Substance P receptor blocker, aprepitant, inhibited cutaneous and other neurogenic inflammation side effects of the EGFR1-TKI, erlotinib.

Cutaneous changes like rash and hair loss, as well as other neurogenic inflammation side effects, occur frequently during anticancer treatment with the epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), erlotinib. These adverse events may be so severe that they impair the patient's compliance with the treatment or even cause its discontinuation. In the current preclinical study, rats (9.2 weeks) were treated with erlotinib (10 mg/kg/day) ± aprepitant (2 mg/kg/day) for 12 weeks. Visual changes in the development of facial skin lesions/hair loss and SP-receptor expression (immunohistochemically) in facial skin tissue were assessed; also changes in plasma magnesium, 8-isoprostane, substance P (SP), neutrophil superoxide production, and cardiac function (echocardiography) were measured. Erlotinib lowered plasma magnesium 14%, elevated SP 65%, caused 3.7-fold higher basal superoxide production, 2.5-fold higher 8-isoprostane levels, 11.6% lower cardiac systolic, and 10.9% lower diastolic function. Facial dermatological changes (alopecia, skin reddening, scabbing, nose crusting) occurred by 4 weeks (± + to ++) in erlotinib-treated rats, and progressively worsened (±++ to +++) by week 12. Facial skin SP-receptor upregulation (78% higher) occurred in epidermal and hair follicle cells. All adverse effects were substantially and significantly mitigated by aprepitant, including a 62% lowering of skin SP-receptors (p < 0.05). Elevated SP levels mediated the side effects of erlotinib treatment, but aprepitant's significant prevention of the systemic and cutaneous adverse events indicates a novel potential therapy against the side effects of this anticancer treatment.

Mg-supplementation attenuated lipogenic and oxidative/nitrosative gene expression caused by Combination Antiretroviral Therapy (cART) in HIV-1-transgenic rats.

We determined if HIV-1 expression in transgenic (HIV-1-Tg) rats enhanced hepatic genomic changes related to oxidative/nitrosative stress and lipogenesis during cART-treatment, and assessed effects of Mg-supplementation. A clinically used cART (atazanavir-ritonavir+Truvada) was given orally to control and HIV-1-Tg rats (18 weeks) with normal or 6-fold dietary-Mg. Oxidative/nitrosative and lipogenic genes were determined by real-time RT-PCR. cART induced a 4-fold upregulation of sterol regulatory element-binding protein-1 (SREBP-1) in HIV-1-Tg-rats, but not in controls; Tg rats displayed a 2.5-fold higher expression. Both were completely prevented by Mg-supplementation. Nrf2 (Nuclear erythroid-derived factor 2), a master transcription factor controlling redox homeostasis, was down-regulated 50% in HIV-Tg rats, and reduced further to 25% in Tg+cART-rats. Two downstream antioxidant genes, heme oxygenase-1(HmOX1) and Glutathione-S-transferase(GST), were elevated in HIV-Tg alone but were suppressed by cART treatment. Decreased Nrf2 in Tg±cART were normalized by Mg-supplementation along with the reversal of altered HmOX1 and GST expression. Concomitantly, iNOS (inducible nitric oxide synthase) was upregulated 2-fold in Tg+cART rats, which was reversed by Mg-supplementation. In parallel, cART-treatment led to substantial increases in plasma 8-isoprostane, nitrotyrosine, and RBC-GSSG (oxidized glutathione) levels in HIV-1-Tg rats; all indices of oxidative/nitrosative stress were suppressed by Mg-supplementation. Both plasma triglyceride and cholesterol levels were elevated in Tg+cART rats, but were lowered by Mg-supplementation. Thus, the synergistic effects of cART and HIV-1 expression on lipogenic and oxidative/nitrosative effects were revealed at the genomic and biochemical levels. Down-regulation of Nrf2 in the Tg+cART rats suggested their antioxidant response was severely compromised; these abnormal metabolic and oxidative stress effects were effectively attenuated by Mg-supplementation at the genomic level.

Combination ART-Induced Oxidative/Nitrosative Stress, Neurogenic Inflammation and Cardiac Dysfunction in HIV-1 Transgenic (Tg) Rats: Protection by Mg.

