Modulation of the Tight Junctions of the Caco-2 Cell Monolayers by H2-antagonists

Purpose. The tight junctions in the intestinal epithelium represent highly specialized intercellular junctions. Ranitidine, an H₂-antagonist, causes a tightening of the tight junctions. Hence, we have investigated the effect of ranitidine and other H₂-antagonists on the function of the intestinal tight junctions. Methods. Effect of the H₂-antagonists on the tight junctions has been investigated using the transepithelial electrical resistance (TEER) and the transport of mannitol across the Caco-2 cell monolayers. Results. Four different H₂-antagonists caused an increase in the TEER across the Caco-2 cell monolayers, accompanied by a decrease in the permeability for mannitol. The effect was concentration-dependent and saturable. Ranitidine and famotidine, caused a decrease in their own transport rate across the Caco-2 cells. Ranitidine competitively inhibited the increase in TEER caused by famotidine, whereas compounds which represent molecular fragments of ranitidine had no effect. The relative potency of the four H₂-antagonists in causing an increase in the TEER correlated inversely with the oral bioavailability of these compounds in humans. Conclusions. We hypothesize that the H₂-antagonists exert their effect on the tight junctions of Caco-2 cells by modulation of interactions among proteins associated with the tight junctional complex.     

Interaction between flurbiprofen and H2-antagonists in rheumatoid arthritis

The non-steroidal anti-inflammatory drug, flurbiprofen, is metabolized by mixed function oxidases. As the H₂-antagonist cimetidine can inhibit the metabolism of some drugs which are metabolized by mixed function oxidases, it was decided to test whether this is also the case for flurbiprofen. Thirty patients with rheumatoid arthritis who were taking flurbiprofen (100 mg tid or 50 mg tid) for 2 weeks were randomly selected and given, for another 2 weeks in addition either cimetidine (300 mg tid) or ranitidine (150 mg bid). An 8 h study of flurbiprofen kinetics (blood sampling at 0, 0.5, 1, 2, 3, 4, 6 and 8 hours) was carried out before the co-administration of the H₂-antagonists and again after 2 weeks of combined treatment. Clinical indices, including Ritchie Index, 50-foot walking time, grip strength and global scores were also assessed on the days of the kinetic studies. Cimetidine increased AUC by 19% (p=0.07), steady-state trough concentrations by 43% (p=0.06) and C_max by 26% (p=0.03). Ranitidine increased AUC by 14% (p=0.03) and trough concentrations by 55% (p=0.001). C_max was not significantly changed (2%;p=0.83). Neither cimetidine nor ranitidine altered the efficacy of flurbiprofen in rheumatoid arthritis.     

A new class of NO-donor H3-antagonists

Synthesis and pharmacological characterisation of a series of compounds obtained by joining, through appropriate spacers, NO-donor furoxan and nitrooxy moieties to the imidazole ring, as well as their structurally related analogues devoid of NO-donating properties are described. All the products were studied for their capacity to interact with H3-receptors present on the guinea-pig ileum and with H2-receptors present on guinea-pig right atrium. The whole series of products displayed reversible H3-antagonistic activity. No activity on H2-receptors was observed when the products were tested at 10 microM concentration. Many of the products were also able to induce partial relaxation when added to the bath after electrical contraction of the guinea-pig ileum during the study of their H3-antagonism. This phenomenon seems to be dependent on various factors; for some compounds it proved to be dependent on NO-mediated sGC activation, for other products it could be due to their weak M3-antagonism. The investigation of the lipophilic-hydrophilic balance of all the products indicates, for many of them, an ideal value to cross the blood-brain barrier.     

