Anaerobic rat heart: mitochondrial role in calcium uptake and contractility.

In order to evaluate the manner by which fumarate enhances contractility in the anaerobic heart, we examined Ca++ movements in isolated heart mitochondria and in the isolated perfused heart. Our experiments showed that in isolated antimycin A plus cyanide treated mitochondria: (a) Ca++ uptake was promoted by electron transport generated by fumarate-dependent oxidation of NADH, (b) Ca++ stimulated fumarate-dependent oxidation of NADH, (c) the ratio of Ca++ uptake:NADH oxidized was 1.7 and (d) the Ca++ sequestered is transiently highly mobile and is rapidly released upon collapse of the membrane potential. In anaerobic hearts perfused with glucose plus fumarate, malate and glutamate Ca++ levels were the same as those in oxygenated hearts while in anaerobic organs perfused with or without glucose Ca++ content was appreciably lower. Succinate production in anaerobic heart perfusions was related to: (a) an increased retention of Ca++ by the heart, (b) a diminution in peak aortic pressure generated by cardiac contractions and (c) an increase in heart rate. The information obtained indicates that mitochondria have a capability for Ca++ movement which be used physiologically, particularly in fumarate perfused anaerobic hearts, to assist the mechanism for contraction and relaxation of the heart.     

Neuroprotection against CCl4 induced brain damage with crocin in Wistar rats

Owing to its lipophilic property, carbon tetrachloride (CCl₄) is rapidly absorbed by both the liver and brain. We investigated the protective effects of crocin against brain damage caused by CCl₄. Fifty rats were divided into five groups of ten: control, corn oil, crocin, CCl₄ and CCl₄ + crocin. CCl₄ administration decreased glutathione (GSH) and total antioxidant status (TAS) levels, and catalase (CAT) activity, while significant increases were observed in malondialdehyde (MDA) and total oxidant status (TOS) levels and superoxide dismutase (SOD) activity. The cerebral cortex nuclear lamina developed a spongy appearance, neuronal degeneration was observed in the hippocampus, and heterochromatic and pyknotic neurons with increased cytoplasmic eosinophilia were observed in the hippocampus after CCl₄ treatment. Because crocin exhibits strong antioxidant properties, crocin treatment increased GSH and TAS levels and CAT activities, and decreased MDA and TOS levels and SOD activity; significant improvements also were observed in histologic architecture. We found that crocin administration nearly eliminated CCl₄ induced brain damage by preventing oxidative stress.     

A critical role for IkappaB kinase beta in metallothionein-1 expression and protection against arsenic toxicity

Arsenic is a widespread environmental toxic agent that has been shown to cause diverse tissue and cell damage and at the same time to be an effective anti-cancer therapeutic agent. The objective of this study is to explore the signaling mechanisms involved in arsenic toxicity. We show that the IkappaB kinase beta (IKKbeta) plays a crucial role in protecting cells from arsenic toxicity. Ikkbeta(-)(/)(-) mouse 3T3 fibroblasts have decreased expression of antioxidant genes, such as metallothionein 1 (Mt1). In contrast to wild type and IKKbeta-reconstituted Ikkbeta(-)(/)(-) cells, IKKbeta-null cells display a marked increase in arsenic-induced reactive oxygen species (ROS) accumulation, which leads to activation of the MKK4-c-Jun NH(2)-terminal kinase (JNK) pathway, c-Jun phosphorylation, and apoptosis. Pretreatment with the antioxidant N-acetylcysteine (NAC) and expression of MT1 in the Ikkbeta(-)(/)(-) cells prevented JNK activation; moreover, NAC pretreatment, MT1 expression, MKK4 ablation, and JNK inhibition all protected cells from death induced by arsenic. Our data show that two signaling pathways appear to be important for modulating arsenic toxicity. First, the IKK-NF-kappaB pathway is crucial for maintaining cellular metallothionein-1 levels to counteract ROS accumulation, and second, when this pathway fails, excessive ROS leads to activation of the MKK4-JNK pathway, resulting in apoptosis.     

Genetically altered mice to evaluate glutathione homeostasis in health and disease

The tripeptide glutathione (GSH) is part of an integrated antioxidant system that protects cells and tissues from oxidative damage. Oxidative stress can result from exposure to excessive amounts of endogenous and exogenous electrophiles. Until recently, animal and cell model systems used to investigate the role of GSH in disease processes had employed chemical agents that deplete cellular GSH by inhibiting GSH synthesis or by reacting chemically with GSH. Such models have proven useful, but questions concerning nonspecific effects of such chemicals remain. Recently, our laboratories and others have developed mouse models with genetic deficiencies in enzymes of the GSH biosynthetic pathway. This review focuses on the regulation of GSH homeostasis and, specifically, the new GSH-deficient mouse models that have been developed. These models will improve our understanding of the role of GSH in animal and human diseases.     

Initial characterization of the glutamate-cysteine ligase modifier subunit Gclm(-/-) knockout mouse. Novel model system for a severely compromised oxidative stress response

Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the GSH biosynthesis pathway. In higher eukaryotes, this enzyme is a heterodimer comprising a catalytic subunit (GCLC) and a modifier subunit (GCLM), which change the catalytic characteristics of the holoenzyme. To define the cellular function of GCLM, we disrupted the mouse Gclm gene to create a null allele. Gclm(-/-) mice are viable and fertile and have no overt phenotype. In liver, lung, pancreas, erythrocytes, and plasma, however, GSH levels in Gclm(-/-) mice were 9-16% of that in Gclm(+/+) littermates. Cysteine levels in Gclm(-/-) mice were 9, 35, and 40% of that in Gclm(+/+) mice in kidney, pancreas, and plasma, respectively, but remained unchanged in the liver and erythrocytes. Comparing the hepatic GCL holoenzyme with GCLC in the genetic absence of GCLM, we found the latter had an approximately 2-fold increase in K(m) for glutamate and a dramatically enhanced sensitivity to GSH inhibition. The major decrease in GSH, combined with diminished GCL activity, rendered Gclm(-/-) fetal fibroblasts strikingly more sensitive to chemical oxidants such as H(2)O(2). We conclude that the Gclm(-/-) mouse represents a model of chronic GSH depletion that will be very useful in evaluating the role of the GCLM subunit and GSH in numerous pathophysiological conditions as well as in environmental toxicity associated with oxidant insult.     