Chronic effects of a combination antiretroviral therapy (cART = tenofovir/emtricitatine + atazanavir/ritonavir) on systemic and cardiac oxidative stress/injury in HIV-1 transgenic (Tg) rats and protection by Mg-supplementation were assessed. cART (low doses) elicited no significant effects in normal rats, but induced time-dependent oxidative/nitrosative stresses: 2.64-fold increased plasma 8-isoprostane, 2.0-fold higher RBC oxidized glutathione (GSSG), 3.2-fold increased plasma 3-nitrotyrosine (NT), and 3-fold elevated basal neutrophil superoxide activity in Tg rats. Increased NT staining occurred within cART-treated HIV-Tg hearts, and significant decreases in cardiac systolic and diastolic contractile function occurred at 12 and 18 weeks. HIV-1 expression alone caused modest levels of oxidative stress and cardiac dysfunction. Significantly, cART caused up to 24% decreases in circulating Mg in HIV-1-Tg rats, associated with elevated renal NT staining, increased creatinine and urea levels, and elevated plasma substance P levels. Strikingly, Mg-supplementation (6-fold) suppressed all oxidative/nitrosative stress indices in the blood, heart and kidney and substantially attenuated contractile dysfunction (>75%) of cART-treated Tg rats. In conclusion, cART caused significant renal and cardiac oxidative/nitrosative stress/injury in Tg-rats, leading to renal Mg wasting and hypomagnesemia, triggering substance P-dependent neurogenic inflammation and cardiac dysfunction. These events were effectively attenuated by Mg-supplementation likely due to its substance P-suppressing and Mg's intrinsic anti-peroxidative/anti-calcium properties.

EGFR-TKI, erlotinib, causes hypomagnesemia, oxidative stress, and cardiac dysfunction: attenuation by NK-1 receptor blockade.

To determine whether the epidermal growth factor receptor tyrosine kinase inhibitor, erlotinib may cause hypomagnesemia, inflammation, and cardiac stress, erlotinib was administered to rats (10 mg · kg(-1)· d(-1)) for 9 weeks. Plasma magnesium decreased progressively between 3 and 9 weeks (-9% to -26%). Modest increases in plasma substance P (SP) occurred at 3 (27%) and 9 (25%) weeks. Neutrophil superoxide-generating activity increased 3-fold, and plasma 8-isoprostane rose 210%, along with noticeable appearance of cardiac perivascular nitrotyrosine. The neurokinin-1 (NK-1) receptor antagonist, aprepitant (2 mg · kg(-1) · d(-1)), attenuated erlotinib-induced hypomagnesemia up to 42%, reduced circulating SP, suppressed neutrophil superoxide activity and 8-isoprostane elevations; cardiac nitrotyrosine was diminished. Echocardiography revealed mild to moderately decreased left ventricular ejection fraction (-11%) and % fractional shortening (-17%) by 7 weeks of erlotinib treatment and significant reduction (-17.5%) in mitral valve E/A ratio at week 9 indicative of systolic and early diastolic dysfunction. Mild thinning of the left ventricular posterior wall suggested early dilated cardiomyopathy. Aprepitant completely prevented the erlotinib-induced systolic and diastolic dysfunction and partially attenuated the anatomical changes. Thus, chronic erlotinib treatment does induce moderate hypomagnesemia, triggering SP-mediated oxidative/inflammation stress and mild-to-moderate cardiac dysfunction, which can largely be corrected by the administration of the SP receptor blocker.

Mg supplementation protects against ritonavir-mediated endothelial oxidative stress and hepatic eNOS downregulation.

Ritonavir (RTV), a prototypical protease inhibitor currently used as a key component of anti-HIV therapy, is known for its endothelial and hepatic toxicity. The effects of RTV and magnesium supplementation on cultured bovine endothelial cell (EC) and rat hepatic endothelial nitric oxide synthase (eNOS) status were investigated. RTV dose-dependently (5-30 µM) decreased EC viability after 48 h; high Mg (2mM) significantly attenuated the lost viability. ECs incubated with 15 µM RTV for 6 to 24h resulted in two- to fourfold elevation of oxidized glutathione and a 25% loss of total glutathione. At 24h, EC superoxide production due to RTV was detected by dihydroethidium staining and increased 41% when quantified by flow cytometry; altered glutathione status and superoxide levels were both substantially reversed by 2mM Mg. RTV reduced eNOS mRNA (-25% at 24 h) and led to decreased eNOS dimer/monomer ratios; nitric oxide-derived products decreased 40%; both changes were attenuated by Mg supplementation. In male Lewis-Brown Norway rats, RTV administration (75 mg/kg/day, 5 weeks) resulted in an 85% increase in plasma 8-isoprostane and a 23% decrease in hepatic eNOS mRNA; concomitantly, eNOS protein decreased 75%, whereas plasma nitrite level was reduced 48%. Dietary Mg supplementation (sixfold higher than control) prevented the eNOS mRNA decrease along with lowering 8-isoprostane and restored the eNOS protein and plasma nitrite levels comparable to controls. In conclusion, Mg attenuates RTV-mediated EC oxidative eNOS dysfunction and downregulation of hepatic eNOS expression; we suggest that Mg can serve as a beneficial adjunct therapeutic against RTV-mediated eNOS toxicity.