In praise of H2O2, the versatile ROS,and its vanadium complexes

Hydrogen peroxide (H₂O₂) is generated in mitochondria in aerobic cells as a minor product of electron transport, is inhibited selectively by phenolic acids (in animals) or salicylhydroxamate (in plants) and is regulated by hormones and environmental conditions. Failure to detect this activity is due to presence of H₂O₂-consuming reactions or inhibitors present in the reaction mixture. H₂O₂ has a role in metabolic regulation and signal transduction reactions. A number of enzymes and cellular activities are modified, mostly by oxidizing the protein-thiol groups, on adding H₂O₂ in mM concentrations. On complexing with vanadate, also occurring in traces, H₂O₂ forms diperoxovanadate (DPV), stable at physiological pH and resistant to degradation by catalase. DPV was found to substitute for H₂O₂ at concentrations orders of magnitude lower, and in presence of catalase, as a substrate for user reaction, horseradish peroxidase (HRP), and in inactivating glyceraldehyde-3-phosphate dehydrogenase. superoxide dismutase (SOD) -sensitive oxidation of NADH was found to operate as peroxovanadate cycle using traces of DPV and decameric vanadate (V₁₀) and reduces O₂ to peroxide (DPV in presence of free vanadate). This offers a model for respiratory burst. Diperoxovanadate reproduces several actions of H₂O₂ at low concentrations: enhances protein tyrosine phosphorylation, activates phospholipase D, produces smooth muscle contraction, and accelerates stress induced premature senescence (SIPS) and rounding in fibroblasts. Peroxovanadates can be useful tools in the studies on H₂O₂ in cellular activities and regulation.     

Carrier-Mediated Transport of H1-Antagonist at the Blood-Brain Barrier: A Common Transport System of H1-Antagonists and Lipophilic Basic Drugs

The blood-brain barrier (BBB) transport system for H₁ antagonists was studied using primary cultured bovine brain capillary endothelial cells (BCEC). The uptake of [³H]mepyramine was inhibited by various H₁-antagonists. Ketotifen competitively inhibited [³H]mepyramine uptake with an inhibition constant (K_i ) of 46.8 µM. Lipophilic basic drugs such as propranolol, lidocaine and imipramine significantly inhibited [³H]mepyramine uptake. In particular, propranolol inhibited [³H]mepyramine uptake competitively at an inhibition constant (K_i) of 51.1 µM. Moreover, in ATP-depleted BCEC, [³H]mepyramine uptake was stimulated by preloading with H₁- antagonists and lipophilic basic drugs. These results indicated that H₁-antagonists are transported across the BBB via a carrier-mediated transport system common to lipophilic basic drugs.     

Effect of the H2-Receptor Antagonist Cimetidine,on the Pharmacokinetics and Pharmacodynamics of the H1-Receptor Antagonists Hydroxyzine and Cetirizine in Rabbits

The effects of coadministration of the H₂-receptor antagonist cimetidine on the pharmacokinetics and pharmacodynamics of the H₁ receptor antagonists hydroxyzine and cetirizine were studied in rabbits. A single dose of hydroxyzine, 10 mg (Experiment A), or cetirizine, 10 mg (Experiment B), was given intravenously on three occasions: 2 weeks before cimetidine administration, after cimetidine, 100 mg/kg, had been given every 12 hr for 1 week, and 2 weeks after the cimetidine was discontinued. Serum concentrations of hydroxyzine and cetirizine, the active metabolite of hydroxyzine arising in vivo (Experiment A), or cetirizine (Experiment B) were measured by HPLC. The pharmacologic effects of hydroxyzine and cetirizine were monitored by measuring the suppression of histamine-induced wheals, using an IBM-PC and digitizer. The hydroxyzine and cetrizine half-life and AUG₀ values were significantly increased and the systemic clearance rates were significantly decreased in the presence of cimetidine. Similar results were obtained when cetirizine was administered de novo. Wheal suppression produced by hydroxyzine or cetirizine was increased and prolonged in the presence of cimetidine. The synergism observed between hydroxyzine or cetirizine and cimetidine in suppression of the histamine-induced cutaneous response may be due to a pharmacokinetic interaction.     

Novel potential nonsedating H1 antagonists related to the gauche rotamer of PROS-NH-histamine

On the basis of the most stable stereorotameric (R) forms of ^πNH-histamine (2), the trans (1-TR) and gauche (1-GR) forms have both been reported to be involved in potentiation of H₁-receptors. Apart from the known classic models of H₁-antagonists that mostly belong to 1-TR, a new topographic receptor map for 1-GR has been postulated. Twenty-seven new compounds pertaining to novel nonclassic molecular models related to 1-GR have been postulated as potential nonsedating, less toxic H₁-antagonists. Representative members of the new agents were investigated for H₁-blocking activity by using isolated segments from guinea pig ileum. Many of the tested new compounds exhibited activities comparable to that of acrivastine as a reference nonsedating drug. The C log P values of the new agents were lower than that of acrivastine (4.34), which might indicate decreased tendency to cross the blood–brain barrier. The most pronounced activity was displayed by the 5-substituted aminomethylenepyrimidine-2,4,6-triones (21, 23) since they displayed nearly equal 50% inhibition concentrations (IC₅₀) (6.12 × 10⁻⁶ M) and lower C log P values.     

