Enhanced tumor necrosis factor-α production following endotoxin challenge in rats is an early event during magnesium deficiency

Magnesium (Mg) plays an essential role in fundamental cellular reactions and the importance of the immuno-inflammatory processes in the pathology of Mg deficiency has been recently reconsidered. The purpose of the present study was to assess the effect of different stages of Mg deficiency on endotoxin response and tumor necrosis factor-α (TNFα) production. Weaning male Wistar rats were pair fed either a Mg-deficient or a control diet. At day 7, lipopolysaccharide (LPS) induced no lethal effects in control rats but resulted in 70% mortality in Mg-deficient rats within 3 h. The vulnerability of Mg-deficient rats to LPS was associated with higher TNFα plasma values. Mg-deficient animals that received magnesium supplementation before endotoxin challenge had significantly increased survival. At day 2, control and Mg-deficient rats were also subjected to endotoxin challenge with or without magnesium pre-treatment. A significant increase in TNFα plasma level was observed in Mg-deficient rats compared to rats fed the control diet. Mg-deficient rats that received magnesium replacement therapy before endotoxin challenge had significantly lower TNFα plasma values than those receiving saline before endotoxin. Thus, the results of this experiment suggest that the activated or primed state of immune cells is an early event occurring in Mg deficiency.     

Inflammatory response following acute magnesium deficiency in the rat

The importance of inflammatory processes in the pathology of Mg deficiency has been recently reconsidered but the sequence of events leading to the inflammatory response remains unclear. Thus, the purpose of the present study was to characterize more precisely the acute phase response following Mg deficiency in the rat. Weaning male Wistar rats were pair-fed either a Mg-deficient or a control diet for either 4 or 8 days. The characteristic allergy-like crisis of Mg-deficient rats was accompanied by a blood leukocyte response and changes in leukocytes subpopulations. A significant increase in interleukin-6 (IL-6) plasma level was observed in Mg-deficient rats compared to rats fed a control diet. The inflammatory process was accompanied by an increase in plasma levels of acute phase proteins. The concentrations of alpha2-macroglobulin and alpha1-acid glycoprotein in the plasma of Mg-deficient rats were higher than in control rats. This was accompanied in the liver by an increase in the level of mRNA coding for these proteins. Moreover, Mg-deficient rats showed a significant increase in plasma fibrinogen and a significant decrease in albumin concentrations. Macrophages found in greater number in the peritoneal cavity of Mg-deficient rats were activated endogenously and appeared to be primed for superoxide production following phorbol myristate acetate stimulation. A high plasma level of IL-6 could be detected as early as day 4 for the Mg-deficient diet. Substance P does not appear to be the initiator of inflammation since IL-6 increase was observed without plasma elevation of this neuropeptide. The fact that the inflammatory response was an early consequence of Mg deficiency suggests that reduced extracellular Mg might be responsible for the activated state of immune cells.     

Magnesium deficiency in vitro enhances free radical-induced intracellular oxidation and cytotoxicity in endothelial cells.

The effect of magnesium (Mg)-deficient culture on endothelial cell susceptibility to oxidative stress was examined. Bovine endothelial cells were cultured in either control sufficient (0.8 mM) or deficient (0.4 mM) levels of MgCl2. Oxygen radicals were produced extracellularly by the addition of dihydroxyfumarate and Fe(3+)-ADP. Isolated Mg-deficient endothelial cells produced 2- to 3-fold higher levels of thiobarbituric acid (TBA)-reactive materials when incubated with this free radical system. Additional studies were performed using digitized video microscopy and 2',7'-dichlorofluorescein diacetate (DCFDA) as an intracellular indicator for oxidative events at the single cell level. In response to the exogenous oxidative stress, endothelial cells exhibited a time-dependent increase in fluorescence, suggestive of intracellular lipid peroxidation. The increase in cellular fluorescence began within 1 min of free radical addition; the Mg-deficient cells exhibited a more rapid increase in fluorescence than that of Mg-sufficient cells. In separate experiments, cellular viability was assessed using the Trypan blue exclusion assay. Mg deficiency increased cytotoxicity of the added oxyradicals, but the loss of cellular viability began to occur only after 15 min of free radical exposure, lagging behind the detection of intracellular oxidation products. These results suggest that increased oxidative endothelial cell injury may contribute to vascular injury during Mg deficiency.     

