Severe Microvesicular Steatosis Research Articles

Clinical Presentation and Gene Expression of Acute Alcohol-Induced Microvesicular Steatosis Mimicking Alcoholic Hepatitis.

Acute alcoholic microvesicular steatosis (MIC) may complicate heavy alcohol intake and present as alcoholic hepatitis (AH) syndrome. However, detailed clinical, biological, and histologic data associated with MIC are scarce. We compared the clinical presentation, histologic features, and hepatic transcriptomic of patients presenting with AH due to either MIC or severe alcoholic steatohepatitis (ASH). In this case‐control study, patients who drank heavily (>100 g/day) with the AH syndrome were included either in the MIC group (>50% severe microvesicular steatosis, no inflammation) or in the severe ASH group (polynuclear neutrophil infiltration, macrosteatosis, ballooned hepatocytes). All patients received standard supportive care plus steroids for those with severe ASH and were followed up for 3 months. Whole‐liver transcriptome profiling was performed on liver snap‐frozen biopsies. Compared to ASH (n = 24, mean age 49.3 years), patients in the MIC group (n = 12, mean age 49.1 years) had a higher reported alcohol intake (P < 0.01), lower Model for End‐Stage Liver Disease score (P < 0.05), lower hepatic venous pressure gradient (P < 0.01), higher alanine aminotransferase (P < 0.02) and gamma‐glutamyltransferase (P < 0.001), higher triglycerides (P < 0.001) and total cholesterol (P < 0.002), but similar bilirubin levels (P = 0.54). At histology, patients with MIC had a lower fibrotic stage compared to those with ASH (P < 0.001). A higher density of megamitochondria was seen in MIC compared to ASH (P < 0.05). During follow‐up, death or transplantation occurred in 4/12 (33%) patients with MIC and 7/24 (29%) patients with severe ASH. Differential hepatic gene expression in MIC compared to ASH included down‐regulation of genes related to inflammation and fibrosis and up‐regulation of genes involved in lipid metabolism and mitochondrial function. Conclusion: MIC is an acute, noninflammatory, potentially severe alcoholic liver injury mimicking ASH, is associated with a lower fibrosis stage, and has a distinct gene expression profile.

Obese rats are more vulnerable to inflammation, genotoxicity and oxidative stress induced by coal dust inhalation than non-obese rats

Obesity is an important nutritional disorder worldwide. Its association with environmental pollution may trigger an increase in oxidative stress and inflammatory parameters. Coal is a resource used throughout the world as an important fuel source for generating electricity. The ashes released by the coal combustion cause serious problems for human health due to their high toxicity and their capacity to bioaccumulate. The aim of this work was to investigate the effects of coal dust inhalation in the organs of obese and non-obese Wistar rats. Pro-inflammatory cytokines, oxidative stress, oxidative damage, histological analysis, comet assay, and micronuclei were investigated. Both obesity and coal dust inhalation increased the pro-inflammatory cytokines IL-1β and TNF-α and decreased HSP70 levels in serum, however, in obese animals that inhaled coal dust these changes were more pronounced. Liver histological analysis showed severe microvesicular steatosis in obese animals that inhaled coal dust. Lung histologic investigation showed abnormalities in lung structure of animals exposed to coal dust and showed severe lung distensibility in obese animals exposed to coal dust. The comet assay showed DNA damage in animals subjected to coal. In addition, there were modulations in enzymatic activities and damage to protein and lipids. Based on our results, the coal dust inhalation can potentiate the pro-inflammatory profile present in obese rats. We also observed an increase in the protein oxidative damage in obese rats that inhaled coal dust. Taken together, our results suggest that the combination of obesity and coal inhalation increased the risks of the development of diseases related to oxidative stress and inflammation.

A systems biology approach utilizing a mouse diversity panel identifies genetic differences influencing isoniazid-induced microvesicular steatosis.