From the H2 receptor gene to reclassification of the H2 receptor antagonists

From previous studies it is known that long-term stimulation of the histamine H₂ receptor results in receptor downregulation. Two different pathways are involved in the downregulation process of the H₂ receptor: a cAMP-dependent and cAMP-independent agonist-dependent pathway. Recently, it became evident that in the absence of an agonist the H₂ receptor expressed in CHO cells already stimulate cAMP production, also referred to as spontaneous activity. The spontaneous activity can be inhibited by several H₂ antagonists, previously thought to act as competitive antagonists, and these antagonists are referred to as inverse agonists. Some antagonists, e.g. burimamide, are not able to inhibit the spontaneous activity and are referred to as neutral antagonists. Inverse agonism appears to be the mechanistic basis of upregulation. Only inverse agonists and not neutral antagonists induce receptor upregulation after long-term treatment as these compounds inhibit the spontaneous receptor activity and thus the basal receptor downregulation. Moreover it might also explain previously reported observations after long-term treatment of gastric ulcers, such as intragastric hyperacidity.     

Comparison of the effects of prostaglandins E2 and I2 on testicular nociceptor activities studied in vitro

Summary Effects of exogenous prostaglandin (PG) E₂ and PGI₂ on testicular polymodal receptor activities were compared in in vitro recordings of single- or multi-fiber discharges from canine testis-spermatic nerve preparations. PGI₂ up to 1.4×10^–6 mol/l (cumulative method) or 1.0×10^–5 mol/l (non-cumulative method) excited only weakly some of the receptors, and similar observations were made with PGE2. Both PGs applied cumulatively or non-cumulatively at concentrations above 1.4×10^–8 mol/l augmented the response to bradykinin (9.4×10^–8 mol/l) in more than half of the cases tested. The augmenting effect of PGE₂ lasted longer than that of PGI₂ both with the cumulative and the non-cumulative method. The degree of augmentation tended to increase dependent on concentration, but some cases showed no further increase or rather a decrease in augmentation by PGs at a ten times higher concentration, especially when PGs were applied cumulatively. A second challenge by PG after a short interval (2 min) did not induce augmentation. These phenomena were considered to be tachyphylaxis to PGs. Cross-tachyphylaxis to PGE₂ and PGI₂ was also observed. There was not much difference in excitatory and augmenting potencies between these two PGs, but there was a clear difference in the concentrations of the PGs necessary to induce excitation of polymodal receptors and to facilitate their response to bradykinin. Send offprint requests to T. Kumazawa at the above address     

Humanized-Single Domain Antibodies (VH/V(H)H) that Bound Specifically to Naja kaouthia Phospholipase A2 and Neutralized the Enzymatic Activity

Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA(2)). The PLA(2) exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/V(H)H) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V(H)H phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-V(H)H, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V(H)H purified from the E. coli homogenates neutralized PLA(2) enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/V(H)H covered the areas around the PLA(2) catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V(H)H would ameliorate/abrogate the principal toxicity of the venom PLA(2) (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.     

In Vitro Antiplasmodial Activity of Phospholipases A2 and a Phospholipase Homologue Isolated from the Venom of the Snake Bothrops asper

The antimicrobial and antiparasite activity of phospholipase A₂ (PLA₂) from snakes and bees has been extensively explored. We studied the antiplasmodial effect of the whole venom of the snake Bothrops asper and of two fractions purified by ion-exchange chromatography: one containing catalytically-active phospholipases A₂ (PLA₂) (fraction V) and another containing a PLA₂ homologue devoid of enzymatic activity (fraction VI). The antiplasmodial effect was assessed on in vitro cultures of Plasmodium falciparum. The whole venom of B. asper, as well as its fractions V and VI, were active against the parasite at 0.13 ± 0.01 µg/mL, 1.42 ± 0.56 µg/mL and 22.89 ± 1.22 µg/mL, respectively. Differences in the cytotoxic activity on peripheral blood mononuclear cells between the whole venom and fractions V and VI were observed, fraction V showing higher toxicity than total venom and fraction VI. Regarding toxicity in mice, the whole venom showed the highest lethal effect in comparison to fractions V and VI. These results suggest that B. asper PLA₂ and its homologue have antiplasmodial potential.     