Glutathione protects chemokine-scavenging and antioxidative defense functions in human RBCs

Oxidant stress, in vivo or in vitro, isknown to induce oxidative changes in human red blood cells (RBCs). Ourobjective was to examine the effect of augmenting RBC glutathione(GSH) synthesis on 1) degenerative protein loss and2) RBC chemokine- and free radical-scavenging functions inthe oxidatively stressed human RBCs by using banked RBCs as a model.Packed RBCs were stored up to 84 days at 1-6°C in Adsol or inthe experimental additive solution (Adsol fortified with glutamine,glycine, and N-acetyl-L-cysteine). Supplementingthe conventional additive with GSH precursor amino acids improved RBCGSH synthesis and maintenance. The rise in RBC -glutamylcysteineligase activity was directly proportional to the GSH content andinversely proportional to extracellular homocysteine concentration,methemoglobin formation, and losses of the RBC proteins band 3, band4.1, band 4.2, glyceraldehyde-3-phosphate dehydrogenase, and Duffyantigen (P < 0.01). Reduced loss of Duffy antigencorrelated well with a decrease in chemokine RANTES (regulated uponactivation, normal T-cell expressed, and secreted) concentration. Weconclude that the concomitant loss of GSH and proteins in oxidatively stressed RBCs can compromise RBC scavenging function. Upregulating GSHsynthesis can protect RBC scavenging (free radical and chemokine) function. These results have implications not only in a transfusion setting but also in conditions like diabetes and sickle cell anemia, inwhich RBCs are subjected to chronic/acute oxidant stresses.

     

Benzo[a]pyrene-induced toxicity: paradoxical protection in Cyp1a1(-/-) knockout mice having increased hepatic BaP-DNA adduct levels.

Previous studies have shown that cytochrome P450 1A1 (CYP1A1), CYP1B1, and prostaglandin-endoperoxide synthase (PTGS2) are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), and all three metabolize BaP to reactive DNA-binding intermediates and excreted products. Because these three enzymes show differing patterns of basal levels, inducibility, and tissue-specific expression, animal studies are necessary to delineate the role of CYP1A1 in BaP-mediated toxicity. In mice receiving large daily doses of BaP (500 mg/kg i.p.), Cyp1a1(-/-) knockout mice are protected by surviving longer than Cyp1a1(+/-) heterozygotes. We found that a single 500 mg/kg dose of BaP induces hepatic CYP1A1 mRNA, protein, and enzyme activity in Cyp1a1(+/-) but not in Cyp1a1(-/-) mice; TCDD pretreatment increases further the CYP1A1 in Cyp1a1(+/-) but not Cyp1a1(-/-) mice. Although a single 500 mg/kg dose of BaP was toxic to Cyp1a1(+/-) mice (serum liver enzyme elevated about 2-fold above control levels at 48 h), Cyp1a1(-/-) mice displayed no hepatotoxicity. Unexpectedly, we found 4-fold higher BaP-DNA adduct levels in Cyp1a1(-/-) than in Cyp1a1(+/-) mice; TCDD pretreatment lowered the levels of BaP-DNA adducts in both genotypes, suggesting the involvement of other TCDD-inducible detoxification enzymes. BaP was cleared from the blood much faster in Cyp1a1(+/-) than Cyp1a1(-/-) mice. Our results suggest that absence of the CYP1A1 enzyme protects the intact animal from BaP-mediated liver toxicity and death, by decreasing the formation of large amounts of toxic metabolites, whereas much slower metabolic clearance of BaP in Cyp1a1(-/-) mice leads to greater formation of BaP-DNA adducts.     

Histopathological effects of cisplatin,doxorubicin and 5-flurouracil (5-FU) on the liver of male albino rats

Cisplatin, doxorubicin and fluorouracil (5-FU), drugs belonging to different chemical classes, have been extensively used for chemotherapy of various cancers. Despite extensive investigations into their hepatotoxicity, there is very limited information on their effects on the structure and ultra-structure of liver cells in vivo. Here, we demonstrate for the first time, the effects of these three anticancer drugs on rat liver toxicity using both light and electron microscopy. Light microscopic observations revealed that higher doses of cisplatin and doxorubicin caused massive hepatotoxicity compared to 5-FU treatment, including dissolution of hepatic cords, focal inflammation and necrotic tissues. Interestingly, low doses also exhibited abnormal changes, including periportal fibrosis, degeneration of hepatic cords and increased apoptosis. These changes were confirmed at ultrastructural level, including vesiculated rough endoplasmic reticulum and atrophied mitochondria with ill-differentiated cisternae, dense collection of macrophages and lymphocytes as well as fibrocytes with collagenous fibrils manifesting early sign of fibrosis, especially in response to cisplatin and doxorubicin -treatment. Our results provide in vivo evidence, at ultrastructural level, of direct hepatotoxicity caused by cisplatin, doxorubicin and 5-FU at both light and electron microscopi. These results can guide the design of appropriate treatment regimen to reduce the hepatotoxic effects of these anticancer drugs.     

Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen

Background  

Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MS^E was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome.