Hepatotoxicity induced by iron overload and alcohol. Studies on the role of chelatable iron, cytochrome P450 2E1 and lipid peroxidation

BACKGROUND/AIMS: Clinical experience and studies with experimental animal models indicate a synergistic hepatotoxic effect of dietary iron overload and chronic alcohol ingestion. In order to elucidate the mechanism underlying this synergism, we examined the hepatic levels of ethanol-inducible cytochrome P450 2E1, glutathione and malondialdehyde, and the effect of iron chelation with desferrioxamine, in livers from rats treated with iron and/or ethanol. METHODS: Animals received diets with or without 2.5-3% carbonyl iron for 6-9 weeks, followed by an ethanol-containing diet or a liquid control diet for 5-9 weeks. Desferrioxamine was administered subcutaneously with mini-osmotic pumps. Alanine aminotransferase activity in serum and hepatic contents of glutathione and malondialdehyde were determined. The hepatic level of cytochrome P450 2E1 was determined with Western Blotting using a specific polyclonal antibody. RESULTS: The combination of iron and alcohol led to a marked increase in serum alanine aminotransferase activity as compared with all other treatment groups, and iron chelation with desferrioxamine reversed these increases. Treatment with alcohol alone led to slightly increased aminotransferases compared with controls. The level of cytochrome P450 2E1 was significantly elevated in microsomes isolated from ethanol-treated rats, but neither additional iron supplementation nor desferrioxamine influenced this level significantly. Glutathione contents were increased in the livers of animals treated with iron and/or ethanol. Malondialdehyde values were increased in iron-treated animals, whereas neither ethanol nor desferrioxamine altered malondialdehyde levels significantly. CONCLUSIONS: The toxic effects exerted by the combination of iron overload and chronic ethanol feeding on rat liver are dependent on a pool of chelatable iron. The hepatic level of cytochrome P450 2E1 is markedly induced by ethanol but not further altered by iron overload. Neither increased lipid peroxidation nor depletion of hepatic glutathione levels can explain the synergistic hepatotoxic effects of iron and ethanol in this model.     

Effect of iron overload and iron deficiency on atherosclerosis in the hypercholesterolemic rabbit

It has been suggested that iron plays an important role in the pathogenesis of atherosclerosis, primarily by acting as a catalyst for the atherogenic modification of LDL. Although some epidemiological data suggest that high stored iron levels are an independent risk factor for coronary artery disease and that iron has been detected in both early and advanced atherosclerotic lesions, the evidence is often contradictory and inconclusive. We used the New Zealand White rabbit to investigate the effects of iron overload (FeO) and iron deficiency (FeD) on atherosclerosis. Groups of 7 rabbits were either iron loaded by injections of iron dextran (FeO group), iron depleted by phlebotomy (FeD group), or given injections of saline (control group) for a total of 9 weeks. All rabbits were fed a chow diet containing 1% (wt/wt) cholesterol for the last 6 weeks of the study. Iron and antioxidant status and cholesterol levels were assayed in plasma before cholesterol feeding (week 3) and at the time that the rabbits were killed (week 9). In addition, the susceptibility of LDL to oxidation was measured and pathological examination of the aortic arch and thoracic aorta performed at the end of the study. FeD significantly decreased the levels of blood hemoglobin, serum iron, and transferrin saturation compared with controls. Conversely, FeO significantly increased transferrin Fe saturation. FeO but not FeD decreased plasma cholesterol levels compared with control animals both before (P < .05) and after (P = .055) cholesterol feeding. Neither FeO nor FeD had a significant effect on the levels of antioxidants and lipid peroxidation products in plasma and aortic tissue or on the susceptibility of LDL to ex-vivo oxidation. FeO significantly decreased aortic arch lesion formation by 56% compared with controls (P < .05), whereas FeD had no significant effect. These results indicate that in this animal model, FeO decreases rather than increases atherosclerosis, likely because iron dextran exerts a hypocholesterolemic effect. Our data do not support the hypotheses that elevation of Fe stores increases or that a reduction of Fe stores by phlebotomy decreases the risk of coronary artery disease.     