Isoniazid (INH), the mainstay therapeutic for tuberculosis infection, has been associated with rare but serious hepatotoxicity in the clinic. However, the mechanisms underlying inter-individual variability in the response to this drug have remained elusive. A genetically diverse mouse population model in combination with a systems biology approach was utilized to identify transcriptional changes, INH-responsive metabolites, and gene variants that contribute to the liver response in genetically sensitive individuals. Sensitive mouse strains developed severe microvesicular steatosis compared with corresponding vehicle control mice following 3 days of oral treatment with INH. Genes involved in mitochondrial dysfunction were enriched among liver transcripts altered with INH treatment. Those associated with INH treatment and susceptibility to INH-induced steatosis in the liver included apolipoprotein A-IV, lysosomal-associated membrane protein 1, and choline phosphotransferase 1. These alterations were accompanied by metabolomic changes including reduced levels of glutathione and the choline metabolites betaine and phosphocholine, suggesting that oxidative stress and reduced lipid export may additionally contribute to INH-induced steatosis. Finally, genome-wide association mapping revealed that polymorphisms in perilipin 2 were linked to increased triglyceride levels following INH treatment, implicating a role for inter-individual differences in lipid packaging in the susceptibility to INH-induced steatosis. Taken together, our data suggest that INH-induced steatosis is caused by not one, but multiple events involving lipid retention in the livers of genetically sensitive individuals. This work also highlights the value of using a mouse diversity panel to investigate drug-induced responses across a diverse population.

CC-003 Abnormal liver function tests and hepatomegaly in a diabetic patient: an under-recognised diagnosis

<h3>Introduction</h3> We report the case of a 29-year-old type 1 diabetic who presented with abnormal liver function tests (LFTs) and RUQ discomfort. At that time she was 7 months post-partum. Physical examination revealed 2 cm tender hepatomegaly. Initial investigations showed the following. (a) Blood tests: bilrubin 5; ALT 256; ALP 137 and GGT 112. A liver screen was essentially negative. (b) Abdominal ultrasound: diffusely enlarged liver. No ascites and vasculature normal. (c) Liver biopsy: reported as showing “diffuse microvesicular change but with no inflammation and mild perisinusoidal fibrosis”. A diagnosis of severe microvesicular steatosis of unknown aetiology was made. <h3>Methods</h3> The patients symptoms of abdominal discomfort gradually settled. Her LFTs also completely settled back to normal in January 2004 and it was felt that whatever the insult was to her liver a few months earlier, it was now settling. The patient continued follow-up at the liver clinic with her LFTs fluctuating but occasionally settling completely back to normal. <h3>Results</h3> The patient was readmitted with complaints of severe abdominal chest pain and was admitted for further investigation. Physical examination revealed marked abdominal distension with the clinical impression of ascites. Blood tests showed deterioration in her LFTs. An urgent CT was undertaken. The CT scan showed a markedly enlarged liver which was not particularly fatty on imaging. We proceeded to a repeat liver biopsy. Hepatic glycogenopathy (glycogenosis) was diagnosed. The biopsy was compared to the previous biopsy. Similar changes were in fact noted in the initial biopsy but, with an overlay of microvesicular and some macrovesicular steatosis, along with a poor appreciation of the disease at that time, led to glycogenosis being overlooked. <h3>Conclusion</h3> This case highlights the importance of considering hepatic glycogenosis in patients with diabetes, hepatomegaly and abnormal LFTs. Hepatic enlargement in diabetes is usually the result of either glycogenosis or non-alcoholic steatohepatitis (NASH). Glycogenosis is the hepatic response to poor glycaemic control in children, adolescence and adults with Type 1 diabetes, while NASH is the more likely diagnosis in adults with type 2 diabetes. Hepatic glycogenosis has been reported previously but remains under-recognised. As mentioned, it is associated with poor glycaemic control (hyperglycaemia and intermittent insulin administration) but is readily treatable by improvement of glycaemic control. It is a benign disease with little chance of progression to fibrosis. Liver biopsy is definitive in the diagnosis of hepatic glycogenosis. An excess of glycogen in the nucleus of the hepatocytes is seen, which are pale and swollen, with peripherally displaced organelles. Steatosis is absent in up to 90% of cases and helps distinguish this condition from NASH. However, our case illustrates that glycogenosis can easily be overlooked probably due to an under appreciation of the condition.