Mg supplementation attenuates ritonavir-induced hyperlipidemia, oxidative stress, and cardiac dysfunction in rats.

Use of protease inhibitors (PI) in HIV patients is associated with hyperlipidemia and increased risk of coronary heart disease. Chronic systemic and cardiac effects of ritonavir (RTV), a universal PI booster, and Mg supplementation were examined. RTV was administered (75 mg·kg(-1)·day(-1) po) to Lewis × Brown-Norway hybrid (LBNF1) rats for up to 8 wk; significant increases in plasma triglyceride and cholesterol occurred from 8 days to 8 wk. At 5 wk, the expression of selected hepatic genes (CYP7A1, CITED2, G6PC, and ME-1), which are key to lipid catabolism/synthesis, were altered toward lipogenesis. Dietary Mg supplementation (six-fold higher) completely reversed the altered expression of these genes and attenuated both hypertriglyceridemia and hypercholesterolemia. Neutrophils isolated from the RTV-treated rats displayed a three-fold higher basal and a twofold higher stimulated superoxide production; plasma isoprostane and red blood cell (RBC) GSSG levels were elevated two- to three-fold. All oxidative indices were normalized by Mg supplementation. After 5 wk, RTV caused significant decreases in cardiac left ventricular (LV) shortening fraction and LV ejection fraction; mitral valve early/late atrial ventricular filling (E/A) ratio was reduced accompanied by LV posterior wall thinning. Immunohistochemical staining revealed significant white blood cell (WBC) infiltration (5 wk) and prominent fibrosis (8 wk) in the RTV hearts. Mg supplementation attenuated RTV-induced declines in systolic and diastolic (improved mitral valve E/A ratio) function (>70%), lessened LV posterior wall thinning (by 75%), and substantially decreased the pathological markers. The known clinical hyperlipidemia effects of RTV can be mimicked in the LBNF1 rats; in association, systemic oxidative stress and progressive cardiac dysfunction occurred. Remarkably, Mg supplementation alone suppressed RTV-mediated hyperlipidemia, oxidative stress, and cardiac dysfunction.

Genomic profiling reveals the potential role of TCL1A and MDR1 deficiency in chemotherapy-induced cardiotoxicity.

BACKGROUND: Anthracyclines, such as doxorubicin (Adriamycin), are highly effective chemotherapeutic agents, but are well known to cause myocardial dysfunction and life-threatening congestive heart failure (CHF) in some patients. METHODS: To generate new hypotheses about its etiology, genome-wide transcript analysis was performed on whole blood RNA from women that received doxorubicin-based chemotherapy and either did, or did not develop CHF, as defined by ejection fractions (EF)≤40%. Women with non-ischemic cardiomyopathy unrelated to chemotherapy were compared to breast cancer patients prior to chemo with normal EF to identify heart failure-related transcripts in women not receiving chemotherapy. Byproducts of oxidative stress in plasma were measured in a subset of patients. RESULTS: The results indicate that patients treated with doxorubicin showed sustained elevations in oxidative byproducts in plasma. At the RNA level, women who exhibited low EFs after chemotherapy had 260 transcripts that differed >2-fold (p<0.05) compared to women who received chemo but maintained normal EFs. Most of these transcripts (201) were not altered in non-chemotherapy patients with low EFs. Pathway analysis of the differentially expressed genes indicated enrichment in apoptosis-related transcripts. Notably, women with chemo-induced low EFs had a 4.8-fold decrease in T-cell leukemia/lymphoma 1A (TCL1A) transcripts. TCL1A is expressed in both cardiac and skeletal muscle, and is a known co-activator for AKT, one of the major pro-survival factors for cardiomyocytes. Further, women who developed low EFs had a 2-fold lower level of ABCB1 transcript, encoding the multidrug resistance protein 1 (MDR1), which is an efflux pump for doxorubicin, potentially leading to higher cardiac levels of drug. In vitro studies confirmed that inhibition of MDR1 by verapamil in rat H9C2 cardiomyocytes increased their susceptibility to doxorubicin-induced toxicity. CONCLUSIONS: It is proposed that chemo-induced cardiomyopathy may be due to a reduction in TCL1A levels, thereby causing increased apoptotic sensitivity, and leading to reduced cardiac MDR1 levels, causing higher cardiac levels of doxorubicin and intracellular free radicals. If so, screening for TCL1A and MDR1 SNPs or expression level in blood, might identify women at greatest risk of chemo-induced heart failure.

