl-p-benzoquinone imine. More recently, it was shown that NAC can also reconstitute mitochondrial GSH levels and scavenge reactive oxygen/peroxynitrite and can support mitochondrial bioenergetics. However, NAC has side effects and may not be efficacious after high overdoses. Repurposing of additional drugs based on their alternate mechanisms of action could be a promising approach. 4-Methylpyrazole (4MP) was shown to be highly effective against APAP toxicity by inhibiting cytochrome P450 enzymes in mice and humans. In addition, 4MP is a potent c-Jun N-terminal kinase inhibitor expanding its therapeutic window. Calmangafodipir (CMFP) is a SOD mimetic, which is well tolerated in patients and has the potential to be effective after severe overdoses. Other drugs approved for humans such as metformin and methylene blue were shown to be protective in mice at high doses or at human therapeutic doses, respectively. Additional protective strategies such as enhancing antioxidant activities, Nrf2-dependent gene induction and autophagy activation by herbal medicine components are being evaluated. However, at this point, their mechanistic insight is limited, and the doses used are high. More rigorous mechanistic studies are needed to advance these herbal compounds. Nevertheless, based on recent studies, 4-methylpyrazole and calmangafodipir have realistic prospects to become complimentary or even alternative antidotes to NAC for APAP overdose.

Cleistanthus collinus poisoning affects mitochondrial respiration and induces oxidative stress in the rat kidney.

Cleistanthus collinus is a poisonous shrub used for deliberate self-harm in rural areas of South India and intake of boiled decoction of leaves is a common method of self-harm. Distal renal tubular acidosis (dRTA) is an important clinical symptom observed in C. collinus poisoning, and renal V-ATPases may be potential targets of damage. However, a lack of understanding of molecular mediators involved hampers medical management, which is mainly supportive. We hypothesized that C. collinus poisoning induces renal oxidative stress; probably by inducing mitochondrial uncoupling, which compromises V-ATPase activity to ultimately produce dRTA. This was tested by exposing renal BBMV, kidney cells in culture, and Wistar rats to C. collinus poisoning. Exposure to C. collinus aqueous extract resulted in significant elevations in the lipid peroxidation marker, conjugated dienes, in cell culture and in vivo. A significant decrease in mitochondrial respiratory control ratio was observed in kidneys from C. collinus-treated animals suggesting that mitochondrial oxidative phosphorylation is uncoupled. This was accompanied by significant increase in ADP levels and a decrease in proton pump activity. Thus, these results demonstrate that C. collinus poisoning induces oxidative stress which influences proton pump activity, probably due to feedback inhibition by elevated ADP levels because of mitochondrial dysfunction in the rat kidney.

The role of apoptosis in acetaminophen hepatotoxicity.

Although necrosis is recognized as the main mode of cell death induced by acetaminophen (APAP) overdose in animals and humans, more recently an increasing number of publications, especially in the herbal medicine and dietary supplement field, claim an important contribution of apoptotic cell death in the pathophysiology. However, most of these conclusions are based on parameters that are not specific for apoptosis. Therefore, the objective of this review was to re-visit the key signaling events of receptor-mediated apoptosis and APAP-induced programmed necrosis and critically analyze the parameters that are being used as evidence for apoptotic cell death. Both qualitative and quantitative comparisons of parameters such as Bax, Bcl-2, caspase processing and DNA fragmentation in both modes of cell death clearly show fundamental differences between apoptosis and cell death induced by APAP. These observations together with the lack of efficacy of pan-caspase inhibitors in the APAP model strongly supports the conclusion that APAP hepatotoxicity is dominated by necrosis or programmed necrosis and does not involve relevant apoptosis. In order not to create a new controversy, it is important to understand how to use these "apoptosis" parameters and properly interpret the data. These issues are discussed in this review.

Enteral glutamine differentially regulates Nrf 2 along the villus-crypt axis of the intestine to enhance glutathione levels.

BACKGROUND AND AIM: Glutamine is an important energy source for the intestinal epithelium, and its supplementation protects intestinal epithelial cells by induction of glutathione. However, mechanisms of glutathione induction in cells at various stages of differentiation along the crypt to villus axis are not well understood. This study examined induction of glutathione in response to glutamine along the intestinal villus-crypt axis and evaluated regulatory mediators involved in the process. METHODS: Animals were administered 4% glutamine in feed for 7 days, following which enterocytes at various stages of differentiation were isolated and glutathione levels and signaling mediators involved in its regulation were studied. RESULTS: In control animals, glutathione levels were higher in the intestinal crypt than in the villus or middle region. This was accompanied by elevated expression of the modifier subunit of glutathione synthetase (GCLM) and the transcription factor Nrf2 when compared with cells from the villus and middle regions. These levels were further enhanced by glutamine throughout the intestine, although the effects were more dramatic in the crypt. In parallel to glutathione induction, glutamine supplementation also altered actin dynamics and proliferation in cells of the crypt. CONCLUSIONS: These results suggest that the variation of glutathione levels along the villus-crypt axis in the intestine is due to gradients in expression of mediators such as glutamate cysteine ligase modifier subunit and Nrf2. The protective effects of glutamine supplementation seem to be most pronounced in the crypt, where it upregulates proliferation, glutathione levels and alters actin dynamics.

Models of drug-induced liver injury for evaluation of phytotherapeutics and other natural products.