Bioactive agents against systemic low-grade inflammation

Introduction: Free radicals and lipid peroxides assign a systemic low-grade inflammation. NF-κB regulated by reactive oxygen radicals, influence the transcription of many target genes, like iNOS, COX-2, and TNF-α. Bioactive agents e.g. vitamins, flavonoids and polyphenols may reduce the inflammation processes. Our aim was to determine the advantages of a functional product containing Beta vulgaris var. rubra and Daucus carota (BVD) in „short term“ alimentary fatty liver rodent model. Methods: Male Wistar (N=30) rats were kept on conventional chow with or without high fat supplementation (2% cholesterol, 0.5% cholic acid, 20% sunflower oil) and 0,1 or 1g/bwkg/day BVD lyofilisate (permission number: 1361/004/2003 BFÁÉÉÁ) was given, in both alimentary groups respectively. After termination on tenth day plasma and liver redox-parameters (spectrophotometry, luminometry) were measured, and histological analysis was carried out. Expression of iNOS, COX-2 and TNF-α were detected with Tachman real-time PCR. Results and discussion: Free radical intensity, lipid peroxidation and decreased antioxidant state showed remarkable changes in steatotic liver (SL) group. The tendency of lipid lowering and antioxidant enhancing effects of the BVD supplementation were detected dose dependently in the plasma as well as in the liver. Histological analysis showed severe microvesicular steatosis. Levels of inflammatory parameters: iNOS, COX-2 and TNF-α, were elevated significantly in SL groups. Beneficial changes of relative expression levels of iNOS, COX-2 and TNF-α could be established after Beta vulgaris var. rubra and Daucus carota treatment dose dependently in group suffered with steatosis. Conclusion: Beta vulgaris var. rubra and Daucus carota may diminish the systemic low-grade inflammation in steatotic liver in applied concentrations.

MORPHOLOGICAL AND FUNCTIONAL CHANGES IN P‐GLYCOPROTEIN DURING DEXAMETHASONE‐INDUCED HEPATOMEGALY

1. The effect of dexamethasone on hepatic and renal P-glycoprotein (P-gp) expression, localization and activity was investigated in rats after 4 days oral administration of two dose regimens (1 or 25 mg/kg per day). Simultaneous increases in liver weight were evaluated by quantitative histological examination. 2. In the liver, dexamethasone pretreatment produced hepatomegaly as a consequence of extensive periportal fat accumulation, which was quantified by densitometry of oil red O-stained liver sections. Quantitative immunohistochemical analysis revealed preferential periportal zonation of P-gp in control animals. Dexamethasone pretreatment resulted in spatially disproportional induction of P-gp protein expression within the liver acinus characterized by preferential increase in pericentral areas, with consequent uniform panlobular distribution. Western blot analysis confirmed these results, showing increases in P-gp protein. Quantitative reverse transcription-polymerase chain reaction analysis revealed no statistically significant change in liver mdr1b mRNA expression after either dexamethasone treatment regimen. The expression of mdr1a mRNA was significantly decreased by 85-87%. 3. In the kidney, dexamethasone reduced mdr1a mRNA expression by 69-89%, whereas mdr1b mRNA expression was increased in a dose-dependent manner. However, despite tendencies, no significant increases in P-gp expression were observed at the protein level. 4. The in vivo function of P-gp was evaluated by measuring renal and biliary secretion of rhodamine-123 (Rho123) under a steady state plasma concentration. The biliary, renal and tubular secretory clearance of Rho123 was significantly increased only after high-dose dexamethasone. 5. In conclusion, the present study suggests that drug interactions observed during corticosteroid therapy may be mediated, at least in part, through increased biliary, and also renal, excretion of P-gp substrates. Expression of P-gp in the liver showed primary periportal zonation with differential changes during induction. Accompanying hepatomegaly may be explained by severe microvesicular steatosis selectively localized to the periportal areas.

Assessment of donor fatty livers for liver transplantation.