Angiotensin II promotes iron accumulation and depresses PGI2 and NO synthesis in endothelial cells: effects of losartan and propranolol analogs.

Angiotensin may promote endothelial dysfunction through iron accumulation. To research this, bovine endothelial cells (ECs) were incubated with iron (30 µmol·L⁻¹) with or without angiotensin II (100 nmol·L⁻¹). After incubation for 6 h, it was observed that the addition of angiotensin enhanced EC iron accumulation by 5.1-fold compared with a 1.8-fold increase for cells incubated with iron only. This enhanced iron uptake was attenuated by losartan (100 nmol·L⁻¹), d-propranolol (10 µmol·L⁻¹), 4-HO-propranolol (5 µmol·L⁻¹), and methylamine, but not by vitamin E or atenolol. After 6 h of incubation, angiotensin plus iron provoked intracellular oxidant formation (2'7'-dichlorofluorescein diacetate (DCF-DA) fluorescence) and elevated oxidized glutathione; significant loss of cell viability occurred at 48 h. Stimulated prostacyclin release decreased by 38% (6 h) and NO synthesis was reduced by 41% (24 h). Both oxidative events and functional impairment were substantially attenuated by losartan or d-propranolol. It is concluded that angiotensin promoted non-transferrin-bound iron uptake via AT-1 receptor activation, leading to EC oxidative functional impairment. The protective effects of d-propranolol and 4-HO-propranolol may be related to their lysosomotropic properties.

d-Propranolol protects against oxidative stress and progressive cardiac dysfunction in iron overloaded rats.

d-Propranolol (d-Pro: 2-8 mg·(kg body mass)(-1)·day(-1)) protected against cardiac dysfunction and oxidative stress during 3-5 weeks of iron overload (2 mg Fe-dextran·(g body mass)(-1)·week(-1)) in Sprague-Dawley rats. At 3 weeks, hearts were perfused in working mode to obtain baseline function; red blood cell glutathione, plasma 8-isoprostane, neutrophil basal superoxide production, lysosomal-derived plasma N-acetyl-ß-galactosaminidase (NAGA) activity, ventricular iron content, and cardiac iron deposition were assessed. Hearts from the Fe-treated group of rats exhibited lower cardiac work (26%) and output (CO, 24%); end-diastolic pressure rose 1.8-fold. Further, glutathione levels increased 2-fold, isoprostane levels increased 2.5-fold, neutrophil superoxide increased 3-fold, NAGA increased 4-fold, ventricular Fe increased 4.9-fold; and substantial atrial and ventricular Fe-deposition occurred. d-Pro (8 mg) restored heart function to the control levels, protected against oxidative stress, and decreased cardiac Fe levels. After 5 weeks of Fe treatment, echocardiography revealed that the following were depressed: percent fractional shortening (%FS, 31% lower); left ventricular (LV) ejection fraction (LVEF, 17%), CO (25%); and aortic pressure maximum (P(max), 24%). Mitral valve E/A declined by 18%, indicating diastolic dysfunction. Cardiac CD11b+ infiltrates were elevated. Low d-Pro (2 mg) provided modest protection, whereas 4-8 mg greatly improved LVEF (54%-75%), %FS (51%-81%), CO (43%-78%), P(max) (56%-100%), and E/A >100%; 8 mg decreased cardiac inflammation. Since d-Pro is an antioxidant and reduces cardiac Fe uptake as well as inflammation, these properties may preserve cardiac function during Fe overload.

The EGFR tyrosine kinase inhibitor tyrphostin AG-1478 causes hypomagnesemia and cardiac dysfunction.

