Lipophilicity is a critical parameter that dominates the efficacy of metalloporphyrins in blocking the development of morphine antinociceptive tolerance through peroxynitrite-mediated pathways

Severe pain syndromes reduce the quality of life of patients with inflammatory and neoplastic diseases, partly because reduced analgesic effectiveness with chronic opiate therapy (i.e., tolerance) leads to escalating doses and distressing side effects. Peroxynitrite-mediated nitroxidative stress in the dorsal horn of the spinal cord plays a critical role in the induction and development of antinociceptive tolerance to morphine. This provides a valid pharmacological basis for developing peroxynitrite scavengers as potent adjuncts to opiates in the management of pain. The cationic Mn(III) ortho-N-alkylpyridylporphyrins MnTE-2-PyP(5+) and MnTnHex-2-PyP(5+) are among the most potent peroxynitrite scavengers, with nearly identical scavenging rate constants (approximately 10(7) M(-1) s(-1)). Yet, MnTnHex-2-PyP(5+) is significantly more lipophilic and more bioavailable and, in turn, was 30-fold more effective in blocking the development of morphine antinociceptive tolerance than MnTE-2-PyP(5+) using the hot-plate test in a well-characterized murine model. The hydrophilic MnTE-2-PyP(5+) and the lipophilic MnTnHex-2-PyP(5+) were 10- and 300-fold, respectively, more effective in inhibiting morphine tolerance than the hydrophilic Fe(III) porphyrin FeTM-4-PyP(5+). Both Mn porphyrins decreased levels of TNF-alpha, IL-1 beta, and IL-6 to normal values. Neither of them affected acute morphine antinociceptive effects nor caused motor function impairment. Also neither was able to reverse already established morphine tolerance. We have recently shown that the anionic porphyrin Mn(III) tetrakis(4-carboxylatophenyl)porphyrin is selective in removing ONOO(-) over O(2)(-), but at approximately 2 orders of magnitude lower efficacy than MnTE-2-PyP(5+) and MnTnHex-2-PyP(5+), which in turn parallels up to 100-fold lower ability to reverse morphine tolerance. These data (1) support the role of peroxynitrite rather than superoxide as a major mechanism in blocking the development of morphine tolerance and (2) show that lipophilicity is a critical parameter in enhancing the potency of such novel peroxynitrite scavengers.     

Manganese-porphyrin reactions with lipids and lipoproteins

Manganese porphyrin complexes serve to catalytically scavenge superoxide, hydrogen peroxide, and peroxynitrite. Herein, reactions of manganese 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)) with lipids and lipid hydroperoxides (LOOH) are examined. In linoleic acid and human low-density lipoprotein (LDL), MnTE-2-PyP(5+) promotes oxidative reactions when biological reductants are not present. By redox cycling between Mn(+3) and Mn(+4) forms, MnTE-2-PyP(5+) initiates lipid peroxidation via decomposition of 13(S)hydroperoxyoctadecadienoic acid [13(S)HPODE], with a second-order rate constant of 8.9 x 10(3) M(-1)s(-1)and k(cat) = 0.32 s(-1). Studies of LDL oxidation demonstrate that: (i) MnTE-2-PyP(5+) can directly oxidize LDL, (ii) MnTE-2-PyP(5+) does not inhibit Cu-induced LDL oxidation, and (iii) MnTE-2-PyP(5+) plus a reductant partially inhibit lipid peroxidation. MnTE-2-PyP(5+) (1-5 microM) also significantly inhibits FeCl(3) plus ascorbate-induced lipid peroxidation of rat brain homogenate. In summary, MnTE-2-PyP(5+) initiates membrane lipid and lipoprotein oxidation in the absence of biological reductants, while MnTE-2-PyP(5+) inhibits lipid oxidation reactions initiated by other oxidants when reductants are present. It is proposed that, as the Mn(+3) resting redox state of MnTE-2-PyP(5+) becomes oxidized to the Mn(+4) redox state, LOOH is decomposed to byproducts that propagate lipid oxidation reactions. When the manganese of MnTE-2-PyP(5+) is reduced to the +2 state by biological reductants, antioxidant reactions of the metalloporphyrin are favored.     