The effect of fatty liver on graft survival, especially with reference to macrovesicular and microvesicular steatosis, is still uncertain. This preliminarily study was designed to create a noninvasive method for the quantification of the hepatic fat content in vivo and to establish provisional criteria for the assessment of fatty donor livers before liver transplantation among transplant surgeons, radiologists, and pathologists. Different degrees of rat fatty liver model were established by feeding rats a diet deficient in choline and methionine for different periods of time. Computed tomography (CT) with test tubes containing variable percentages of fat equivalent substance were used to assess the severity of fatty change of the rat liver. This was then correlated with the histological classification, level of hepatic enzymes, and graft survival. Linear correlation between the fat volume fraction added to the test tubes and CT density were found. The process of producing a fatty liver via diet alteration peaked at week 3. At this time hepatic enzymes, radiological fat content, and posttransplantation survival were worse (P=0.013), compared with other time points. Radiological assessment of fatty liver correlated well with survival and serum glutamic oxaloacetic transaminase and glutamic pyruvate transaminase levels. Severe microvesicular steatosis does not influence recipient survival, however, macrovesicular steatosis affects graft survival. Caliber CT is a practical and simple method that allows an accurate noninvasive quantitative assessment of hepatic fatty infiltration. It has potential to be a useful parameter for the assessment of donor livers for clinical liver transplantation.

Delayed liver regeneration, increased injury and mortality in heterozyogous or homozygous obmutant mice

Background: Hepatic steatosis is thought to contribute to primary nonfunction (PNF)after orthotopic liver transplantation (OLT). Since ischemiareperfusion injury is inevitable during OLT, recovery requires hepatic regeneration. Thus, PNF may develop in fatty livers because regeneration is impaired. Aim: To determine if regeneration is inhibited in fatty livers. Methods: Regeneration after 70% partial hepatectomy (PH) in male C57BU 6 obese (ob/ob) mice with fatty livers was compared to ageand sex-matched lean, heterozygous (-lob) litter mates and wild-type (WT) C57BU6 mice. Both control groups had normal basal liver histology. Results: WT mice had < 10% mortality during the first 48h after PH. BrdV pulse-labeling demonstrated replicating hepatocytes at various times after PH: 2% BrdV(+) hepatocytes at 6 and l2h, 8% at 24h, 33% at 36h, and 25% at 48h. Almost 60% of the initial liver weight was regained by 48h. In contrast, PH mortality was increased in lean, -lob mice (33% by 48h) and in obese, oblob mice (68% by 48h), ps .OOI. Liver regeneration was delayed in -lob mice (2:!:1% BrdV(+ )hepatocytes at 24h and 29:!:3% at 48h) and never occurred in oblob mice. Liver mass restoration also was decreased in -lob (l 4::!: 5%)and oblob (-2:!:6%) groups. FACS analysis of propidium iodide-stained liver nuclei not only confirmed the BrdV results but also demonstrated a basal shift in the cell cycle distribution of oblob hepatocytes. In oblob livers, 46:!:6% hepatocytes were in GO-GI and 52:!:6% in G2; however, in -lob livers, 71:!:6% hepatocytes were in GO-G I and 27:!:6% in G2 (psO.05 for both). After PH, in oblob fatty livers after PH, there were extensive areas of hemorrhage, dead hepatocytes, and inflammation. Heterozygous, -lob, mice developed severe microvesicular steatosis, with islands of dead hepatocytes (without much associated hemorrhage or inflammation) by 24-48h after PH. In WT mice after PH, mild microvesicular steatosis occurred at 6-12h, and minor focal liver injury rarely was noted at 24-48h. Conclusions: After PH, obese, oblob mice with fatty livers do not regenerate their livers and develop extensive liver injury with high mortality. Delayed regeneration and increased liver injury also occur post-PH in lean, -lob mice. Although these heterozygotes have non-fatty livers pre-PH, they develop severe steatosis post-PH. The inverse relationship between hepatic steatosis and regeneration supports the concept that inhibited regeneration contributes to PNF of fatty allografts after OLT.

Use of livers with microvesicular fat safely expands the donor pool.