Real-time three-dimensional echocardiography for determining right ventricular stroke volume in an animal model of chronic right ventricular volume overload

BACKGROUND: The lack of a suitable noninvasive method for assessing right ventricular (RV) volume and function has been a major deficiency of two-dimensional (2D) echocardiography. The aim of our animal study was to test a new real-time three-dimensional (3D) echo imaging system for evaluating RV stroke volumes. METHODS AND RESULTS: Three to 6 months before hemodynamic and 3D ultrasonic study, the pulmonary valve was excised from 6 sheep (31 to 59 kg) to induce RV volume overload. At the subsequent session, a total of 14 different steady-state hemodynamic conditions were studied. Electromagnetic (EM) flow probes were used for obtaining aortic and pulmonic flows. A unique phased-array volumetric 3D imaging system developed at the Duke University Center for Emerging Cardiovascular Technology was used for ultrasonic imaging. Real-time volumetric images of the RV were digitally stored, and RV stroke volumes were determined by use of parallel slices of the 3D RV data set and subtraction of end-systolic cavity volumes from end-diastolic cavity volumes. Multiple regression analyses showed a good correlation and agreement between the EM-obtained RV stroke volumes (range, 16 to 42 mL/beat) and those obtained by the new real-time 3D method (r=0.80; mean difference, -2.7+/-6.4 mL/beat). CONCLUSIONS: The real-time 3D system provided good estimation of strictly quantified reference RV stroke volumes, suggesting an important application of this new 3D method.     

The combined effects of memantine and fluoxetine on an animal model of obsessive compulsive disorder.

Obsessive Compulsive Disorder (OCD) is currently treated with behavioral modification and psychotropic medications, with varying degrees of success. The most popular drugs for the treatment of OCD are the selective serotonin reuptake inhibitors (SSRIs). Another drug, the N-methyl-d-aspartate antagonist memantine, has recently been tested in the treatment of OCD. The present study investigates the effect of fluoxetine and memantine alone and in combination in a mouse model of compulsive behavior. In this model, compulsive scratching is induced by a subcutaneous injection of serotonin or a serotonin releasing agent, compound 48–80, in the back of the neck. The effects of the memantine and fluoxetine combination were found to synergistic, specifically as defined by an isobologram. The results of the present investigation suggest the potential of a more effective management of the symptoms of OCD. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Hyperferritinaemia in the absence of iron overload

We present a case of a 3-month-old boy presenting with fulminating meningococcal septicaemia associated with extensive peripheral gangrene requiring amputation of three limbs. The surgical management options and the role of early fasciotomy are discussed.     

Tuberculosis and iron overload in Africa: a review

Both pulmonary tuberculosis and dietary iron overload are common conditions in sub-Saharan Africa. The incidence of tuberculosis has increased markedly over the last decade, primarily as a result of the rapid spread of infection with the human immunodeficiency virus (HIV). Dietary iron overload affects up to 10% of adults in rural populations and is characterized by heavy iron deposition both in parenchymal cells and in macrophages. Mycobacterium tuberculosis grows within macrophages and, at the same time, the antimicrobial function of macrophages is important in the body's defence against tuberculosis. In vitro, the loading of macrophages with iron reduces the response of these cells to activation by interferon-gamma and diminishes their toxicity against micro-organisms. In the clinical setting, dietary iron overload appears to increase the risk for death from tuberculosis even in the absence of the acquired immunodeficiency syndrome. The combination of dietary iron overload and infection with the HIV, with impaired function of both macrophages and T-cells, may make patients especially vulnerable to tuberculosis. It is possible that the prevention and treatment of dietary iron overload could contribute to the control of tuberculosis in African populations.     

alpha-Cell neogenesis in an animal model of IDDM

Currently there is debate regarding the capacity of pancreatic islets to regenerate in adult animals. Because pancreatic endocrine cells are thought to arise from duct cells, we examined the pancreatic ductal epithelium of the diabetic NOD mouse for evidence of islet neogenesis. We have evidence of duct proliferation as well as ductal cell differentiation, as suggested by bromodeoxyuridine-labeling and the presence of glucagon-containing cells within these ducts. In addition, the ductal epithelia in diabetic NOD mice expressed the neuroendocrine markers neuropeptide Y and tyrosine hydroxylase. These ducts also expressed the homeobox gene product, insulin promoter factor 1. Ductal cell proliferation and expression of these markers was not observed in transgenic NOD mice (NOD-E), which do not develop clinical or histopathological symptoms of IDDM. This suggests that the observed ductal cell proliferation and differentiation was a direct result of beta-cell destruction and insulin insufficiency in these adult diabetic mice, which further suggests that these events are recapitulating islet ontogeny observed during embryogenesis. It is possible that comparable processes occur in the human diabetic pancreas.     