Cytotoxic effects of Mn(III) N-alkylpyridylporphyrins in the presence of cellular reductant, ascorbate

Due to the ability to easily accept and donate electrons Mn(III)N-alkylpyridylporphyrins (MnPs) can dismute O(2)(·-), reduce peroxynitrite, but also generate reactive species and behave as pro-oxidants if conditions favour such action. Herein two ortho isomers, MnTE-2-PyP(5+), MnTnHex-2-PyP(5+), and a meta isomer MnTnHex-3-PyP(5+), which differ greatly with regard to their metal-centered reduction potential, E(1/2) (Mn(III)P/Mn(II)P) and lipophilicity, were explored. Employing Mn(III)P/Mn(II)P redox system for coupling with ascorbate, these MnPs catalyze ascorbate oxidation and thus peroxide production. Consequently, cancer oxidative burden may be enhanced, which in turn would suppress its growth. Cytotoxic effects on Caco-2, Hela, 4T1, HCT116 and SUM149 were studied. When combined with ascorbate, MnPs killed cancer cells via peroxide produced outside of the cell. MnTE-2-PyP(5+) was the most efficacious catalyst for peroxide production, while MnTnHex-3-PyP(5+) is most prone to oxidative degradation with H(2) , and thus the least efficacious. A 4T1 breast cancer mouse study of limited scope and success was conducted. The tumour oxidative stress was enhanced and its microvessel density reduced when mice were treated either with ascorbate or MnP/ascorbate; the trend towards tumour growth suppression was detected.     

Manganese porphyrin, MnTE-2-PyP5+, Acts as a pro-oxidant to potentiate glucocorticoid-induced apoptosis in lymphoma cells

Using current chemotherapy protocols, over 55% of lymphoma patients fail treatment. Novel agents are needed to improve lymphoma survival. The manganese porphyrin, MnTE-2-PyP(5+), augments glucocorticoid-induced apoptosis in WEHI7.2 murine thymic lymphoma cells, suggesting that it may have potential as a lymphoma therapeutic. However, the mechanism by which MnTE-2-PyP(5+) potentiates glucocorticoid-induced apoptosis is unknown. Previously, we showed that glucocorticoid treatment increases the steady state levels of hydrogen peroxide ([H(2)O(2)](ss)) and oxidizes the redox environment in WEHI7.2 cells. In the current study, we found that when MnTE-2-PyP(5+) is combined with glucocorticoids, it augments dexamethasone-induced oxidative stress however, it does not augment the [H(2)O(2)](ss) levels. The combined treatment depletes GSH, oxidizes the 2GSH:GSSG ratio, and causes protein glutathionylation to a greater extent than glucocorticoid treatment alone. Removal of the glucocorticoid-generated H(2)O(2) or depletion of glutathione by BSO prevents MnTE-2-PyP(5+) from augmenting glucocorticoid-induced apoptosis. In combination with glucocorticoids, MnTE-2-PyP(5+) glutathionylates p65 NF-κB and inhibits NF-κB activity. Inhibition of NF-κB with SN50, an NF- κB inhibitor, enhances glucocorticoid-induced apoptosis to the same extent as MnTE-2-PyP(5+). Taken together, these findings indicate that: 1) H(2)O(2) is important for MnTE-2-PyP(5+) activity; 2) Mn-TE-2-PyP(5+) cycles with GSH; and 3) MnTE-2-PyP(5+) potentiates glucocorticoid-induced apoptosis by glutathionylating and inhibiting critical survival proteins, including NF-κB. In the clinic, over-expression of NF-κB is associated with a poor prognosis in lymphoma. MnTE-2-PyP(5+) may therefore, synergize with glucocorticoids to inhibit NF-κB and improve current treatment.     

Comparison of two Mn porphyrin-based mimics of superoxide dismutase in pulmonary radioprotection