Enhancement of endotoxin-induced vascular hyporeactivity to phenylephrine in the thoracic aortas of Mg-deficient rats ex vivo

Since endotoxin lethality is enhanced by Mg deficiency in animals, we determined whether endotoxin-induced vascular hyporeactivity to phenylephrine (PE) is enhanced in Mg-deficient rats. Normal and Mg-deficient adult male Wistar rats were injected with Escherichia coli 011: B4 lipopolysaccharide (1 or 5 mg/kg, i.p.). Six h later, rings prepared from their thoracic aortas showed severe hyporeactivity to PE. This was more pronounced in the Mg-deficient rats, and was reversed by in vitro treatment with a highly selective inducible nitric oxide (NO) synthase inhibitor, 1400 W, or a highly selective soluble guanylyl cyclase inhibitor, ODQ. However, reversal required high doses of both inhibitors in Mg-deficient rats. Endotoxemia for 6 h was associated with elevated serum interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha levels, and strong TNF receptor mRNA expression in the abdominal aortas, which were significantly greater in the Mg-deficient rats. Treatment of the thoracic aortas, isolated from control and Mg-deficient rats before endotoxic challenge, with IL-1beta or TNF-alpha for 6 h in vitro caused hyporeactivity to PE, but its severity did not differ significantly between the two groups. These results suggest that high serum IL-1beta and TNF-alpha levels, and increased TNF receptor production in the vascular tissue, contribute to vascular hyporeactivity to PE in endotoxemia, and to its enhancement in Mg-deficient rats, via NO/cGMP signaling.     

Genome-wide transcriptional response of Yersinia pestis to stressful conditions simulating phagolysosomal environments

Yersinia pestis is a Gram-negative coccobacillus causing the dangerous disease, plague. Survival of Y. pestis within host macrophages is important in the initial stages of infection. In our present work, DNA microarray was used to determine the expression profiles of Y. pestis strain 201 in response to in vitro simulating conditions of Mg(2+) limitation, polymyxin treatment and oxidative stress that could be found in phagolysosomal environment. It was demonstrated that Y. pestis made appropriate adaptive/protective responses to survive the stressful environments. There are the induced expression of antiphagocytic factors and Mg(2+) transporters under Mg(2+) limitation condition, the stimulation of drug/analogue sensitivity and glycerol assimilation after polymyxin treatment, and the differential expression in genes encoding stress-responsive proteins, components of cell envelope, iron assimilation and regulatory functions in response to both Mg(2+) limitation and polymyxin treatment. Under oxidative stress, Y. pestis uses several mechanisms, especially including the induced expression of detoxification enzymes and DNA repair proteins, to protect from or repair the oxidative cell damages. This microarray analysis would provide the candidates for identifying genes or pathways required for growth and proliferation of Y. pestis in macrophages.     

The Effects of Magnesium Deficiency on Molybdenum Metabolism in Rats

Our previous report indicated that magnesium (Mg) deficiency increased molybdenum (Mo) concentration in the rat liver, suggesting the possibility that Mg deficiency affects Mo metabolism. Growing male rats were given a control diet or a Mg-deficient diet for 4 weeks. Urine and feces were collected during the second and fourth weeks of the feeding trial. The liver, kidney, spleen, skeletal muscle, and blood were collected at the end of the feeding trial. Mg deficiency did not affect the apparent absorption of Mo, but it reduced urinary excretion of Mo. The retention of Mo tended to be higher in the Mg-deficient group than in the control group. Hepatic Mo concentration was higher in the Mg-deficient group than in the control group, but Mg deficiency did not affect Mo concentration in other tissues and plasma. Mg deficiency downregulated the mRNA expression of Mo transporter 2 (MOT2) in the liver, but not in the kidney. These results suggest that Mg deficiency decreases urinary Mo excretion, which is too slight to affect plasma Mo concentration, and that Mg deficiency selectively disturbs the homeostatic mechanism of Mo in the liver, which is not related to the mRNA expression of MOT2 in the liver.     