Proconvulsive effects of histamine H1-antagonists on electrically-induced seizure in developing mice

The purpose of this study was to investigate the developmental differences in seizure susceptibility in mice and the roles of the histaminergic neuron system in inhibition of convulsions in development. First, we studied developmental differences in electrically-induced seizures. Since the 14-day-old mice showed a different seizure pattern from that of older mice, we evaluated the seizure susceptibility of mice older than 21 days. The durations of all the convulsive phases were significantly increased in 21- and 30-day-old mice, compared with older mice. Second, pyrilamine (or mepyramine), ketotifen, andd-chlorpheniramine, centrally-acting H₁-antagonists, increased the durations of all the convulsive phases in the 21- and 30-day-old mice, but did not increase duration in 42-day-old mice. Terfenadine and astemizole, H₁-antagonists that hardly enter the brain, did not affect the durations of all the convulsive phases in 21-, 30- and 42-day-old mice. The proconvulsant effect of centrally-acting H₁-antagonists in 21- and 30-day-old mice were considered to be mediated via the central H₁-receptors. Thus, the histaminergic neuron system may have an important physiological role in inhibition of seizures in 21- and 30-day-old mice which have higher seizure susceptibility. This would compensate for the immaturity of the other protective neuron systems such as NMDA receptor complexes and GABA receptors. In conclusion, the present findings support the view that the central histaminergic system plays a role in inhibition of convulsions.     

Histamine H1- and H2-receptors in the gastrointestinal circulation

Summary Evidence for the presence of specific histamine H₁- and H₂-receptors in the gastrointestinal circulation was obtained using histamine, 2-methylhistamine (a specific H₁-agonist), 4-methylhistamine (a specific H₂-agonist), and selective H₁- and H₂-receptor antagonists in the anesthetized dog. Histamine and 2-methylhistamine increased conductance in the vascular beds of the superior mesenteric artery, the left gastric artery and the common hepatic artery, whereas 4-methylhistamine mainly enhanced conductance in the vascular beds of the left gastric artery and the common hepatic artery. All three agents depressed systemic arterial blood pressure. The vasodilatory effect of histamine and 2-methylhistamine on the superior mesenteric artery bed occurred earlier and was of shorter duration than their effect on the two other vessels. The H₁-receptor antagonists mepyramine and clemastine blocked the response of the superior mesenteric artery bed to histamine, but had a lesser inhibitory effect on the histamine response of the common hepatic artery and the left gastric artery. The addition of the H₂-receptor antagonist cimetidine to mepyramine blockade augmented the inhibiting effect of mepyramine on the common hepatic artery. Cimetidine bolus injections prevented enhancement of vascular conductance by 4-methylhistamine, but did not influence conductance enhancement by histamine or 2-methylhistamine. These data demonstrate there are separable histamine H₁- and H₂-receptors in the gastrointestinal circulation which are distinguished by anatomic location, temporal relationships of receptor response, and response to specific histamine H₁- and H₂-agonists and antagonists.     

Identification of endothelial H1, vascular H2 and cardiac presynaptic H3 receptors in the pithed rat

Summary In pithed and vagotomized rats the effects of the H₃ receptor agonist R-(–)--methylhistamine, the H₁ receptor agonist 2-(2-thiazolyl)ethylamine and the H₂ receptor agonist dimaprit on basal diastolic blood pressure, basal heart rate and the electrically induced rise in heart rate were examined.Basal diastolic blood pressure was not altered by low, but increased by high doses of R-(–)--methylhistamine; the latter effect was not affected by selective H₁, H₂ or H₃ receptor antagonists and by prazosin, but was attenuated by rauwolscine. Rauwolscine also unmasked a vasodepressor response to R-(–)--methylhistamine not affected by the H₃ receptor antagonist thioperamide, but counteracted by the H₁ receptor antagonist dimetindene or the H₂ receptor antagonist ranitidine. The vasodepressor responses to 2-(2-thiazolyl)ethylamine and dimaprit were antagonized by dimetindene and ranitidine, respectively. The vasodepressor response to 2-(2-thiazolyl)ethylamine was not altered by indomethacin, but reduced by an inhibitor of endothelial nitric oxide synthase, N-nitro-L-arginine methyl ester (which, by itself, markedly increased blood pressure). Both drug tools did not alter the effect of dimaprit. Basal heart rate was not affected by 2-(2-thiazolyl)ethylamine (examined after administration of propranolol), dimaprit and R-(–)--methylhistamine. The electrically induced increase in heart rate (studied in animals which had received rauwolscine) was decreased by R-(–)--methylhistamine but not affected by 2-(2-thiazolyl)ethylamine and dimaprit. The effect of R-(–)--methylhistamine was abolished by thioperamide. R-(–)--methylhistamine did not influence the increase in heart rate produced by isoprenaline.In conclusion, the pithed rat offers the opportunity to study cardiac presynaptic H₃ receptors, endothelial H₁ receptors and vascular H₂ receptors in the same experimental model. Cardiac presynaptic H₁ and H₂ receptors as well as postsynaptic H₃ receptors in the heart and in the resistance vessels were not found. R-(–)--methylhistamine is a weak agonist at ₂, H₁ and H₂ receptors.Correspondence to E. Schlicker at the above address     