Development of radiation therapy (RT)-induced lung injury is associated with chronic production of reactive oxygen and nitrogen species (ROS/RNS). MnTE-2-PyP5+ is a catalytic Mn porphyrin mimic of SOD, already shown to protect lungs from RT-induced injury by scavenging ROS/RNS. The purpose of this study was to compare MnTE-2-PyP5+ with a newly introduced analogue MnTnHex-2-PyP5+, which is expected to be a more effective radioprotector due to its lipophilic properties. This study shows that Fischer rats which were irradiated to their right hemithorax (28 Gy) have less pulmonary injury as measured using breathing frequencies when treated with daily subcutaneous injections of MnTE-2-PyP5+ (3 and 6 mg/kg) or MnTnHex-2-PyP5+ (0.3, 0.6, or 1.0 mg/kg) for 2 weeks after RT. However, at 16 weeks post-RT, only MnTE-2-PyP5+ at a dose of 6 mg/kg is able to ameliorate oxidative damage, block activation of HIF-1alpha and TGF-beta, and impair upregulation of CA-IX and VEGF. MnTnHex-2-PyP5+ at a dose of 0.3 mg/kg is effective only in reducing RT-induced TGF-beta and CA-IX expression. Significant loss of body weight was observed in animals receiving MnTnHex-2-PyP5+ (0.3 and 0.6 mg/kg). MnTnHex-2-PyP5+ has the ability to dissolve lipid membranes, causing local irritation/necrosis at injection sites if given at doses of 1 mg/kg or higher. In conclusion, both compounds show an ability to ameliorate lung damage as measured using breathing frequencies and histopathologic evaluation. However, MnTE-2-PyP5+ at 6 mg/kg proved to be more effective in reducing expression of key molecular factors known to play an important role in radiation-induced lung injury.     

Peroxynitrite flux-mediated LDL oxidation is inhibited by manganese porphyrins in the presence of uric acid

We have studied the role of three Mn(III)porphyrins differing in charge, alkyl substituent length and reactivity, on LDL exposed to low fluxes of peroxynitrite (PN) in the presence of uric acid. Mn(III)porphyrins (5 microM, MnTE-2-PyP(5+), MnTnOct-2-PyP(5+), and MnTCPP(3-)) plus uric acid (300 microM) inhibited cholesteryl ester hydroperoxide formation, changes in REM as well as spared alpha- and gamma-tocopherol. MnTnOct-2-PyP(5+), the more lipophilic compound, was the most effective in protecting LDL lipids, while MnTCPP(3-) exerted the lesser protection. Mn(III)porphyrins react fast with PN ( approximately 10(5)-10(7) M(-1) s(-1)) to yield a O=Mn(IV) complex. The stoichiometry of uric acid consumption was approximately 1.7 moles per mol of PN, in agreement with reactions with both the O=Mn(IV) complex and nitrogen dioxide. A shift from an anti- to a pro-oxidant action of the Mn(III)porphyrin was observed after uric acid was significantly consumed, supporting competition reactions between LDL targets and uric acid for the O=Mn(IV) complex. Overall, the data is consistent with the catalytic reduction of PN in a cycle that involves a one electron oxidation of Mn(III) to Mn(IV) by PN followed by the reduction back to Mn(III) by uric acid. These antioxidant effects should predominate under in vivo conditions having plasma uric acid concentration range between 150 and 500 microM.     

Pure MnTBAP selectively scavenges peroxynitrite over superoxide: Comparison of pure and commercial MnTBAP samples to MnTE-2-PyP in two models of oxidative stress injury,an SOD-specific Escherichia coli model and carrageenan-induced pleurisy