Proteasome inhibition by lactacystin in primary neuronal cells induces both potentially neuroprotective and pro-apoptotic transcriptional responses: a microarray analysis

Although inhibition of the ubiquitin proteasome system has been postulated to play a key role in the pathogenesis of neurodegenerative diseases, studies have also shown that proteasome inhibition can induce increased expression of neuroprotective heat-shock proteins (HSPs). The global gene expression of primary neurons in response to treatment with the proteasome inhibitor lactacystin was studied to identify the widest range of possible pathways affected. Our results showed changes in mRNA abundance, both at different time points after lactacystin treatment and at different lactacystin concentrations. Genes that were differentially up-regulated at the early time point but not when most cells were undergoing apoptosis might be involved in an attempt to reverse proteasome inhibitor-mediated apoptosis and include HSP70, HSP22 and cell cycle inhibitors. The up-regulation of HSP70 and HSP22 appeared specific towards proteasome inhibitor-mediated cell death. Overexpression of HSP22 was found to protect against proteasome inhibitor-mediated loss of viability by up to 25%. Genes involved in oxidative stress and the inflammatory response were also up-regulated. These data suggest an initial neuroprotective pathway involving HSPs, antioxidants and cell cycle inhibitors, followed by a pro-apoptotic response possibly mediated by inflammation, oxidative stress and aberrant activation of cell cycle proteins.     

Role of magnesium in carbon partitioning and alleviating photooxidative damage

Magnesium (Mg) deficiency exerts a major influence on the partitioning of dry matter and carbohydrates between shoots and roots. One of the very early reactions of plants to Mg deficiency stress is the marked increase in the shoot-to-root dry weight ratio, which is associated with a massive accumulation of carbohydrates in source leaves, especially of sucrose and starch. These higher concentrations of carbohydrates in Mg-deficient leaves together with the accompanying increase in shoot-to-root dry weight ratio are indicative of a severe impairment in phloem export of photoassimilates from source leaves. Studies with common bean and sugar beet plants have shown that Mg plays a fundamental role in phloem loading of sucrose. At a very early stage of Mg deficiency, phloem export of sucrose is severely impaired, an effect that occurs before any noticeable changes in shoot growth, Chl concentration or photosynthetic activity. These findings suggest that accumulation of carbohydrates in Mg-deficient leaves is caused directly by Mg deficiency stress and not as a consequence of reduced sink activity. The role of Mg in the phloem-loading process seems to be specific; resupplying Mg for 12 or 24 h to Mg-deficient plants resulted in a very rapid recovery of sucrose export. It appears that the massive accumulation of carbohydrates and related impairment in photosynthetic CO2 fixation in Mg-deficient leaves cause an over-reduction in the photosynthetic electron transport chain that potentiates the generation of highly reactive O2 species (ROS). Plants respond to Mg deficiency stress by marked increases in antioxidative capacity of leaves, especially under high light intensity, suggesting that ROS generation is stimulated by Mg deficiency in chloroplasts. Accordingly, it has been found that Mg-deficient plants are very susceptible to high light intensity. Exposure of Mg-deficient plants to high light intensity rapidly induced leaf chlorosis and necrosis, an outcome that was effectively delayed by partial shading of the leaf blade, although the Mg concentrations in different parts of the leaf blade were unaffected by shading. The results indicate that photooxidative damage contributes to development of leaf chlorosis under Mg deficiency, suggesting that plants under high-light conditions have a higher physiological requirement for Mg. Maintenance of a high Mg nutritional status of plants is, thus, essential in the avoidance of ROS generation, which occurs at the expense of inhibited phloem export of sugars and impairment of CO2 fixation, particularly under high-light conditions.     