Antioxidant ameliorating effects against H2O2-induced cytotoxicity in primary endometrial cells

Oxidative stress and a disrupted antioxidant system are involved in a variety of pregnancy complications. In the present study, the role of vitamin E (Vit E) and folate as radical scavengers on the GSH homeostasis in stress oxidative induced in rat endometrial cells was investigated. Primary endometrial stromal cell cultures treated with 50 and 200?µM of H₂O₂ and evaluated the cytoprotective effects of Vit E (5?µM) and folate (0.01?µM) in H₂O₂-treated cells for 24?h. Following the exposure of endometrial cells to H₂O₂ alone and in the presence of Vit E and/or folate, cell survival, glutathione peroxidase (GPx) and glutathione reductase activities and the level of reduced glutathione (GSH) were measured. Cell adhesions comprise of cell attachment and spreading on collagen were determined. Flow cytometric analysis using annexin V was used to measure apoptosis. H₂O₂ treatment showed a marked decrease in cell viability, GPx and GR activities and the level of GSH. Although Vit E or folate had some protective effect, combination therapy with Vit E and folate attenuated all the changes due to H₂O₂ toxicity. An increasing number of alive cells was showed in the cells exposed to H₂O₂ (50?µM) accompanied by co-treatment with Vit E and folic acid. The present findings indicate that co-administration of Vit E and folate before and during pregnancy may maintain a viable pregnancy and contribute to its clinical efficacy for the treatment of some idiopathic infertility.     

Involvement of Thromboxane A2 in the Modulation of Pacemaker Activity of Interstitial Cells of Cajal of Mouse Intestine

Although many studies show that thromboxane A₂ (TXA₂) has the action of gastrointestinal (GI) motility using GI muscle cells and tissue, there are no reports on the effects of TXA₂ on interstitial cells of Cajal (ICC) that function as pacemaker cells in GI tract. So, we studied the modulation of pacemaker activities by TXA₂ in ICC with whole cell patch-clamp technique. Externally applied TXA₂ (5µM) produced membrane depolarization in current-clamp mode and increased tonic inward pacemaker currents in voltage-clamp mode. The tonic inward currents by TXA₂ were inhibited by intracellular application of GDP-β-S. The pretreatment of ICC with Ca²⁺ free solution and thapsigargin, a Ca²⁺-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the TXA₂-induced tonic inward currents. However, chelerythrine or calphostin C, protein kinase C inhibitors, did not block the TXA₂-induced effects on pacemaker currents. These results suggest that TXA₂ can regulate intestinal motility through the modulation of ICC pacemaker activities. This modulation of pacemaker activities by TXA₂ may occur by the activation of G protein and PKC independent pathway via extra and intracellular Ca²⁺ modulation.     

Regulation of sterol synthesis by histamine in human mononuclear leukocytes: Roles of H1- and H2-receptors

Summary The effect of histamine on sterol synthesis has been investigated in freshly isolated human mononuclear leukocytes from healthy subjects.Incubation of cells for 6 h in a medium containing lipid depleted serum led to a threefold increase in the incorporation of (¹⁴C)-acetate or tritiated water into sterols. Histamine 0.3 µM added to the incubation medium at zero time inhibited this induction by 35% with a sigmoidal log dose-effect curve.The receptors mediating this action were characterised pharmacologically by using selective H₁- and H₂-agonists and -antagonists. The H₂-agonists impromidine and 4-methylhistamine mimicked the effect of histamine on sterol synthesis, the suppression being 42% and 31%, respectively, at a concentration of 1 µM. In contrast, the H₁-agonist 2-pyridylethylamine did not affect the pathway. The H₂-antagonist cimetidine (10 µM) but not the H₁-antagonist mepyramine (10 µM) totally reversed the inhibition of sterol synthesis by histamine.The results provide evidence that sterol synthesis in human mononuclear leukocytes is regulated by histamine, which appears to act predominantly via H₂-receptors.     