MnTBAP is often referred to as an SOD mimic in numerous models of oxidative stress. We have recently reported that pure MnTBAP does not dismute superoxide, but commercial or poorly purified samples are able to perform O₂^·?dismutation with low-to-moderate efficacy via non-innocent Mn-containing impurities. Herein, we show that neither commercial nor pure MnTBAP could substitute for SOD enzyme in a SOD-deficient Escherichia coli model, whereas MnTE-2-PyP-treated SOD-deficient E. coli grew as well as a wild-type strain. This SOD-specific system indicates that MnTBAP does not act as an SOD mimic in vivo. In another model, carrageenan-induced pleurisy in mice, inflammation was evidenced by increased pleural fluid exudate and neutrophil infiltration and activation: these events were blocked by 0.3 mg/kg MnTE-2-PyP and, to a slightly lesser extent, by 10 mg/kg of either MnTBAP. Also, 3-nitrotyrosine formation, an indication of peroxynitrite existence in vivo, was blocked by both compounds; again MnTE-2-PyP was 33-fold more effective. Pleurisy model data indicate that MnTBAP exerts some protective actions in common with MnTE-2-PyP, which are not O₂^·? related and can be fully rationalized if one considers that the common biological role shared by MnTBAP and MnTE-2-PyP is related to their reduction of peroxynitrite and carbonate radical, the latter arising from ONOOCO₂ adduct. The log k_cat (O₂^·?) value for MnTBAP is estimated to be about 3.16, which is ~ 5 and ~ 6 orders of magnitude smaller than the SOD activities of the potent SOD mimic MnTE-2-PyP and Cu,Zn-SOD, respectively. This very low value indicates that MnTBAP is too inefficient at dismuting superoxide to be of any biological impact, which was confirmed in the SOD-deficient E. coli model. The peroxynitrite scavenging ability of MnTBAP, however, is only ~ 2.5 orders of magnitude smaller than that of MnTE-2-PyP and is not significantly affected by the presence of the SOD-active impurities in the commercial MnTBAP sample (log k_red (ONOO^?) = 5.06 for pure and 4.97 for commercial sample). The reduction of carbonate radical is equally fast with MnTBAP and MnTE-2-PyP. The dose of MnTBAP required to yield oxidative stress protection and block nitrotyrosine formation in the pleurisy model is > 1.5 orders of magnitude higher than that of MnTE-2-PyP, which could be related to the lower ability of MnTBAP to scavenge peroxynitrite. The slightly better protection observed with the commercial MnTBAP sample (relative to the pure MnTBAP) could arise from its impurities, which, by scavenging O₂^·?, reduce consequently the overall peroxynitrite and secondary ROS/RNS levels. These observations have profound biological repercussions as they may suggest that the effect of MnTBAP observed in numerous studies may conceivably relate to peroxynitrite scavenging. Moreover, provided that pure MnTBAP is unable to dismute superoxide at any significant extent, but is able to partially scavenge peroxynitrite and carbonate radical, this compound may prove valuable in distinguishing ONOO^?/CO₃^·? from O₂^·? pathways.     

Comprehensive pharmacokinetic studies and oral bioavailability of two Mn porphyrin-based SOD mimics,MnTE-2-PyP5+ and MnTnHex-2-PyP5+

The cationic, ortho Mn(III) N-alkylpyridylporphyrins (alkyl=ethyl, E, and n-hexyl, nHex) MnTE-2-PyP⁵⁺ (AEOL10113, FBC-007) and MnTnHex-2-PyP⁵⁺ have proven efficacious in numerous in vivo animal models of diseases having oxidative stress in common. The remarkable therapeutic efficacy observed is due to their: (1) ability to catalytically remove O₂^•− and ONOO⁻ and other reactive species; (2) ability to modulate redox-based signaling pathways; (3) accumulation within critical cellular compartments, i.e., mitochondria; and (4) ability to cross the blood–brain barrier. The similar redox activities of both compounds are related to the similar electronic and electrostatic environments around the metal active sites, whereas their different bioavailabilities are presumably influenced by the differences in lipophilicity, bulkiness, and shape. Both porphyrins are water soluble, but MnTnHex-2-PyP⁵⁺ is approximately 4 orders of magnitude more lipophilic than MnTE-2-PyP⁵⁺, which should positively affect its ability to pass through biological membranes, making it more efficacious in vivo at lower doses. To gain insight into the in vivo tissue distribution of Mn porphyrins and its impact upon their therapeutic efficacy and mechanistic aspects of action, as well as to provide data that would ensure proper dosing regimens, we conducted comprehensive pharmacokinetic (PK) studies for 24 h after single-dose drug administration. The porphyrins were administered intravenously (iv), intraperitoneally (ip), and via oral gavage at the following doses: 10 mg/kg MnTE-2-PyP⁵⁺ and 0.5 or 2 mg/kg MnTnHex-2-PyP⁵⁺. Drug levels in plasma and various organs (liver, kidney, spleen, heart, lung, brain) were determined and PK parameters calculated (C_max, C_24 h, t_max, and AUC). Regardless of high water solubility and pentacationic charge of these Mn porphyrins, they are orally available. The oral availability (based on plasma AUC_oral/AUC_iv) is 23% for MnTE-2-PyP⁵⁺ and 21% for MnTnHex-2-PyP⁵⁺. Despite the fivefold lower dose administered, the AUC values for liver, heart, and spleen are higher for MnTnHex-2-PyP⁵⁺ than for MnTE-2-PyP⁵⁺ (and comparable for other organs), clearly demonstrating the better tissue penetration and tissue retention of the more lipophilic MnTnHex-2-PyP⁵⁺.     