We determined whether the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) N-​(3-​chlorophenyl)-​6,​7-​dimethoxy-​4-​quinazolinamine (tyrphostin AG-1478) causes hypomagnesemia and cardiac dysfunction in rats. Tyrphostin was administered (3 times per week, intraperitoneal injection, to achieve 21.4 mg·(kg body mass)(-1)·day(-1)) to normomagnesemic rats for 5 weeks. Levels of magnesium in the plasma of the tyrphostin-treated rats decreased significantly by the following amount: 17% at week 1, 27% at week 2, and 26%-35% between weeks 3 to 5. Levels of the plasma lipid peroxidation marker 8-isoprostane rose significantly: by 58% at week 1, 168% at week 3, and 113% at week 5. At week 5, blood neutrophils from the tyrphostin-treated group displayed a 2.26-fold higher basal level of O(2)(·-) generation; the ratio of oxidized glutathione (glutathione disulfide; GSSG) to reduced glutathione (GSH) in the red blood cells increased 2.5-fold. At week 5, echocardiography revealed that TKI treatment resulted in significant cardiac systolic dysfunction, with impaired diastolic function and dilated cardiomyopathy. Since hypomagnesemia alone can trigger oxidative stress and cardiac injury, we suggest that inhibition of EGFR-TK caused magnesium wasting, which partly contributed to decreased cardiac contractility.

Loss of neutral endopeptidase activity contributes to neutrophil activation and cardiac dysfunction during chronic hypomagnesemia: Protection by substance P receptor blockade.

BACKGROUND/OBJECTIVE: Hypomagnesemia (Hypo-Mg) in rodents leads to neurogenic inflammation associated with substance P (SP) elevations; neutral endopeptidase (NEP) is a principle cell surface proteolytic enzyme, which degrades SP. The effects of chronic Hypo-Mg on neutrophil NEP activity, cell activation and the associated cardiac dysfunction were examined. METHODS/RESULTS: Male Sprague-Dawley rats (180 g) were fed Mg-sufficient or Mg-deficient (Hypo-Mg) diets for five weeks. Enriched blood neutrophils were isolated at the end of one, three and five weeks by step gradient centrifugation. NEP enzymatic activity decreased by 20% (P value was nonsignificant), 50% (P<0.025) and 57% (P<0.01), respectively, for week 1, 3 and 5 Hypo-Mg rats. In association, neutrophil basal superoxide (•O(2) (-))-generating activities were elevated: 30% at week 1 (P value was nonsignificant), and fourfold to sevenfold for weeks 3 to 5 (P<0.01). Maximal phorbol myristate acetate-stimulated •O(2) (-) production by Hypo-Mg neutrophils increased twofold at week 5. Also, plasma 8-isoprostane levels were elevated twofold to threefold, and red blood cell glutathione decreased by 50% (P<0.01) after three to five weeks of chronic Hypo-Mg. When Hypo-Mg rats were treated with the SP receptor blocker (L-703,606), neutrophil NEP activities were retained at 75% (week 3) and 77% (week 5) (P<0.05); activation of neutrophil •O(2) (-) and other oxidative indexes were also significantly (P<0.05) attenuated. After five weeks, histochemical (hematoxylin and eosin) staining of Hypo-Mg-treated rat ventricles revealed significant white blood cell infiltration, which was substantially reduced by L-703,606. Echocardiography after three weeks of Hypo-Mg only showed modest diastolic impairment, but five weeks resulted in significant (P<0.05) depression in both left ventricular systolic and diastolic functions; changes in these functional parameters were attenuated by L-703,606. CONCLUSION: NEP activity regulates neutrophil •O(2) (-) formation by controlling SP bioavailability. When oxidative inactivation of NEP is prevented by SP receptor blockade, partial protection is afforded against cardiac contractile dysfunction.

Cardiovascular and intestinal responses to oxidative and nitrosative stress during prolonged magnesium deficiency.

In rodents with dietary magnesium deficiency (Mg deficiency), hypomagnesemia, occurs leading to a rise in circulating substance P from neuronal tissues to trigger systemic inflammatory stress in cardiac and intestinal tissues. Sustained elevations of substance P may result from impaired neutral endopeptidase (NEP) activity due to reactive oxygen and reactive nitrogen species. Associated increase in intestinal permeability includes infiltration of WBC and endotoxemia, which can further amplify the systemic inflammatory response that leads to impaired contractile function associated with up-regulation of the cardiac CD14 endotoxin receptor. The neurogenic signal transduction pathways that we have identified in the pro-oxidant/pro-inflammatory processes found with prolonged hypomagnesemia are described in this report.

Neutral endopeptidase inhibition enhances substance P mediated inflammation due to hypomagnesemia.