Extracts from medicinal plants, many of which have been used for centuries, are increasingly tested in models of hepatotoxicity. One of the most popular models to evaluate the hepatoprotective potential of natural products is acetaminophen (APAP)-induced liver injury, although other hepatotoxicity models such as carbon tetrachloride, thioacetamide, ethanol and endotoxin are occasionally used. APAP overdose is a clinically relevant model of drug-induced liver injury. Critical mechanisms and signaling pathways, which trigger necrotic cell death and sterile inflammation, are discussed. Although there is increasing understanding of the pathophysiology of APAP-induced liver injury, the mechanism is complex and prone to misinterpretation, especially when unknown chemicals such as plant extracts are tested. This review discusses the fundamental aspects that need to be considered when using this model, such as selection of the animal species or in vitro system, timing and dose-responses of signaling events, metabolic activation and protein adduct formation, the role of lipid peroxidation and apoptotic versus necrotic cell death, and the impact of the ensuing sterile inflammatory response. The goal is to enable researchers to select the appropriate model and experimental conditions for testing of natural products that will yield clinically relevant results and allow valid interpretations of the pharmacological mechanisms.

Mechanisms of toxicity of Cleistanthus collinus: vacuolar ATPases are a putative target.

Ingestion of Cleistanthus collinus, a shrub native to South India, either intentionally or accidentally, is a common cause of death in the area. Consumption of a boiled decoction of leaves is highly toxic, but medical management of patients is mainly supportive because the molecular mechanisms of toxin action are unknown. Distal renal tubular acidosis is one of the symptoms of poisoning in patients and adenosine triphosphate (ATP) requiring proton pumps is important for acid secretion in the kidney. Hence, we hypothesized that these may be putative targets for C. collinus action and we tested this by exposing rat renal brush border membrane (BBM) as well as cultured kidney cells to a boiled decoction of C. collinus. Exposure to the C. collinus decoction resulted in significant inhibition of vacuolar type H(+)-ATPase (V-ATPase) activity in renal BBM as well as blocking of the proton pump in renal BBM vesicles. C. collinus decoction was also found to inhibit acidification of intracellular organelles in cells in culture, similar to the effect seen with either bafilomycin or concanamycin - specific inhibitors of the V-ATPase. This was accompanied by a decrease in V-ATPase activity, but an increase in protein levels. These results demonstrate that the V-ATPase in renal cells is a putative target for the toxins in C. collinus and the inhibition of this important proton pump probably plays a role in the development of distal renal tubular acidosis and subsequent renal failure seen in poisoned patients.

Locally made ready to use therapeutic food for treatment of malnutrition a randomized controlled trial.

OBJECTIVE: To evaluate the effectiveness of a locally made ready-to-use therapeutic food (RUTF) in decreasing mild to moderate malnutrition. DESIGN: A randomized open label, controlled trial. SETTING: Pre-schools run by the Department of Community Health in Kaniyambadi administrative block, Vellore, India; duration of follow-up 3 months from the date of recruitment. PARTICIPANTS: Pupils aged 18 -60 months with Weight-for-Age 2 SD. INTERVENTIONS: A locally produced energy-dense supplement (RUTF), and the current standard of care [teaching caregivers how to make a fortified cereal-milk supplement called High Calorie Cereal Milk (HCCM)]. MAIN OUTCOME MEASURES: Increase in weight-for-age status; increase in levels of plasma zinc, vitamin B12, serum albumin and haemoglobin. RESULTS: The Mean (SD) weight gain at 3 months was higher in the RUTF group: RUTF (n=51): 0.54 kg; (SE = 0.05; 95% CI = 0.44 - 0.65) vs HCCM (n=45): 0.38 kg;(SE = 0.06; 95% CI = 0.25 - 0.51), P = 0.047. The weight gain per kilogram of body weight was directly proportional to the severity of malnutrition. CONCLUSIONS: Community-based treatment showed weight gain in both groups, the gain being higher with RUTF.

Mild whole-body heat stress alters retinoid metabolism in the rat small intestine.

Mild heat treatment can modulate metabolism and prevent stress-induced alterations in cells and tissues. Retinoids are known to influence cellular metabolism and are essential for growth and differentiation, particularly of epithelial tissue. This study examines the effect of mild heat treatment on retinoid alterations in enterocytes in the rat small intestine. Heat treatment changed the differentiation pattern of enterocytes along the villus-crypt axis, accompanied by increases in retinol, retinaldehyde, and retinoic acid in proliferating crypt cells. Activities of retinoid metabolizing enzymes such as retinaldehyde oxidase and retinaldehyde reductase were also increased. These results suggest that mild heat treatment can alter retinoid metabolism in the small intestine, which might influence epithelial cell proliferation and differentiation.

Intestinal mucosal alterations in rats with carbon tetrachloride-induced cirrhosis: changes in glycosylation and luminal bacteria.

Spontaneous bacterial peritonitis is a major cause of mortality after liver cirrhosis. Altered permeability of the mucosa and deficiencies in host immune defenses through bacterial translocation from the intestine due to intestinal bacterial overgrowth have been implicated in the development of this complication. Molecular mechanisms underlying the process are not well known. In order to understand mechanisms involved in translocation of bacteria, this study explored the role of oxidative stress in mediating changes in intestinal mucosal glycosylation and luminal bacterial content during cirrhosis. CCl4-induced cirrhosis in rats led to prolonged oxidative stress in the intestine, accompanied by increased sugar content of both intestinal brush border and surfactant layers. This was accompanied by changes in bacterial flora in the gut, which showed increased hydrophobicity and adherence to the mucosa. Inhibition of xanthine oxidase using sodium tungstate or antioxidant supplementation using vitamin E reversed the oxidative stress, changes in brush border membrane sugar content, and bacterial adherence. In conclusion, oxidative stress in the intestine during cirrhosis alters mucosal glycosylation, accompanied by an increased hydrophobicity of luminal bacteria, enabling increased bacterial adherence onto epithelial cells. This might facilitate translocation across the mucosa, resulting in complications such as spontaneous bacterial peritonitis.