Decreased hepatic ischemia-reperfusion injury by manganese-porphyrin complexes

Reactive oxygen and nitrogen species have been implicated in ischemia-reperfusion (I/R) injury. Metalloporphyrins (MP) are stable catalytic antioxidants that can scavenge superoxide, hydrogen peroxide, peroxynitrite and lipid peroxyl radicals. Studies were conducted with three manganese-porphyrin (MnP) complexes with varying superoxide dimutase (SOD) and catalase catalytic activity to determine if the MnP attenuates I/R injury in isolated perfused rat livers. The release of the hepatocellular enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) was maximal at 1 min reperfusion, decreased rapidly and increased gradually by 90 min. Manganese tetrakis-(N-ethyl-2 pyridyl) porphyrin (MnTE-2-PyP) decreased ALT, AST, LDH at 1-90 min reperfusion, while manganese tetrakis-(N-methyl-2 pyridyl) porphyrin (MnTM-2-PyP) and manganese tetrakis-(ethoxycarbonyl) porphyrin (MnTECP) decreased ALT and LDH from 5 to 90 min reperfusion. The release of thiobarbituric acid-reacting substances (TBARS) was diminished by MnTE-2-PyP and MnTM-2-PyP at 90 min. The extent of protein nitration (nitrotyrosine, NT) was decreased in all three MnPs treated livers. These results demonstrate that MnP complexes can attenuate hepatic I/R injury and may have therapeutic implications in disease states involving oxidants.     

Reduction of manganese porphyrins by flavoenzymes and submitochondrial particles: a catalytic cycle for the reduction of peroxynitrite

The reduction of manganese(III) meso-tetrakis((N-ethyl)pyridinium-2-yl)porphyrin (MnTE-2-PyP) to manganese(II) was catalyzed by flavoenzymes such as xanthine oxidase and glucose oxidase, and by Complex I and Complex II of the mitochondrial electron transport chain. The reduced manganese porphyrin has been previously shown to react rapidly with superoxide and carbonate radical anion. Herein, we describe the reaction of a reduced manganese porphyrin with peroxynitrite that proceeds as a two-electron process, has a rate constant greater than 7 x 10(6) M(-1) s(-1) (at pH 7.25 and 37 degrees C), and produces nitrite and the Mn(IV)Porphyrin. The Mn(II)/Mn(IV) redox cycle was used to divert peroxynitrite from the inactivation of succinate dehydrogenase. In a typical experiment, 5 microM MnTE-2-PyP in the presence of excess succinate was able to protect the succinate dehydrogenase and succinate oxidase activities of submitochondrial particles challenged with a cumulative dose of 140 microM peroxynitrite infused in the course of 2 h. Other MnPorphyrins that are reduced more slowly do not provide as much protection underscoring the rate limiting character of the reduction step. The data presented here serve to rationalize the pharmacological action of MnPorphyrins as peroxynitrite reduction catalysts in vivo and opens avenues for the development of MnPorphyrins to protect mitochondria from oxidative damage.     

Levels and distribution of zinc,copper, magnesium,and calcium in rats fed different levels of dietary zinc

Effects of altered dietary zinc on levels of zinc, copper, magnesium, and calcium in organ and peripheral tissues were studied. When rats fed a zinc-deficient diet (1.3 μg Zn/g) for 28 d were compared with rats fed a control diet (37.5 μg Zn/g), levels of zinc were slightly lower in plasma, hair, and skin and 50% lower in femur and pancreas, whereas the levels of copper were higher in all tissue except plasma. Magnesium levels were higher than controls in the heart and lower in the spleen, whereas the calcium levels were lower in plasma, lung, spleen, kidney, and skin and strikingly higher in brain, hair, and femur. When rats fed a zinc-supplemented diet (1.0 mg Zn/g) were compared to the same conrols, levels of zinc in these were higher in all organs and peripheral tissues studied, except heart, lung, and liver; copper levels were higher in liver, kidney, and spleen; magnesium levels were significantly higher in the spleen, but were little affected in other tissues, although calcium levels were higher in pancreas, spleen, kidney, and skin and lower in plasma and hair. These data indicate that overall copper organ and peripheral tissue levels are affected inversely, and zinc and calcium levels directly, by zinc nutriture.     