Magnesium deficiency in pregnant rats alters methylation of specific cytosines in the hepatic hydroxysteroid dehydrogenase-2 promoter of the offspring

Prenatal under-nutrition involves changes in the epigenetic regulation of specific genes. Maternal magnesium (Mg) deficiency affects maternal glucocorticoid metabolism, but the mechanisms underlying changes in glucocorticoid homeostasis of offspring are not well understood. In this study, we investigated the effects of feeding pregnant rats a Mg-deficient diet (0.003% magnesium) on the methylation of cytosine-guanine (CpG) dinucleotides in hepatic glucocorticoid genes of neonatal offspring, compared with controls (0.082% magnesium). Methylation of CpG dinucleotides in the peroxisome proliferator-activated receptor α (Ppara), glucocorticoid receptor (Nr3c1) and 11β-hydroxysteroid dehydrogenase-2 (Hsd11b2) promoters in the liver were measured by pyrosequencing. Quantitative real-time PCR was used to assess hepatic mRNA expression of each gene. Mean methylation of the Hsd11b2 promoter in the Mg-deficient offspring (33.2%) was higher than in controls (10.4%). This was due to a specific increase at CpG dinucleotides 1 (20.0% vs. control 10.1%), 2 (58.8% vs. 17.0%), 3 (29.7% vs. 6.2%) and 4 (38.7% vs. 8.8%) (p < 0.05). Ppara and Nr3c1 methylation status and expression did not differ between the groups. No significant difference was noted between male and female pups, which were equally represented. Therefore, a Mg-deficient diet alters glucocorticoid metabolism, predicting higher hepatic intracellular glucocorticoid concentrations, and is possibly a key mechanism that induces the metabolic complications of Mg deficiency.     

Changes in N-acetyl-beta-D-glucosaminidase activity in the urine and urinary albumin excretion in magnesium deficient rats

To discover the details of the effects of magnesium (Mg) deficiency on kidney function, the course of changes in N-acetyl-beta-D-glucosaminidase (NAG) activity in the urine and in urinary albumin excretion were examined in rats fed a Mg-deficient diet. NAG activity in the urine and urinary albumin excretion in rats fed the Mg-deficient diet significantly increased from 7 d until the end of the feeding period. We suggest that Mg-deficient diet rapidly induces kidney function insufficiency.     

Alleviation of Aflatoxin B1‐Induced Genomic Damage by Proanthocyanidins via Modulation of DNA Repair

In order to study the mechanisms underlying the alleviation of aflatoxin B1‐induced genomic damage by proanthocyanidins (PAs), we examined the modulation of oxidative DNA damage induced by aflatoxin B1 in PAs‐pretreated animals. The effects of PAs on changes in the expression of DNA damage and repair genes induced by aflatoxin B1 were also evaluated in rat marrow cells. Administration of PAs before aflatoxin B1 significantly mitigated aflatoxin B1‐induced oxidative DNA damage in a dose‐dependent manner. Aflatoxin B1 treatment induced significant alterations in the expression of specific DNA repair genes, and the pre‐treatment of rats with PAs ameliorated the altered expression of these genes. Conclusively, PAs protect against aflatoxin B1‐induced oxidative DNA damage in rats. These protective effects are attributed to the antioxidant effects of PA and enhanced DNA repair through modulation of DNA repair gene expression. Therefore, PAs are a promising chemoprotective agent for averting genotoxic risks associated with aflatoxin B1 exposure.     

The characterization of central neuromediation in rats fed with magnesium-deprived diet before and after magnesium replenishment

