The Effects of Chronic Iron Overload in Rats with Acute Acetaminophen Overdose.

BACKGROUND AND AIMS: Rats are resistant to acetaminophen (APAP) hepatotoxicity. In this study, we evaluated whether by augmentation of the hepatic oxidative stress, through the induction of hepatic iron overload (IO), it will be feasible to overcome the resistance of rats to the toxic effects of APAP. METHOD: Rats with no or increased hepatic IO. RESULTS: Providing iron by diet induced hepatocellular IO, while parenteral iron administration induced combined hepatocellular and sinusoidal cell IO. APAP administration to rats with no IO caused an increase in hepatic oxidative stress and a decrease in the hepatic antioxidative markers but no hepatic cell damage. APAP administration to rats with hepatocellular IO further amplified the hepatic oxidative stress but induced only hepatocyte feathery degeneration without any increase in serum aminotransaminases. APAP administration to rats with combined hepatocellular and sinusoidal cell IO caused an unexpected decrease in hepatic oxidative stress and increase in the hepatic antioxidative markers and no hepatic cell damage. No hepatic expression of activated c-jun-N-terminal kinase was detected in any of the rats. CONCLUSIONS: The hepatic distribution of iron may affect its oxidative/antioxidative milieu. Augmentation of hepatic oxidative stress did not increase the rats' vulnerability to APAP.

Liver toxicity of thioacetamide is increased by hepatocellular iron overload.

An increase in hepatic iron concentration might exacerbate liver injury. However, it is unknown whether hepatic iron overload may exacerbate acute liver injury from various toxins. Therefore, we evaluated how manipulations to increase hepatic iron concentration affected the extent of acute liver injury from thioacetamide. In this study, we used rats with either "normal" or increased hepatic iron concentration. Iron overload was induced by either providing excess iron in the diet or by injecting iron subcutaneously. Both routes of providing excess iron induced an increase in hepatic iron overload. Meanwhile, the subcutaneous route induced both hepatocellular and sinusoidal cell iron deposition; the oral route induced lesser degree of hepatic iron concentration and only hepatocellular iron overload. Thioacetamide administration to the rats with "normal" hepatic iron concentration induced hepatic cell necrosis and apoptosis associated with a remarkable increase in serum aminotransaminases and depletion of hepatic glutathione and other antioxidative indices. Thioacetamide administration to the iron-overloaded rats exacerbated the extent of liver injury only in the rats orally induced with iron overload. In the rats subcutaneously induced with iron overload, the extent of liver injury from thioacetamide was not different from that observed in the rats with "normal" iron overload. It was concluded that the outcome of thioacetamide-induced acute liver injury may depend on both the level of hepatic iron concentration and on the cellular distribution of iron. While isolated hepatocellular iron overload may exacerbate thioacetamide-induced acute liver injury, a combined hepatocellular and sinusoidal cell iron deposition, even at high hepatic iron concentration, had no such an effect.

Screening for active small molecules in mitochondrial complex I deficient patient's fibroblasts, reveals AICAR as the most beneficial compound.

Congenital deficiency of the mitochondrial respiratory chain complex I (CI) is a common defect of oxidative phosphorylation (OXPHOS). Despite major advances in the biochemical and molecular diagnostics and the deciphering of CI structure, function assembly and pathomechanism, there is currently no satisfactory cure for patients with mitochondrial complex I defects. Small molecules provide one feasible therapeutic option, however their use has not been systematically evaluated using a standardized experimental system. In order to evaluate potentially therapeutic compounds, we set up a relatively simple system measuring different parameters using only a small amount of patient's fibroblasts, in glucose free medium, where growth is highly OXPOS dependent. Ten different compounds were screened using fibroblasts derived from seven CI patients, harboring different mutations.5-Aminoimidazole-4-carboxamide ribotide (AICAR) was found to be the most beneficial compound improving growth and ATP content while decreasing ROS production. AICAR also increased mitochondrial biogenesis without altering mitochondrial membrane potential (Δψ). Fluorescence microscopy data supported increased mitochondrial biogenesis and activation of the AMP activated protein kinase (AMPK). Other compounds such as; bezafibrate and oltipraz were rated as favorable while polyphenolic phytochemicals (resverastrol, grape seed extract, genistein and epigallocatechin gallate) were found not significant or detrimental. Although the results have to be verified by more thorough investigation of additional OXPHOS parameters, preliminary rapid screening of potential therapeutic compounds in individual patient's fibroblasts could direct and advance personalized medical treatment.

Iron-chelator complexes as iron sources for early developing human erythroid precursors.

Developing erythroid cells are dependent on transferrin (Tf) to acquire iron in amounts sufficient for hemoglobin production. Previously, we showed that although these cells cannot grow in culture in the absence of Tf, ferritin (Ft) can substitute Tf to some extent and support the development of hemoglobin-containing cells. In the current study, we investigated the ability of various iron sources to replace Tf in cultures of normal human erythroid precursors. The results showed that whereas Ft and hemin supported erythroid cell proliferation and hemoglobinization in Tf-free cultures to some extent, ferric amonium citrate and iron complexed with several chelators had little or no effect. Although salicylaldehyde-isonicotinoyl-hydrazone, which is a tridentate lipid-soluble chelator, complexed with iron increased both cytosolic and mitochondrial labile iron pools, it failed to support heme synthesis and did not decrease the surface Tf receptors, suggesting that its iron is not recognized by the cells. Moreover, this iron-chelator complex did not support erythroid precursor proliferation and hemoglobinization. Thus, although under normal conditions, Tf is the major route of iron uptake, Ft and hemin, but not iron-chelator complexes, may serve as alternative iron sources under Tf-poor conditions.

Effects of amlodipine, captopril, and bezafibrate on oxidative milieu in rats with fatty liver.

Oxidative stress may initiate significant hepatocyte injury in subjects with fatty liver. We characterized changes in hepatic oxidative anti-oxidative parameters in rats given a fructose-enriched diet (FED) with and without medications to reduce blood pressure or plasma triglycerides. FED rats had an increase in malondialdehyde (MDA) concentration, a reduction in alpha-tocopherol concentration, a reduction in paraoxonase (PON) activity, an increase in glutathione peroxidase (GSH-Px), and glutathione reductase (GSSG-R) activity. Amlodipine increased PON and GSH-Px, but decreased GSSG-R activity and alpha-tocopherol concentration. Captopril decreased MDA concentration and the activity of both GSH-Px and GSSG-R, but increased alpha-tocopherol concentration and PON activity. Bezafibrate increased alpha-tocopherol concentration and PON activity, but decreased the activity of GSSG-R. Animals with fatty liver exhibit an increase in peroxidative stress but also a defect in anti-oxidative pathways. Drugs administered to treat hypertension and hypertriglyceridemia could lead to a variety of changes in the hepatic oxidative, anti-oxidative milieu.

The synthetic cannabinoid HU-210 attenuates neural damage in diabetic mice and hyperglycemic pheochromocytoma PC12 cells.

Diabetic neuropathy (DN) is a common complication of diabetes mellitus resulting in cognitive dysfunction and synaptic plasticity impairment. Hyperglycemia plays a critical role in the development and progression of DN, through a number of mechanisms including increased oxidative stress. Cannabinoids are a diverse family of compounds which can act as antioxidative agents and exhibit neuroprotective properties. We investigated the effect of the synthetic cannabinoid HU-210 on brain function of streptozotocin (STZ)-induced diabetic mice. These animals exhibit hyperglycemia, increased cerebral oxidative stress and impaired brain function. HU-210, through a receptor independent pathway, alleviates the oxidative damage and cognitive impairment without affecting glycemic control. To study the neuroprotective mechanism(s) involved, we cultured PC12 cells under hyperglycemic conditions. Hyperglycemia enhanced oxidative stress and cellular injuries were all counteracted by HU-210-in a dose dependent manner. These results suggest cannabinoids might have a therapeutic role in the management of the neurological complications of diabetes.

Effect of hepatic iron concentration reduction on hepatic fibrosis and damage in rats with cholestatic liver disease.

AIM: To assess the effect of iron reduction after phlebotomy in rats with "normal" hepatic iron concentration (HIC) on the progression of hepatic fibrosis, as a result of bile duct ligation (BDL). METHODS: Rats underwent phlebotomy before or after sham operation or BDL. Animals undergone only BDL or sham operation served as controls. Two weeks after surgery, indices of hepatic damage and fibrosis were evaluated. RESULTS: Phlebotomy lowered HIC. Phlebotomy after BDL was associated with body weight increase, lower hepatic weight, less portal hypertension, less periportal necrosis, less portal inflammation, lower hepatic activity index score and higher albumin levels. On the other hand, phlebotomy before BDL was associated with body weight decrease and hepatic activity index score increase. Phlebotomy after sham operation was not associated with any hepatic or systemic adverse effects. CONCLUSION: Reduction of HIC after induction of liver damage may have beneficial effects in BDL rats. However, iron deficiency could induce impairment of liver function and may make the liver more susceptible to insults like BDL.

Objectives and mechanism of iron chelation therapy.

Prevention of cardiac mortality is the most important beneficial effect of iron chelation therapy. Unfortunately, compliance with the rigorous requirements of daily subcutaneous deferoxamine (DFO) infusions is still a serious limiting factor in treatment success. The development of orally effective iron chelators such as deferiprone and ICL670 is intended to improve compliance. Although total iron excretion with deferiprone is somewhat less than with DFO, deferiprone may have a better cardioprotective effect than DFO due to deferiprone's ability to penetrate cell membranes. Recent clinical studies indicate that oral ICL670 treatment is well tolerated and is as effective as parenteral DFO used at the standard dose of 40 mg/kg of body weight/day. Thus, for the patient with transfusional iron overload in whom results of DFO treatment are unsatisfactory, several orally effective agents are now available to avoid serious organ damage. Finally, combined chelation treatment is emerging as a reasonable alternative to chelator monotherapy. Combining a weak chelator that has a better ability to penetrate cells with a stronger chelator that penetrates cells poorly but has a more efficient urinary excretion may result in improved therapeutic effect through iron shuttling between the two compounds. The efficacy of combined chelation treatment is additive and offers an increased likelihood of success in patients previously failing DFO or deferiprone monotherapy.