The safety of transplanting livers with moderate to severe microvesicular steatosis is unknown. Livers that appear fatty are often abandoned at the donor hospital. We have recently used frozen-section biopsy to distinguish between microvesicular and macrovesicular steatosis. We present here our single-center experience with transplantation of 40 allografts with moderate or severe microvesicular steatosis. We reviewed our data on 426 transplants and identified 40 cases in which the donor liver contained at least 30% microvesicular steatosis. Early graft function, patient and graft survival, and donor risk factors for steatosis were examined, and results in this cohort were compared with results in all other patients who received liver transplants at our center during the same time period. We also analyzed the reliability of donor frozen-section biopsies in quantitating microsteatosis. Persistence of steatosis was assessed on the basis of 1-year follow-up biopsies. The incidence of primary nonfunction and poor early graft function was 5% and 10%, respectively. One-year patient and graft survival rates were 80% and 72.5%, respectively. Donor obesity and traumatic death were commonly identified risk factors for microvesicular steatosis. Frozen-section biopsy was reliable for pretransplant decision-making about the use of potential grafts, and the steatosis had disappeared from the graft at 1 year in the majority of cases. Livers with even severe microvesicular steatosis can be reliably used for transplantation without the fear of high rates of primary nonfunction. There was a significant incidence of poor early graft function, but this did not affect outcome. Microsteatosis is usually associated with some underlying risk factor in the donor and is reversible, as demonstrated by follow-up biopsies after transplant.

Fatal hyperammonemia following orthotopic lung transplantation

There have been anecdotes of unexplained coma and death in patients after otherwise successful orthotopic lung transplantation. A patient with primary pulmonary hypertension who underwent a technically uncomplicated single orthotopic lung transplantation is described. The patient developed intractable status epilepticus 4 days after surgery in association with the presence of a markedly elevated plasma ammonium level. Despite multiple therapeutic interventions, the hyperammonemia ultimately resulted in the patient's death. Both metabolic and enzymatic studies showed that the unique physiological disturbance in this disorder results at least in part from defective in vivo conversion of waste nitrogen to urea and increased production of waste nitrogen. Although the rate of hepatic ureagenesis was therefore insufficient to prevent accumulation of ammonium, the cause was not severe liver disease. Liver histology showed widespread, microvesicular steatosis on light-microscopic examination, but only electron- microscopic examination showed severe microvesicular steatosis with severe mitochondrial injury. As in Reye's syndrome, it was unclear whether the hepatic mitochondrial injury played a role in development or if it was the result of hyperammonemia. We recommended that any patient with an unexplained alteration of mental status after solid organ transplantation be evaluated for hyperammonemia. (Gastroenterology 1997 Jan;112(1):236-40)

Inhibition of the mitochondrial oxidation of fatty acids by tetracycline in mice and in man: possible role in microvesicular steatosis induced by this antibiotic.

Intravenous administration of high doses of tetracycline may produce severe microvesicular steatosis of the liver in man. A similar disease is observed after ingestion of drugs which inhibit hepatic mitochondrial fatty acid beta-oxidation and in subjects with various inborn defects in this metabolic pathway. We therefore determined the effects of tetracycline on the mitochondrial oxidation of fatty acids in mice and in man. In vitro, addition of tetracycline 0.25, 0.5, 1 or 2 mM inhibited by 15, 38, 56 and 65%, respectively, the formation of beta-oxidation products during incubation of palmitic acid with mouse liver mitochondria and the various cofactors necessary for beta-oxidation. Inhibition was reversible. Inhibition appeared even greater with human liver mitochondria. Tricarboxylic acid cycle activity, assessed by the in vitro formation of [14C]CO2 from [1-14C]acetylcoenzyme A by mouse liver mitochondria, was inhibited by 25, 32 and 43%, respectively, in the presence of 0.5, 1 or 2 mM of tetracycline. In vivo, administration of tetracycline, 0.25 or 1 mmole per kg, inhibited by 53 and 84%, respectively, the exhalation of [14C]CO2 during the first 3 hours following the administration of a tracer dose of [U-14C]palmitic acid. Administration of tetracycline, 0.0625, 0.25 or 1 mmole per kg, 6 hr before the measurement, increased hepatic triglycerides by 100, 170 and 250%, respectively. After 1 mmole per kg, accumulation of hepatic triglycerides was maximum at 24 hr, reaching 9-fold the control value; liver histology showed microvesicular steatosis at 6 and 24 hr. We conclude that tetracycline inhibits the mitochondrial oxidation of fatty acids in mice and in man.(ABSTRACT TRUNCATED AT 250 WORDS)