Magnesium (Mg) has been proposed to take part in biochemical dysregulation contributing to psychiatric disorders. The aims of this study was to estimate acute behavioural responses to clonidine (0.1 mg/kg i.p.), d-amphetamine (5 mg/kg, i.p), arecoline (15 mg/kg i.p), nicotine (6 mg/kg i.p.), apomorphine (1.5 mg/kg i.p.) and L-5-hydroxytryptophan (300 mg/kg i.p.) in rats fed with Mg-deprivated diet for 49 days and then treated with organic and inorganic Mg salts (50 mg Mg per kg) ether alone or in combination with pyridoxine (5 mg vitamin B6 per kg). In our study Mg-deficient rats were more sensitive to d-amphetamine-induced motor stereotypes compared with control rats; time of onset of the stereotypies insignificantly decreased by 14.89% and duration of the stereotypies significantly increased by 19.44% (320.36 +/- 19.90 vs. 268.23 +/- 8.17 minutes; p = 0.043). Mg deficiency did not modulate sensitivity to nicotine-induced seizure. The time between nicotine injection and emergence of clonic seizure (seizure latency) in the controls and Mg-deficient rats were 0.80 +/- 0.26 and 0.96 +/- 0.21 minutes respectively. Duration of the seizures in the controls and Mg-deficient rats were 64.93 +/- 7.20 and 79.32 +/- 8.13 minutes. In our study, Mg deficiency did not affect on clonidine- and apomorphine-induced hypothermia. Clonidine produced similar decreases in rectal temperature in controls and Mg-deficient group. In experiments using apomorphine, values of hypothermia were similar to those observed with clonidine. Mg deficiency antagonized 5-hydroxytryptophan-induced head-twitch response. The number of head twitches produced by 5-hydroxytryptophan was significantly (p = 0.49) decreased: twofold in magnesium-deficient rats (1.23 +/- 0.44 per minute) as compared with controls (2.42 +/- 0.52 per minute). Arecoline-induced tremor was comparably less expressed in Mg-deficient rats than in controls. The time between arecoline injection and time of onset of the tremor in the controls and Mg-deficient rats were 92.75 +/- 19.35 and 245.17 +/- 121.86 seconds respectively (p < or = 0.035). Duration of the tremors in the controls and Mg-deficient rats were 1175.58 +/- 127.87 and 703.83 +/- 89.33 seconds (p = 0.015). Magnesium salts (Mg chloride, Mg L-aspartate alone and in combination with B6) were administered through gastric tube during twenty days up to complete compensation oferythrocyte and plasma Mg levels in all experimental groups. In our study administration of Mg salts resulted in normalization of acute behavioural responses in Mg-deficient rats to d-amphetamine, arecoline, and L-5-hydroxytryptophan. Behavioural responses in rats treated with both Mg chloride and Mg L-aspartate in combinations with B6 were comparable with those observed in MagneB6 treatment.     

Impact of seasonal variation on HSP70 expression quantitated in stressed fish hepatocytes

Heat shock protein 70 (HSP70) is an effective molecular chaperone, playing a role in cell protection from damage in response to stress stimuli. Here, we report the impact of environmental stress on hepatocyte HSP70 expression in Mugil cephalus living in either a contaminated (Ennore) or uncontaminated (Kovalam) estuary over the course of two seasons. Oxidative and nitrative stress was determined along with quantification of HSP70 by enzyme-linked immunosorbent assay (ELISA) after electroelution from polyacrylamide gels. Fish from Ennore showed significantly higher levels of oxidative and nitrative stress and HSP70 expression than fish from Kovalam. Also, there was significant seasonal variation in all oxidative, nitrative stress marker levels and HSP70 expression which peaked during summer. These results provide direct evidence that HSP70 overexpression in fish hepatocytes under stress may aid cell survival by protecting against oxidative and nitrative stress-induced changes. In addition, seasonal variation may have a significant impact on HSP70 expression.     

Depressed antioxidant defense in rat heart in experimental magnesium deficiency implications for the pathogenesis of myocardial lesions

Magnesium (Mg) deficiency has been shown to produce myocardial lesions in different experimental models. Based on several lines of evidence, it has been proposed that oxidative injury to the cardiac muscle may explain the pathobiology of such lesions. In pursuance of this postulation, the present study examined the effect of dietary deficiency of Mg on the activity of the antioxidant enzymes, Superoxide dismutase (SOD) and catalase, in rat heart. This article reports a significant lowering of the activity of both these enzymes in the cardiac tissue in Mg-deficient rats. Since depressed antioxidant defense in the heart may enhance myocardial susceptibility to oxidative injury, the observation is of possible relevance to the pathogenesis of cardiac lesions in Mg deficiency.