Mn porphyrin-based superoxide dismutase (SOD) mimic, MnIIITE-2-PyP5+, targets mouse heart mitochondria

The Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnIIITE-2-PyP5+ (AEOL-10113) has proven effective in treating oxidative stress-induced conditions including cancer, radiation damage, diabetes, and central nervous system trauma. The ortho cationic pyridyl nitrogens of MnTE-2-PyP5+ are essential for its high antioxidant potency. The exceptional ability of MnIIITE-2-PyP5+ to dismute O2.- parallels its ability to reduce ONOO- and CO3-. Decreasing levels of these species are considered its predominant mode of action, which may also involve redox regulation of signaling pathways. Recently, Ferrer-Sueta at al. (Free Radic. Biol. Med. 41:503-512; 2006) showed, with submitochondrial particles, that>or=3 microM MnIIITE-2-PyP5+ was able to protect components of the mitochondrial electron transport chain from peroxynitrite-mediated damage. Our study complements their data in showing, for the first time that micromolar mitochondrial concentrations of MnIIITE-2-PyP5+ are obtainable in vivo. For this study we have developed a new and sensitive method for MnIIITE-2-PyP5+ determination in tissues. The method is based on the exchange of porphyrin Mn2+ with Zn2+, followed by the HPLC/fluorescence detection of ZnIITE-2-PyP4+. At 4 and 7 h after a single 10 mg/kg intraperitoneal administration of MnIIITE-2-PyP5+, the mice (8 in total) were anesthetized and perfused with saline. Mitochondria were then isolated by the method of Mela and Seitz (Methods Enzymol.55:39-46; 1979). We found MnIIITE-2-PyP5+ localized in heart mitochondria to 2.95 ng/mg protein. Given the average value of mitochondrial volume of 0.6 microL/mg protein, the calculated MnIIITE-2-PyP5+ concentration is 5.1 microM, which is sufficient to protect mitochondria from oxidative damage. This study establishes, for the first time, that MnIIITE-2-PyP5+, a highly charged metalloporphyrin, is capable of entering mitochondria in vivo at levels sufficient to exert there its antioxidant action; such a result encourages its development as a prospective therapeutic agent.     

Effect of whole-body gamma irradiation on lipid peroxidation in rat tissues

In this work, we studied the influence of wholebody gamma irradiation (800 rads) upon malonaldehyde (MDA) content in plasma, erythrocyte, brain, heart, lung, kidney, spleen, liver, thymus and bone marrow. MDA levels were increased in all studied samples, except lung; the highest increases were observed in the most radiosensitive organs (bone marrow, thymus, spleen) and not in those continuously exposed to high concentrations of molecular oxygen (lungs, erythrocytes). Comparison of the variations of MDA levels in plasma, kidneys and spleen to those in the other tissues lead to the hypothesis that MDA is released from tissues in plasma and trapped from plasma in kidney and spleen. The variations in plasma and erythrocyte were found not to be related to each other.     

Plasma dependent and independent accumulation of betaine in male and female rat tissues

Tissue betaine is an intracellular osmolyte that also provides a store of labile methyl groups. Despite these important biological roles, there are few data regarding tissue betaine content. We measured the betaine concentration of plasma and various tissues (brain, heart, lungs, liver, kidney, spleen, intestine, reproductive tissues, skeletal muscle and skin) in male and female rats and assessed whether there were any gender-specific differences in betaine content or distribution and whether there was any relationship between tissue accumulation and plasma levels. Betaine was highest in the liver and kidney with values ranging from 1.6 to 9.5 mmol/l and 2.0 to 5.4 mmol/l, respectively. Plasma betaine concentrations were significantly lower than tissue levels except in the brain (? 25 % of plasma) and skeletal muscle (similar to plasma). Regression analysis of the combined male and female data revealed a significant plasma-related accumulation of betaine in the heart, skin and skeletal muscle, while the lung, liver, kidney, spleen, and intestine showed significant plasma-related and plasma-independent accumulations of betaine. The betaine content of the skin, liver and kidney was not significantly different between males and females, but in plasma and all tissues analyzed it was significantly higher in males (P<0.01).     