Inhibitory effect of piracetam on platelet-rich thrombus formation in an animal model

Intravenous administration of piracetam to hamsters reduced the formation of a platelet-rich venous thrombus induced by a standardised crush injury, in a dose-dependent fashion with an IC50 of 68 +/- 8 mg/kg. 200 mg/kg piracetam also significantly reduced in vivo thrombus formation in rats. However, in vitro aggregation of rat platelets was only inhibited with piracetam-concentrations at least 10-fold higher than plasma concentrations (6.2 +/- 1.1 mM) obtained in the treated animals. No effects were seen on clotting tests. In vitro human platelet aggregation, induced by a variety of agonists, was inhibited by piracetam, with IC50's of 25-60 mM. The broad inhibition spectrum could be explained by the capacity of piracetam to prevent fibrinogen binding to activated human platelets. Ex vivo aggregations and bleeding times were only minimally affected after administration of 400 mg/kg piracetam i.v. to healthy male volunteers, resulting in peak plasma levels of 5.8 +/- 0.3 mM. A possible antiplatelet effect of piracetam could be due to the documented beneficial effect on red blood cell deformability leading to a putative reduction of ADP release by damaged erythrocytes. However similarly high concentrations were needed to prevent stirring-induced "spontaneous" platelet aggregation in human whole blood. It is concluded that the observed antithrombotic action of piracetam cannot satisfactorily be explained by an isolated direct effect on platelets. An additional influence of piracetam on the rheology of the circulating blood and/or on the vessel wall itself must therefore be taken into consideration.     

The effect of radial keratotomy on ocular integrity in an animal model

The safety of deep corneal incisions in radial keratotomy was evaluated in a porcine model of blunt trauma. One eye of each enucleated pair (right and left) of porcine eyes was subjected to a variation of radial keratotomy; the fellow eyes served as unoperated-on controls. All eyes were subjected to a standard injury. Control eyes ruptured at the equatorial sclera. Eyes with radial incisions cut through approximately 70% of corneal thickness also ruptured at the equator. When incisions of this depth (70%) were extended across the limbus (rather than to the corneal-scleral junction), all ruptures occurred at the limbal incisions. Eyes cut 95% to 100% of corneal thickness tended to rupture at the incisions. The safety of deep radial keratotomy incisions with respect to ocular integrity is discussed.     

Characterization of an animal model of ventilator-acquired pneumonia

To develop an experimental model of ventilator-acquired pneumonia (VAP), we investigated whether healthy piglets could develop endogenously acquired pulmonary infection as a result of prolonged mechanical ventilation (MV). Thirty-three piglets underwent MV with anesthesia, analgesia, and paralysis produced by continuous infusion of midazolam, fentanyl, and pancuronium bromide. Ten animals received antibioprophylaxis with ceftriaxone (ATB group) and 23 received no antibiotics (control group). Eighteen control animals and 9 ceftriaxone-treated animals completed the 4-day study protocol. The presence of pneumonia on day 4 was ascertained by multiple pulmonary biopsy specimens, processed for microscopic examination and quantitative cultures. The anesthetic regimen provided satisfactory electrolyte balance and cardiovascular stability. Under these circumstances, 17 of 18 animals and 4 of 9 animals developed VAP in the control and the ATB groups, respectively. Lesions of different grades of severity were unevenly distributed through both lungs with a predominance and a higher severity in dependent lung segments. Noninfectious lesions frequently associated with VAP in humans were not observed. Pneumonia was usually polymicrobial with a predominance of Gram-negative organisms. Most of the causative organisms originated from the oropharynx. Histologic lesions and lung bacterial concentrations were less in the ATB group than in control animals. We then investigated the effects of intrabronchial challenge with bacterial pathogens in the absence of MV. Intrabronchial bacterial inoculation resulted in the development of pneumonia that spontaneously resolved even when using very highly titrated inocula. Therefore, MV seems to be the main predisposing factor in the development of pneumonia in this model. This model that resembles human VAP in its histologic, bacteriologic, and pathogenic aspects may be useful to further study pathogenesis, diagnosis, prevention, and therapy of VAP.     

