Exposing cells to H2O2: a quantitative comparison between continuous low-dose and one-time high-dose treatments.

Most studies investigating the influence of H2O2 on cells in culture apply nonphysiological concentrations over nonphysiological time periods (i.e., a one-time bolus that is metabolized in minutes). As an alternative, the glucose oxidase/catalase (GOX/CAT) system allows application of physiologically relevant H2O2 concentrations (300nM-10µM) over physiologically relevant time periods (up to 24h). Recent findings suggest that bolus and GOX/CAT treatments can lead to opposing cellular responses, thus warranting a quantitative comparison between the two approaches. First, we established a reaction-diffusion model that can predict the behavior of the GOX/CAT system with spatiotemporal resolution, thus aiding selection of optimal experimental conditions for its application. Measurements of H2O2 concentration in the cellular supernatant with the luminol/hypochlorite system were consistent with the predictions of the model. Second, we compared the impact of bolus and GOX/CAT treatments on cytosolic H2O2 levels over time. Intracellular H2O2 was monitored by the response of the thiol peroxidase Prx2 and the H2O2 sensor roGFP2-Orp1. We found that Prx2 rapidly and reversibly responds to submicromolar H2O2 levels and accurately reflects kinetic competition with cellular catalase. Our measurements reveal fundamental differences in the dynamic response of cellular H2O2 concentrations following either bolus or GOX/CAT treatments. Thus, different, or even opposing, biological outcomes from differing means of H2O2 delivery may be expected. Cellular responses induced by bolus treatment may not occur under GOX/CAT conditions, and vice versa.

In vitro-targeted gene identification in patients with hepatitis C using a genome-wide microarray technology.

UNLABELLED: Iron in association with reactive oxygen species (ROS) is highly toxic, aggravating oxidative stress reactions. Increased iron not only plays an important role in the progression of hereditary hemochromatosis (HH) but also in common liver diseases such as chronic hepatitis C. The underlying mechanisms of hepatitis C virus (HCV)-mediated iron accumulation, however, are poorly understood. We introduce an in vitro-targeted approach to identify ROS/iron-regulated genes in patients with HCV using a genome-wide DNA microarray. The sensitivity of the 32,231 complementary DNA clone-carrying microarray was approximately 20% as estimated by detecting target genes of the genome-wide transcription factor hypoxia inducible factor 1alpha. Upon in vitro challenge to iron and oxidative stress, 265 iron-related and 1326 ROS-related genes could be identified in HepG2 cells; 233 significantly regulated genes were found in patients with mild (HCV) or severe (HH) iron deposition. Notably, 17 of the in vitro-selected genes corresponded to the genes identified in patients with HCV or HH. Among them, natriuretic peptide precursor B (NPPB) was the only iron-regulated gene identified in vitro that was differentially regulated between HCV and HH. Reverse-transcription polymerase chain reaction confirmed most of the microarray-identified genes in an even larger group of patients (n = 12). In patients with HCV, these included genes that are associated with RNA processing (MED9/NFAT, NSUN2), proliferation, differentiation, hypoxia, or iron metabolism (ISG20, MIG6, HIG2, CA9, NDRG1), whereas none of the nine known iron-related genes showed significant differences between HCV and HH. CONCLUSION: Although high-density microarray technology is less suitable for routine liver diagnosis, its use in combination with prior in vitro selection is a powerful approach to identify candidate genes relevant for liver disease.

Bactobilia after liver transplantation: frequency and antibiotic susceptibility.

After liver transplantation (LT), bactobilia occurs frequently in patients, leading in some cases to cholangitis and biliary sepsis. The present study is the first to investigate bactobilia after LT, and it gives an overview of predisposing factors for bactobilia, the microbial spectrum in the bile of LT patients, and the antibiotic susceptibility. A total of 172 endoscopic retrograde cholangiography (ERC) procedures were performed in 66 LT patients between 1 month and 5.8 years after LT. Bile samples were examined microbiologically. Sixty-eight nontransplanted patients without cholestasis, but requiring ERC for other reasons served as a control group. Of 172 samples obtained from LT patients, 126 (73.3%) were positive for microbes. A total of 236 organisms were isolated: 114 (48.3%) gram-positive bacteria, 92 (39.0%) aerobic gram-negative, 8 (3.4%) anaerobes, and 22 (9.3%) fungi. Ciprofloxacin and amoxycillin/clavulanic acid showed the best susceptibility results among oral antibiotics and piperacillin/tazobactam and imipenem/cilastatin among intravenous preparations. In contrast, only 15.7% of non-LT patients showed bactobilia. In conclusion, our study shows that bactobilia is a problem in patients after LT and that it is not only a contamination from endoscopic intervention. Mechanical obstruction, plastic stents, gallstones, and papillotomy increase the risk of bactobilia significantly. In our cohort we had the best antibiotic susceptibility results for positive cultures in LT patients with piperacillin/tazobactam, ciprofloxacin, or amoxycillin/clavulanic acid.

Alendronate in combination with calcium and vitamin D prevents bone loss after orthotopic liver transplantation: a prospective single-center study.

Bone loss is a common complication in patients before and after liver transplantation (LT). The aim of this study was to investigate the efficacy of prophylactic treatment with bisphosphonates after LT in preventing progressive bone loss in LT patients. We included 136 patients with end-stage liver diseases awaiting LT. Bone mineral density (BMD) (by dual X-ray absorptiometry) and markers of bone metabolism were determined before, and 4, 12, 24, 36, and 48 months after LT. All patients received vitamin D and calcium supplementation before and after LT, those with osteopenia or osteoporosis prior to LT were additionally treated with alendronate following LT. Decreased BMD was seen in a high percentage of patients undergoing LT (osteopenia 48.5%, osteoporosis 23.5%). Reduced BMD before LT was not related to gender, underlying liver disease, or Child-Turcotte-Pugh classification. Body mass index (BMI) prior to LT, however, correlated significantly with the fracture risk. Alendronate prevented the ubiquitously observed bone loss after LT in patients with osteoporosis and osteopenia and, in addition, led to an increase in BMD in patients with osteoporosis within 24 months after LT. In conclusion, our study suggests that alendronate is efficacious in preventing the natural course of bone loss associated with LT.

Herbal hepatotoxicity: acute hepatitis caused by a Noni preparation (Morinda citrifolia).

A 45-year-old patient was sent to our department because of highly elevated transaminases and elevated lactate dehydrogenase. His medical history was unremarkable and he took no medication on regular basis. Physical examination did not detect any abnormalities. There was no evidence for viral hepatitis, Epstein-Barr virus or cytomegalovirus, autoimmune hepatitis, Budd-Chiari syndrome, haemochromatosis or Wilson's disease. During the interview he admitted that for 'prophylactic reasons' he had been drinking the juice of Noni (Morinda citrifolia), a Polynesian herbal remedy made from a tropical fruit, during the preceding 3 weeks. This gave rise to the suspicion of herbal toxicity, which was confirmed by a liver biopsy. After ceasing the ingestion of Noni, transaminase levels normalized quickly and were within normal ranges 1 month after the first presentation. To our knowledge, this is the first report of hepatotoxicity caused by this herbal remedy, which has been highly praised in the tabloid press.