During dietary deficiency of magnesium neurogenic inflammation is mediated, primarily, by elevated levels of substance P (SP). The enzyme most specific for degrading this neuropeptide is neutral endopeptidase (NEP). In recent studies we found that pharmacological inhibition of NEP by phosphoramidon resulted in elevated plasma levels of SP and greater oxidative stress. We also observed that hypomagnesemia reduced cardiac and intestinal expression of NEP. In these magnesium-deficient rats increased intestinal permeability and impaired cardiac contractility occurred. In our colony of genetically-engineered NEP knockout mice that have reduced ability to degrade SP, we found increased oxidative stress that was prevented by SP (neurokinin-1) receptor blockade. Thus, we submit that inhibition of NEP by pharmacological, genetic and dietary approaches (magnesium restriction), causes greater neurogenic inflammation that may result in increased intestinal and cardiac dysfunction.

Neurogenic inflammation and cardiac dysfunction due to hypomagnesemia.

Hypomagnesemia continues to be a significant clinical disorder that is present in patients with diabetes mellitus, alcoholism, and treatment with magnesuric drugs (diuretics, cancer chemotherapy agents, etc.). To determine the role of magnesium in cardiovascular pathophysiology, we have used dietary restriction of this cation in animal models. This review highlights some key observations that helped formulate the hypothesis that release of substance P (SP) during experimental dietary Mg deficiency (MgD) may initiate a cascade of deleterious inflammatory, oxidative, and nitrosative events, which ultimately promote cardiomyopathy, in situ cardiac dysfunction, and myocardial intolerance to secondary stresses. SP acts primarily through neurokinin-1 receptors of inflammatory and endothelial cells, and may induce production of reactive oxygen and nitrogen species (superoxide anion, NO*, peroxynitrite, hydroxyl radical), leading to enhanced consumption of tissue antioxidants; stimulate release of inflammatory mediators; promote tissue adhesion molecule expression; and enhance inflammatory cell tissue infiltration and cardiovascular lesion formation. These SP-mediated events may predispose the heart to injury if faced with subsequent oxidative stressors (ischemia/reperfusion, certain drugs) or facilitate development of in situ cardiac dysfunction, especially with prolonged dietary Mg restriction. Significant protection against most of these MgD-mediated events has been observed with interventions that modulate neuronal SP release or its bioactivity, and with several antioxidants (vitamin E, probucol, epicaptopril, d-propranolol). In view of the clinical prevalence of hypomagnesemia, new treatments, beyond magnesium repletion, may be needed to diminish deleterious neurogenic and prooxidative components described in this article.

AZT-induced oxidative cardiovascular toxicity: attenuation by Mg-supplementation.

Cardiovascular effects of chronic AZT treatment on SD male rats (185 g) fed either a normal Mg diet (0.1% MgO) or a high Mg diet (0.6% MgO) were examined. AZT treatment (1 mg/ml drinking water) for 3 weeks led to a 5.5-fold (0.88 +/- 0.11 nmol/min/10(6) cells, P < 0.05) elevation in neutrophil basal activity of O2(-) production versus controls (0.16 +/- 0.03 nmol/min, assayed ex vivo as SOD-inhibitable cytochrome c reduction). Concomitantly, plasma 8-isoprostane and PGE(2) levels rose 2.1-fold and 3-fold (both P < 0.05), respectively, compared to control; however, RBC GSH decreased 28% (P < 0.02) with GSSG content increased 3-fold, indicative of systemic oxidative stress. High Mg diet substantially attenuated the AZT-induced neutrophil activation by 70% (0.26 +/- 0.05 nmol/min, P < 0.05); reduced plasma 8-isoprostane and PGE(2) to levels comparable to normal; and RBC GSH was restored back to 92% of control. AZT alone caused moderate, but significant vascular inflammatory lesions in the heart (assessed by H&E staining). Immunohistochemical staining revealed significantly higher (about 4-fold) infiltration of CD11b positive cells (WBC surface marker) in the atria and ventricles of AZT-treated rats. However, these inflammatory pathological markers were minimal in samples of rats treated with AZT plus high Mg diet. Moreover, AZT alone significantly (P < 0.02) decreased rat weight gain by 21% at 3 weeks; Mg-supplementation completely prevented (P < 0.05) the weight gain loss due to AZT intake. It is concluded that high dietary Mg may provide beneficial effects against AZT toxicity due to its systemic antioxidative/anti-inflammatory properties.