Studies on induction hardening of gray iron and ductile iron

The effect of induction case hardening of a gray cast iron (FG 260) and SG iron (600/3) as a function of applied induction power has been studied. The influence of various operating parameters on the penetration depth has been analysed. The case depth as a function of applied power and the associated changes in microstructure has been investigated. The case depth of SG iron was found to be twice than the gray iron due to higher resistivity of the material and increase in depth of penetration. Both hardness and the depth of penetration increased with increase in applied power associated with martensitic case formation. The surface hardness of both the irons varies between 600 to 800 VHN. The core microstructure in both the irons displayed pearlitic matrix. In the case of SG iron, the nodule size, sphericity and nodularity have reduced in the induction hardened case compared to the core.     

Etiological role of cadmium in hypertension in an animal model

Dahl hypertension-resistant (R) and hypertension-sensitive (S) lines of rats were used to determine whether cadmium plays an etiological role in hypertension. In Study I, weanling (3-week-old) R and S rats of both sexes were given a low-salt (0.4% NaCl) diet and were divided into two groups. Rats in the cadmium group were injected with cadmium (2 mg/kg body weight, ip), whereas the controls received identical volumes of saline. Three weeks after the first injection, no elevations of systolic blood pressure were detected. A second dose of cadmium (1 mg/kg) produced hypertension in S females but not in S males or in R rats of either sex. Also, female S cadmium rats manifested significant (p less than 0.01) mild to moderate renal vascular changes. The concentrations of cadmium in hepatic and renal tissues of S cadmium rats were significantly higher (p less than 0.001) than in R rats. In Study II, weanling (3-week-old) female S rats on a high-salt (4% NaCl) diet were given cadmium (2 mg/kg body weight, ip) at week 3 followed by second and third injections of cadmium (1 mg/kg) at weeks 6 and 23. S controls received the same volumes of saline. Cadmium enhanced the rate and the degree of salt-induced hypertension development. Pathological lesions of periarteritis nodosa in the mesenteric arteries and renal vascular lesions occurred to the same extent in the cadmium and control groups. These data indicate that differences in genetic background influence the development of cadmium-induced hypertension in weanling rats, and that cadmium exacerbates the severity of salt-induced hypertension.     

Effects of iron deficiency and iron overload on manganese uptake and deposition in the brain and other organs of the rat

Manganese (Mn) is an essential trace element at low concentrations, but at higher concentrations is neurotoxic. It has several chemical and biochemical properties similar to iron (Fe), and there is evidence of metabolic interaction between the two metals, particularly at the level of absorption from the intestine. The aim of this investigation was to determine whether Mn and Fe interact during the processes involved in uptake from the plasma by the brain and other organs of the rat. Dams were fed control (70 mg Fe/kg), Fe-deficient (5-10 mg Fe/kg), or Fe-loaded (20 g carbonyl Fe/kg) diets, with or without Mn-loaded drinking water (2 g Mn/L), from day 18-19 of pregnancy, and, after weaning the young rats, were continued on the same dietary regimens. Measurements of brain, liver, and kidney Mn and nonheme Fe levels, and the uptake of 54Mn and 59Fe from the plasma by these organs and the femurs, were made when the rats were aged 15 and 63 d. Organ nonheme Fe levels were much higher than Mn levels, and in the liver and kidney increased much more with Fe loading than did Mn levels with Mn loading. However, in the brain the increases were greater for Mn. Both Fe depletion and loading led to increased brain Mn concentrations in the 15-d/rats, while Fe loading also had this effect at 63 d. Mn loading did not have significant effects on the nonheme Fe concentrations. 54Mn, injected as MnCl2 mixed with serum, was cleared more rapidly from the circulation than was 59Fe, injected in the form of diferric transferrin. In the 15-d-rats, the uptake of 54Mn by brain, liver, kidneys, and femurs was increased by Fe loading, but this was not seen in the 63-d rats. Mn supplementation led to increased 59Fe uptake by the brain, liver, and kidneys of the rats fed the control and Fe-deficient diets, but not in the Fe-loaded rats. It is concluded that Mn and Fe interact during transfer from the plasma to the brain and other organs and that this interaction is synergistic rather than competitive in nature. Hence, excessive intake of Fe plus Mn may accentuate the risk of tissue damage caused by one metal alone, particularly in the brain.     