Fetal and maternal tissue distribution of the new fluoroquinolone DW-116 in pregnant rats

We investigated maternal and fetal tissue distribution of DW-116, a newly developed fluoroquinolone with a broad antibacterial spectrum against both G(+) and G(-) bacteria, in pregnant rats. After oral administration of [14C]-DW-116 (labeled 1 mg and unlabeled 500 mg/kg) to female rats on the 18th day of gestational, groups of three rats were killed at various time points up to 24 h, and plasma and tissues were collected, processed and analyzed. [14C]-DW-116 was rapidly absorbed, and distributed into the maternal and fetal tissues, and it declined in a biphasic manner with elimination half-lives (t(1/2)) of 10-15 h and mean residence times (MRT(0-24 h)) of 4-9 h. The radioactivity in most tissues of both dams and fetus reached its peak within 1 h and radioactivity levels of up to 10-25% of the peak level were maintained until 24 h after dosing. Among various tissues, the radioactivity in the maternal lungs was the highest (27 times that of plasma) at the C(max). Radioactivity in other tissues including liver, kidney, heart, lung, brain, spleen, mammary gland, placenta, ovary and uterus was higher than that in the maternal plasma (one- to three-fold). The tissue-to-plasma partition coefficient (K(p), AUC(0-24 h,tissue)/AUC(0-24 h,plasma)) of [14C]-DW-116 in maternal tissues was highest in the lung (K(p)=3.7), followed by the spleen (2.2), kidney (2.0), liver (1.8), heart (1.5), placenta (1.3), brain (1.3), ovary (1.1), uterus (1.1), and mammary gland (1.0). The tissue-to-plasma partition coefficient values in fetal tissues were heart (K(p)=2.2), kidney (2.1), liver (1.9), lung (1.6) and brain (1.4). When lactating rats were given a single oral dose of [14C]-DW-116, the radioactivity was rapidly secreted into the milk with K(p) of 1.7 at T(max) (0.5 h). These results indicate that DW-116 or its related metabolite(s) rapidly cross the blood-placenta and blood-milk barrier, extensively distribute into the fetal tissues, and are eliminated from the body in a prolonged manner. This study sheds insights into the maternal and fetal tissue distribution of DW-116 and will be useful for assessing both therapeutic and toxicological relevance of DW-116 in pregnant subjects.     

Effect of age and dietary protein level on tissue mineral levels in female rats

Mineral (phosphorus, sulfur, potassium, calcium, magnesium, iron, zinc, copper, and manganese) concentrations were measured in plasma, and several tissues from female Wistar rats (young: 3-wk-old; mature: 6-mo-old) were fed on a dietary regimen designed to study the combined or singular effects of age and dietary protein on mineral status. Three diets, respectively, contained 5, 15, and 20% of bovine milk casein. Nephrocalcinosis chemically diagnosed by increased calcium and phosphorus in kidney was prevented in rats fed a 5% protein diet. Renal calcium and phosphorus were more accumulated in young rats than mature rats. A 5% protein diet decreased hemoglobin and blood iron. The hepatic and splenic iron was increased by a 5% protein diet in mature rats but was not altered in young rats. Mature rats had higher iron in brain, lung, heart, liver, spleen, kidney, muscle, and tibia than young rats. A 5% protein diet decreased zinc in plasma and liver. Zinc in tibia was increased with dietary protein level in young rats but was not changed in mature rats. A 5% protein diet decreased copper concentration in plasma of young rats but not in mature rats. Mature rats had higher copper in plasma, blood, brain, lung, heart, liver, spleen, and kidney than young rats. With age, manganese concentration was increased in brain but decreased in lung, heart, liver, kidney, and muscle. These results suggest that the response to dietary protein regarding mineral status varies with age.     