Comparison of the effects of MnO2-NPs and MnO2-MPs on mitochondrial complexes in different organs

Today, nanoparticles (NPs) have been widely used in various fields. Manganese oxide nanoparticles have attracted a lot of attention due to many applications. One of the major concerns regarding the widespread use of various NPs is the exposure and accumulation in human organs and finally toxicity. The generation of reactive oxygen species (ROS) by mitochondria is one of the most important mechanisms of toxicity suggested by published studies induced by other NPs. However, limited studies have been conducted on the mechanism of toxicity of MnO₂-NPs and MnO₂-microparticles (MnO₂-MPs). In this study, we compared the accumulation of MnO₂-NPs and MnO₂-MPs in different tissues and evaluated their effects on mitochondrial complexes in isolated mitochondria. Our results showed that intravascular (iv) administration of the MnO₂-NPs in the same dose compared to the MnO₂-MPs resulted in more accumulation in the C57 mouse female tissues. The effect of MnO₂-NPs and MnO₂-MPs in mitochondria showed that complexes I and III play an important role in increasing ROS generation and this effect is related to type of tissue. Also, our results showed that exposure to MnO₂-NPs and MnO₂-MPs reduced the activity of mitochondrial complexes II and IV. Our results suggest that the toxicity of the MnO₂-NPs is higher than that of the MnO₂-MPs and can lead to the depletion of antioxidant status, likely induction of apoptosis, cancer, and neurodegenerative disease.

Abbreviations: NPs: nanoparticles; ROS: reactive oxygen species; SDH: succinate dehydrogenase; DCFH-DA: dichloro-dihydro-fluorescein diacetate; ELISA: enzyme-linked immunosorbent assay; MnO2-NPs: manganese oxide nanoparticles     

A manganese-porphyrin complex decomposes H(2)O(2), inhibits apoptosis, and acts as a radiation mitigator in vivo

Ionizing radiation triggers mitochondrial overproduction of H(2)O(2) with concomitant induction of intrinsic apoptosis, whereby clearance of H(2)O(2) upon overexpression of mitochondrial catalase increases radioresistance in vitro and in vivo. As an alternative to gene therapy, we tested the potential of Mn((III))-porphyrin complexes to clear mitochondrial H(2)O(2). We report that triphenyl-[(2E)-2-[4-[(1Z,4Z,9Z,15Z)-10,15,20-tris(4-aminophenyl)-21,23-dihydroporphyrin-5-yl]phenyl]iminoethyl]phosphonium-Mn((III)) compartmentalizes preferentially into mitochondria of mouse embryonic cells, reacts with H(2)O(2), impedes γ-ray-induced mitochondrial apoptosis, and increases the survival of mice exposed to whole body irradiation with γ-rays.     

Investigation of the role of extracellular H2O2 and transition metal ions in the genotoxic action of ascorbic acid in cell culture models

In the presence of oxygen, ascorbic acid (AA) is unstable in aqueous media and oxidises to dehydroascorbate (DHA), generating reactive intermediates such as ascorbate free radical and H2O2. It is proposed that the cytotoxicity of AA is due to the extracellular production of H2O2 and that this is mediated by transition metal ions present in cell media. Here we investigate the role of extracellular H2O2 and metal ions in the genotoxicity of AA in cell culture models. Our preliminary results confirmed that physiological concentrations of AA were not toxic to confluent human fibroblasts, although they inhibited the proliferation of cells at low density. No inhibition was observed with ascorbic acid 2-phosphate (AA2P), a vitamin C derivative that remains stable in culture media. Furthermore, high concentrations of AA induced DNA strand breakage in a dose-dependent manner, whereas DHA and AA2P were not genotoxic. The genotoxic effect of AA was transient, required the formation of extracellular H2O2 and the presence of intracellular iron, but not of extracellular transition metal ions. These observations further clarify the pro-oxidant effect of AA solutions in cell culture models. The possibility that intravenous administration of high-dose AA may cause a similar genotoxic effect in vivo is discussed.