Iron overload and chelation.

Iron is one of the most common elements in nature. As a transition metal it is very efficient in electron transport and redox reactions. The proteins and enzymes in which iron is an essential component play a key role in respiration, energy production, detoxification of harmful oxygen species and cell replication. Despite the abundance of iron in nature, the solubility of its stable ferric form is extremely low. Hence, living organisms were compelled to develop efficient mechanisms for iron transport and storage.

Fructose-induced fatty liver disease: hepatic effects of blood pressure and plasma triglyceride reduction.

The most known risk factor for nonalcoholic fatty liver disease (NAFLD) is the metabolic syndrome. In this study, we characterized changes in liver pathology, hepatic lipid composition, and hepatic iron concentration (HIC) occurring in rats given fructose-enriched diet (FED), with and without therapeutic maneuvers to reduce blood pressure and plasma triglycerides. Rats were given FED or standard rat chow for 5 weeks. Rats on FED were divided into 4 groups: receiving amlodipine (15 mg/kg per day), captopril (90 mg/kg per day), bezafibrate (10 mg/kg per day) in the last 2 weeks, or a control group that received FED only. FED rats had hepatic macrovesicular and microvesicular fat deposits develop, with increase in hepatic triglycerides (+198%) and hepatic cholesterol (+89%), but a decrease in hepatic phospholipids (-36%), hypertriglyceridemia (+223%), and hypertension (+15%), without increase in HIC. Amlodipine reduced blood pressure (-18%), plasma triglycerides (-12%), but there was no change in hepatic triglycerides and phospholipids concentrations. Captopril reduced blood pressure (-24%), plasma triglycerides (-36%), hepatic triglycerides (-51%), and hepatic macrovesicular fat (-51%), but increased HIC (+23%), with a borderline increase in hepatic fibrosis. Bezafibrate reduced plasma triglycerides (-49%), hepatic triglycerides (-78%), hepatic macrovesicular fat (-90%), and blood pressure (-11%). We conclude that FED rats can be a suitable model for human NAFLD. Drugs administered to treat various aspects of the metabolic syndrome could have hepatic effects. An increase in HIC in rats with NAFLD could be associated with increased hepatic fibrosis.

Iron chelation therapy.

Although iron chelation therapy with deferoxamine (DFO) has changed life expectancy in thalassemic patients, compliance with the rigorous requirements of long-term subcutaneous DFO infusions is unsatisfactory. This problem underlines the current efforts for developing alternative, orally effective chelators to improve compliance and treatment results. For the patient with transfusional iron overload in whom results of DFO treatment are unsatisfactory, several orally effective agents are now available. The most important of the new generation of oral chelators are deferiprone and ICL670. Total iron excretion with deferiprone is less than with DFO, but deferiprone has a better ability to penetrate cell membranes and may have a better cardioprotective effect than DFO. Current studies of the clinical efficacy and tolerability of ICL670 indicate that at a single oral dose of 20 mg/kg daily, it may be as effective as parenteral DFO used at the standard dose of 40 mg/kg daily. Combined chelation treatment, employing a weak chelator that penetrates cells better, and a stronger chelator with efficient urinary excretion, may result in improved therapeutic effect through iron shuttling between the two compounds. The efficacy of combined chelation treatment is additive and offers an increased likelihood of success in patients previously failing DFO or deferiprone monotherapy.

Effects of combined chelation treatment with pyridoxal isonicotinoyl hydrazone analogs and deferoxamine in hypertransfused rats and in iron-loaded rat heart cells.

Although iron chelation therapy with deferoxamine (DFO) results in improved life expectancy of patients with thalassemia, compliance with parenteral DFO treatment is unsatisfactory, underlining the need for alternative drugs and innovative ways of drug administration. We examined the chelating potential of pyridoxal isonicotinoyl hydrazone (PIH) analogs, alone or in combination with DFO, using hypertransfused rats with labeled hepatocellular iron stores and cultured iron-loaded rat heart cells. Our in vivo studies using 2 representative PIH analogs, 108-o and 109-o, have shown that PIH analogs given orally are 2.6 to 2.8 times more effective in mobilizing hepatocellular iron in rats, on a weight-per-weight basis, than parenteral DFO administered intraperitoneally. The combined effect of DFO and 108-o on hepatocellular iron excretion was additive, and response at a dose range of 25 to 200 mg/kg was linear. In vitro studies in heart cells showed that DFO was more effective in heart cell iron mobilization than all PIH analogs studied. Response to joint chelation with DFO and PIH analogs was similar to an increase in the equivalent molar dose of DFO alone, rather than the sum of the separate effects of the PIH analog and DFO. This finding was most likely the result of iron transfer from PIH analogs to DFO, a conclusion supported directly by iron-shuttle experiments using fluorescent DFO. These findings provide a rationale for the combined, simultaneous use of iron-chelating drugs and may have useful, practical implications for designing novel strategies of iron chelation therapy.