Neurogenically mediated cystitis in rats: an animal model

PURPOSE: Pseudorabies virus (PRV) is a useful tool for mapping the control circuitry of the spinal cord. In the process of mapping CNS regulatory pathways for the lower urinary tract, a hemorrhagic change in the bladder was observed that was not overtly evident in other pelvic organs. The relationship between the appearance of hemorrhagic changes in the bladder and the evolution of PRV induced changes in the spinal cord was therefore explored. MATERIALS AND METHODS: Sprague-Dawley rats were injected with PRV into the ACD tail-muscle. Bladder and CNS fixation were achieved by transcardial perfusion with formaldehyde. Multi-level sections were obtained from T8 through S4. Fixed tissue was stained and evaluated by light microscopy. Immunohistochemical stains were carried out for PRV and iNOS on spinal cord tissue. We were therefore able to evaluate the relationship between the manifestation of the hemorrhagic cystitis, appearance of the PRV in the spinal cord and evidence of CNS inflammation. RESULTS: The evolution of hemorrhagic cystitis paralleled the evidence of inflammation in the thoraco-lumbar and sacral cord. These bladders contained 5 to 9 ml. of bloody urine (a normal rat bladder contains 1 to 2 ml.). On cystomanometry (CMG) the bladders were acontractile. No PRV could be cultured in the hemorrhagic bladders. The histological changes observed in the bladder represent true inflammation. CONCLUSION: There was no obvious explanation for these changes other than the associated inflammatory changes in the spinal cord. The findings are consistent with the hypothesis that a spinal cord stress, via an unknown metabolic pathway, can result in dramatic, neurogenically mediated changes in the bladder.     

Metachromatic leukodystrophy: molecular genetics and an animal model

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulphatase A (ASA; EC 3.1.6.8). Deficiency of this enzyme causes intralysosomal storage of the sphingolipid cerebroside sulphate. This lipid is abundant in myelin and it may thus not be surprising that storage mainly affects oligodendrocytes. Patients suffer from a progressive demyelination causing various neurological symptoms. The disease is fatal and treatment is not available. The human ASA gene has been cloned and more than 40 mutations have been analysed that cause metachromatic leukodystrophy. Few of these alleles are frequent among patients, whereas most mutant alleles have only been found in single families. Since MLD has only been described in humans and no naturally occurring animal model has been described, ASA-deficient mice have been generated by homologous recombination. The ASA knockout mice are unable to degrade sulphatide and store the lipid intralysosomally. The pattern of lipid storage in neuronal and non-neuronal tissues resembles that described for patients. In the nervous system, lipid storage is found in oligodendrocytes, astrocytes and some neurons. Animals display an astrogliosis and a decreased average axonal diameter. Purkinje cells and Bergmann glia of the cerebellum are morphologically aberrant. Demyelination is seen in the acoustic ganglion and occurs between the ages of 6 and 12 months. The animals are deaf at this age and display various neuromotor abnormalities. However, compared to humans the mice have a surprisingly mild phenotype, since they have a normal life span and do not develop widespread demyelination. ASA-deficient mice have been transplanted with bone marrow, which was transduced with a retroviral vector expressing arylsulphatase A. The majority of transplanted animals display sustained expression of arylsulphatase A from the retroviral construct up to 5 months after transplantation. However, preliminary data suggest that this therapeutic approach does not reduce storage material.     