Mitochondrial oxidative stress-induced apoptosis and radioprotection in proton-irradiated rat retina

There is concern about possible radiation damage to the eyes from occupational exposure and medical procedures. In this study, molecular mechanisms of proton radiation-induced oxidative damage to retinal cells were evaluated, with and without a cell-permeable superoxide dismutase (SOD) mimetic, metalloporphyrin compound (MnTE-2-PyP). Retinal mitochondria-associated genes and protein expression profiles were studied. Rats were treated with MnTE-2-PyP at 2.5 μg/injection into one eye 1 h before irradiation. Proton irradiation was delivered to the same eye at doses of 1 or 4 Gy and assays were done at 6 h. Levels of Bax, Bcl-2 and Sod2 proteins were evaluated by Western blot and caspase-3 immunohistochemistry was performed to confirm the occurrence of apoptosis. Expression of several genes playing central roles in regulating the mitochondrial apoptotic pathway were significantly increased after radiation exposure, including Bbc3, Bax, Bak1, Bid, and Bcl2. Among genes involved in radiation-induced oxidative stress, Sod2, Gpx and Ucp3 were up-regulated, whereas Ucp2 was down-regulated. In addition, irradiation caused changes in various proteins involved in apoptosis (caspase-3, Bax and Bcl2). Reduction in pro-apoptotic and increase in anti-apoptotic protein levels were documented after treatment with MnTE-2-PyP. Decreased activity of cytochrome c, which is involved in initiation of mitochondrial apoptosis, was also revealed after irradiation and MnTE-2-PyP. Data demonstrated that proton radiation induced mitochondrial apoptosis and altered mitochondrial function in retina. MnTE-2-PyP protected, or at least ameliorated, radiation-induced oxidative damage. These insights prompt further study of this compound as a potential therapeutic candidate for retinal protection against degenerative ocular damage induced by ionizing radiation.     

The nature of the collagenolytic cathepsin of rat liver and its distribution in other rat tissues

1. An enzyme present in rat liver extracts degraded insoluble collagen maximally at pH3.5. Collagenolytic activity was more abundant in kidney, spleen and bone marrow and was also present in decreasing concentrations in ileum, lung, heart, skin and muscle. 2. The crude collagenolytic cathepsin was activated by cysteine and dithiothreitol, but not by 2-mercaptoethanol. Iodoacetamide, p-chloromercuribenzoate and 7-amino-1-chloro-3-l-tosylamidoheptan-2-one hydrochloride inhibited the enzyme. Zn(2+), Fe(3+) and Hg(2+) ions were strongly inhibitory, but Ca(2+), Co(2+), Mg(2+) and Fe(2+) ions had little or no effect. EDTA was an activator of the enzyme. Inhibitors of cathepsin B were found to enhance collagenolysis, but phenylpyruvic acid, a cathepsin D inhibitor, inhibited the enzyme. Di-isopropyl phosphorofluoridate had no effect. 3. Collagenolysis at pH3.5 and 28 degrees C was restricted to cleavage of the telopeptide region in insoluble collagen, and the material that was solubilized consisted mostly of alpha-chains. 4. The collagenolytic cathepsin was separated from cathepsins B2 and D by fractionation on Sephadex G-100 and a partial separation from cathepsin B1 was obtained by chromatography on DEAE-Sephadex. 5. The function of the collagenolytic cathepsin in the catabolism of collagen is discussed in relation to the action of the other lysosomal proteinases and the neutral collagenase.     

Characterization of Na/Ca exchange in plasmalemmal vesicles from zona fasciculata cells of the bovine adrenal gland

The presence of an Na/Ca exchange system in fasciculata cells of the bovine adrenal gland was tested using isolated plasmalemmal vesicles. In the presence of an outwardly Na(+) gradient, Ca(2+) uptake was about 2-fold higher than in K(+) condition. Li(+) did not substitute for Na(+) and 5 mM Ni(2+) inhibited Ca(2+) uptake. Ca(2+) efflux from Ca(2+)-loaded vesicles was Na(+)-stimulated and Ni(2+)-inhibited. The saturable part of Na(+)-dependent Ca(2+) uptake displayed Michaelis-Menten kinetics. The relationship of Na(+)-dependent Ca(2+) uptake versus intravesicular Na(+) concentration was sigmoid (apparent K(0.5) approximately 24 mM; Hill number approximately 3) and Na(+) acted on V(max) without significant effect on K(m). Na(+)-stimulated Ca(2+) uptake was temperature-dependent (apparent Q(10) approximately 2.2). The inhibition properties of several divalent cations (Cd(2+), Sr(2+), Ni(2+), Ba(2+), Mn(2+), Mg(2+)) were tested and were similar to those observed in kidney basolateral membrane. The above results indicate the presence of an Na/Ca exchanger located on plasma membrane of zona fasciculata cells of bovine adrenal gland. This exchanger displays similarities with that of renal basolateral cell membrane.