Antioxidant and lysosomotropic properties of acute D-propranolol underlies its cardioprotection of postischemic hearts from moderate iron-overloaded rats.

The benefits of acute D-propranolol (D-Pro, non-beta-adrenergic receptor blocker) pretreatment against enhanced ischemia/reperfusion (I/R) injury of hearts from moderate iron-overloaded rats were examined. Perfused hearts from iron-dextran-treated rats (450 mg/kg/week for 3 weeks, intraperitoneal administration) exhibited normal control function, despite iron treatment that elevated plasma iron and conjugated diene levels by 8.1-and 2.5-fold, respectively. However, these hearts were more susceptible to 25 mins of global I/R stress compared with non-loaded hearts; the coronary flow rate, aortic output, cardiac work, left ventricular systolic pressure, positive differential left ventricular pressure (dP/dt), and left ventricular developed pressure displayed 38%, 60%, 55%, 13%, 41%, and 15% lower recoveries, respectively, and a 6.5-fold increase in left ventricular end-diastolic pressure. Postischemic hearts from iron-loaded rats also exhibited 5.6-, 3.48-, 2.43-, and 3.45-fold increases in total effluent iron content, conjugated diene levels, lactate dehydrogenase (LDH) activity, and lysosomal N-acetyl-beta-glucosaminidase (NAGA) activity, respectively, compared with similarly stressed non-loaded hearts. A comparison of detection time profiles during reperfusion suggests that most of the oxidative injury (conjugated diene) in hearts from iron-loaded rats occurred at later times of reperfusion (8.5-15 mins), and this corresponded with heightened tissue iron and NAGA release. D-Pro (2 microM infused for 30 mins) pretreatment before ischemia protected all parameters compared with the untreated iron-loaded group; pressure indices improved 1.2- to 1.6-fold, flow parameters improved 1.70- to 2.96-fold, cardiac work improved 2.87-fold, and end-diastolic pressure was reduced 56%. D-Pro lowered total release of tissue iron, conjugated diene content, LDH activity, and NAGA activity 4.59-, 2.55-, 3.04-, and 4.14-fold, respectively, in the effluent of I/R hearts from the iron-loaded group. These findings suggest that the enhanced postischemic dysfunction and tissue injury of hearts from iron-loaded rats was caused by excessive iron-catalyzed free radical stress, and that the membrane antioxidant properties of D-Pro and its stabilization of sequestered lysosomal iron by D-Pro may contribute to the cardioprotective actions of D-Pro.

D-propranolol attenuates lysosomal iron accumulation and oxidative injury in endothelial cells.

The influence of selected beta-receptor blockers on iron overload and oxidative stress in endothelial cells (ECs) was assessed. Confluent bovine ECs were loaded with iron dextran (15 muM) for 24 h and then exposed to dihydroxyfumarate (DHF), a source of reactive oxygen species, for up to 2 h. Intracellular oxidant formation, monitored by fluorescence of 2',7'-dichlorofluorescin (DCF; 30 microM), increased and peaked at 30 min; total glutathione decreased by 52 +/- 5% (p < 0.01) at 60 min. When the ECs were pretreated 30 min before iron loading with 1.25 to 10 microM d-propranolol, glutathione losses were attenuated 15 to 80%, with EC(50) = 3.1 microM. d-Propranolol partially inhibited the DCF intensity increase, but atenolol up to 10 microM was ineffective. At 2 h, caspase 3 activity was elevated 3.2 +/- 0.3-fold (p < 0.01) in the iron-loaded and DHF-treated ECs, and cell survival, determined 24 h later, decreased 47 +/- 6% (p < 0.01). Ten micromoles of d-propranolol suppressed the caspase 3 activation by 63% (p < 0.05) and preserved cell survival back to 88% of control (p < 0.01). In separate experiments, 24-h iron loading resulted in a 3.6 +/- 0.8-fold increase in total EC iron determined by atomic absorption spectroscopy; d-propranolol at 5 microM reduced this increase to 1.5 +/- 0.4-fold (p < 0.01) of controls. Microscopic observation by Perls' staining revealed that the excessive iron accumulated in vesicular endosomal/lysosomal structures, which were substantially diminished by d-propranolol. We previously showed that propranolol could readily concentrate into the lysosomes and raise the intralysosomal pH; it is suggested that the lysosomotropic properties of d-propranolol retarded the EC iron accumulation and thereby conferred the protective effects against iron load-mediated cytotoxicity.

Intestinal and cardiac inflammatory response shows enhanced endotoxin receptor (CD14) expression in magnesium deficiency.