Dietary alpha-tocopherol supplementation on antioxidant defenses after in vivo iron overload in rats

In the present study we have examined the effects and mechanisms of endothelin-1 (ET-1) on arachidonic acid (AA) release and prostaglandin (PG) synthesis in human ciliary muscle (HCM) cells. ET-1 stimulated AA release in a time (t1/2=1.5 min) and concentration-dependent (EC50=5 nM) manner, which is primarily mediated through the ETA receptor subtype. The AA liberated by ET-1 appears to derive mainly from the phosphoinositides and phosphatidylcholine. Our data show that phospholipase A2 (PLA2), but not phospholipase C (PLC), plays an important role in ET-1-induced AA release. This conclusion is supported by the following findings: (1) ET-1-evoked AA release was inhibited by the PLA2 inhibitors dexamethasone, mepacrine and manoalide in a concentration-dependent manner. Conversion of AA into PGE2 was inhibited by the cyclooxygenase inhibitors in the following order: Indomethacin>naproxen >ibuprofen>NS-398>aspirin. (2) The phorbol ester, PDBu, an activator of protein kinase C, potentiated ET-1-induced AA release by 39%, but inhibited that of inositol phosphates formation by 62%. (3) Pretreatment of the labeled cells with isoproterenol lowered ET-1-induced inositol phosphates production, but had no effect on AA release. (4) U71322, a PLC inhibitor, inhibited ET-1-induced inositol phosphates production, but had no effect on that of AA release. (5) Pretreatment of the cells with pertussis toxin (0.1 microg ml-1) attenuated the stimulatory effects of ET-1 on AA release and PGE2 formation. These data demonstrate that ET-1 is a potent agonist for AA release and PG synthesis in HCM cells, and that PLA2, but not PLC, plays an important role in ET-1-induced AA release and PG synthesis. In ciliary muscle, AA and its metabolites play important roles in intracellular signalling, modulation of physiological processes, and regulation of intraocular pressure.     

The effects of overuse combined with intrinsic or extrinsic alterations in an animal model of rotator cuff tendinosis

An in vivo animal model was used to evaluate overuse and overuse plus intrinsic tendon injury or extrinsic tendon compression in the development of rotator cuff injury. Forty-four male Sprague-Dawley rats were divided into groups of 22. Each left shoulder received an intrinsic or extrinsic injury plus overuse (treadmill running), and each right shoulder received only overuse. Eleven rats from each group were sacrificed at 4 and 8 weeks. Supraspinatus tendons were evaluated histologically or geometrically and biomechanically. Ten rats constituted a cage-activity control group. Both supraspinatus tendons of the experimental groups had increases in cellularity and collagen disorganization and changes in cell shape compared with control tendons. Tendons with injury plus overuse exhibited a worse histologic grade than those with overuse alone. The cross-sectional area of both supraspinatus tendons of the experimental rats was significantly more than in control tendons. The area of the injury plus overuse tendons was increased on average compared with overuse-alone tendons. Biomechanically, the tissue moduli of overuse/intrinsic injury tendons at 4 weeks and those of the overuse/extrinsic injury tendons at 8 weeks were significantly lower than in control tendons. Tissue moduli of the overuse/injury tendons were significantly lower than in the overuse-alone tendons at 8 weeks. This study demonstrated that damage to the supraspinatus tendon can be caused by overuse and intrinsic injury, overuse and extrinsic compression, and overuse alone.     

Effect of alcohol consumption on adult and aged bone: a histomorphometric study of the rat animal model

The adult and aged skeleton exist in a time when osteoporosis and age-related bone loss is at a maximum, and it is modified by lifestyle factors such as alcohol. To determine the effect of life-long alcohol consumption on the adult and aged rat model, 4-week-old female Sprague-Dawley rats were divided into three diet groups. Alcohol-treated animals were fed a modified Lieber-DeCarli diet ad libitum containing 35% ethanol-derived calories, whereas the pair-fed animals (weight-matched to ethanol rats) received an isocaloric liquid diet in which maltose-dextrin substituted calories supplied by ethanol. Chow animals were fed a standard rat chow ad libitum. Proximal tibiae were removed and prepared for histomorphometric analysis after 3, 6, 9, 12, or 18 months on the diets. Previous studies, with young animals, showed that chronic alcohol consumption during the age of bone development reduced bone volume and trabecular number in cancellous bone. The present study demonstrates that these reductions last throughout life. The rate of bone formation is reduced in alcohol-fed animals, but most bone cell parameters are relative normal, except for wall thickness, indicating a reduced osteoblast activity.