Substance P is elevated in plasma and in other tissues during Mg-deficiency, and was found localised to neuronal C-fibres of cardiac and intestinal tissues, where it could promote neurogenic inflammation. Plasma prostaglandin E2 (PGE2), indicative of systemic inflammation, rose significantly (>or=4 fold, p<0.01) after 1 week and remained elevated through week 2 and 3 in rat on the Mg-deficient (MgD) diet. Concomitantly, total blood glutathione decreased by 50%. Immunohistochemical staining for endotoxin (lipopolysaccaride, LPS) receptor, CD14 was prominent in macrophage-type cells in intestinal tissue; more importantly, cardiac tissue revealed both CD11b (monocyte/macrophage surface protein) and CD14 positive cells after 3 weeks in rats on MgD diet. Western blot analysis indicated a significant increase in the endotoxin receptor protein level in the 3 week MgD hearts. Since CD14 is known to be up-regulated in cells exposed to LPS, these observations suggest that prolonged Mg-deficiency results in increased intestinal permeability to bacterial products that induce the endotoxin receptor in cells localized to myocardial and intestinal tissues. These CD14 positive cells may amplify the cardiomyopathic inflammatory process by stimulating TNF-alpha and other pro-inflammatory cytokines.

Pro-oxidant properties and cytotoxicity of AZT-monophosphate and AZT.

The effects of zidovudine (AZT) and AZT-monophosphate (AZT-MP) on lipid peroxidation and oxidative cell injury were studied. When microsomal membranes from rat livers were peroxidized by a superoxide-driven, Fe-catalyzed oxy-radical system (ORS), both AZT-MP and, to a lesser extent AZT, but not thymidine, concentration dependently (2-100 microM) enhanced lipid peroxidation (TBARS formation) up to 51% above control. Significance (p < 0.05) was achieved by 6.7 microM AZT-MP. When cultured bovine aortic endothelial cells were incubated with the ORS for 60 min, total glutathione (GSH) decreased by 40% and 24-h cell survival, determined by the tetrazolium salt MTT assay, decreased by 38%. Using this cell system, AZT-MP (7-100 microM) promoted cell death further; at 20 microM 50% (p < 0.01), cell death was induced. In comparison, AZT was less effective. Concurrently, AZT-MP significantly promoted ORS-mediated loss of GSH. These cytotoxic effects were further exacerbated by low extracellular magnesium. Interestingly, when the endothelial cells were exposed to an iron-independent peroxynitrite generating system (SIN-1), the AZT-MP effects were absent. We propose that these pro-oxidant properties of AZT-MP are iron dependent. Because AZT-MP is a major phosphorylated metabolite, the data suggest that potential pro-oxidative activities may be associated with AZT use when catalytic iron is present.

Cardiac pathologic effects of azidothymidine (AZT) in Mg-deficient mice.

Treatment of HIV with AZT (zidovudine) may have toxic side effects as a result of multiple mechanisms. It is known that patients with AIDS may suffer from magnesium deficiency (MgD). We studied selected biochemical and histopathologic consequences of AZT administration (0.7 mg/mL in drinking water) with concurrent Mg-deficient (20% of normal) diet in male C57Bl/6N mice for 3 wk. Significant decreases in red blood cell glutathione (GSH) were evident in the Mg-deficient mice with or without AZT treatment, suggesting compromised antioxidant capacity in the blood. Although MgD alone led to a 1.9-fold increase in plasma thromboxane B(2) (TXB(2), derived from the highly vasoconstrictive TXA(2)), AZT + MgD increased the TXB(2) level 3.5-fold. AZT (+/-MgD) provoked prominent hepatic damage expressed by distortion of lobular architecture, nuclear and cellular swelling, and inflammatory lesions and loss of hepatocytes. AZT alone caused mild cardiac lesions, resulting in partial cardiac fibrosis, especially in the atrium. AZT + MgD caused only scattered small-size cardiac lesions consisting of microscopic foci of inflammatory infiltrates in the ventricles but led to more prominent lesions, fibrosis, and scars in the atrium. MgD or AZT alone caused varying degrees of skeletal muscle degeneration; in combination, more intense degeneration and regeneration of muscle cells were evident. In conclusion, it is suggested that both the decreased blood GSH and elevated plasma TXA(2) might contribute, at least in part, to the aggravated pathological damages observed in the atrium and skeletal muscle of the AZT-treated Mg-deficient mice.