In vivo radioprotection by alpha-TMG: preliminary studies

alpha-TMG is a novel water-soluble derivative of Vitamin E that has shown excellent antioxidant activity. The parent compound has demonstrated protection against radiation induced chromosomal damage in vivo. Hence, the preliminary experiments to determine the radioprotective activity of alpha-TMG were carried out in adult Swiss albino mice. Acute toxicity of the drug was studied taking 24h, 72 h and 30 day mortality after a single intraperitoneal injection of 500-2000 mg/kg body weight of the drug. The drug LD(50) for 24h and 72 h/30 day survival were found to be 1120 and 1000 mg/kg body weight, respectively. The optimum time of drug administration and drug dose-dependent effect on in vivo radiation protection of bone marrow chromosomes was studied in mice. Injection of 600 mg/kg of the drug 15 min before or within 5, 15 or 30min after 3Gy whole body gamma radiation resulted in a significant decrease in the aberrant metaphases percent at 24h post-irradiation; the maximum effect was seen when the drug was given immediately after irradiation. Injection of 200-800 mg/kg TMG within 5 min of irradiation with 3 Gy produced a significant dose-dependent reduction in the radiation induced percent aberrant metaphases and in the frequency of micronucleated erythrocytes at 24h after exposure, with a corresponding decrease in the different types of aberrations. The optimum dose for protection without drug toxicity was 600 mg/kg body weight. At this dose, TMG produced 70 and >60% reduction in the radiation induced percent aberrant metaphases and micronucleated erythrocytes, respectively. The high water solubility and effectiveness when administered post-irradiation favor TMG as a likely candidate for protection in case of accidental exposures.     

In vivo radioprotection by 5-androstenediol: stimulation of the innate immune system

We showed previously that 5-androstenediol stimulates myelopoiesis, increases the numbers of circulating neutrophils and platelets, and enhances resistance to infection in gamma-irradiated mice. We have extended those studies to include monocytes, natural killer (NK) cells, eosinophils and basophils, and we have measured the activation marker CD11b using flow cytometry. Androstenediol (160 mg/kg) was administered subcutaneously to female B6D2F1 mice 24 h before whole-body gamma irradiation. Androstenediol treatments increased the blood levels of neutrophils, monocytes and NK cells in unirradiated animals; decreased the numbers of circulating eosinophils; and ameliorated radiation-induced decreases in neutrophils, monocytes, NK cells, erythrocytes and platelets. The androstenediol treatments had no significant effect on the numbers of circulating B cells or T cells. CD11b labeling intensity on monocytes was decreased slightly after androstenediol treatment. In contrast, radiation or androstenediol alone caused increases in CD11b labeling intensity on NK cells. Androstenediol and radiation combined caused a marked increase in NK cell CD11b. The results indicate that androstenediol increases the numbers of the three major cell types of the innate immune system (neutrophils, monocytes and NK cells), that androstenediol-induced changes in blood elements in irradiated animals persist for at least several weeks, and that there is a significant positive interaction between radiation and administration of androstenediol in the activation of NK cells.     

Skin radioprotection by 5-thio-D-glucose

The radioprotective effect of 5-thio-D-glucose on mouse skin was studied. Intraperitoneal injection of A/J mice with 1.5 g/kg of 5-thio-D-glucose 2 hr prior to X irradiation of the foot reduced early foot skin damage through Day 40 postirradiation by a dose modification factor of 1.3 +/- 0.1. Similarly, late foot deformity during Days 60-90 postirradiation was reduced by a factor of 1.2 +/- 0.1. The radioprotective effect of 5-thio-D-glucose was also compared with that of WR-2721, an aminothiol radioprotector, in CDF1 mice. An intraperitoneal injection of 1.5 g/kg of 5-thio-D-glucose reduced early radiation-induced skin damage by a dose modification factor of 1.2 +/- 0.1 as compared to that of 1.5 +/- 0.2 by 0.65 g/kg of WR-2721 in this strain of mice. 5-Thio-D-glucose is also known to specifically kill and radiosensitize hypoxic tumor cells. Consequently, this drug may be a useful radiotherapy adjuvant, reducing normal tissue damage and enhancing tumor control by minimizing hypoxic protection.     

Induction of chromosomal damage in mammalian cells in vitro and in vivo by sulfapyridine or 5-aminosalicylic acid.

Sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA) are the two primary metabolites of the anti-inflammatory drug salicylazosulfapyridine (SASP). These two metabolites were studied for induction of chromosomal damage in mammalian cells, in vitro and in vivo, in an attempt to understand better the genetic effects produced by SASP in humans and laboratory mice. To this end, SP and 5-ASA were tested for induction of sister-chromatid exchanges (SCE) and chromosomal aberrations (Abs) in Chinese hamster ovary (CHO) cells in vitro. In addition, they were tested in vivo for induction of micronuclei (MN) in mouse bone marrow polychromatic erythrocytes (PCE). SP gave positive results in the in vitro SCE test and the in vivo MN test, and negative results in the in vitro Abs test. 5-ASA was negative in all three tests. These results indicate that it is the SP metabolite of SASP that is necessary for the induction of chromosomal damage reported to occur in humans and mice after treatment with SASP.     

Ulcerative colitis impairs the acylethanolamide-based anti-inflammatory system reversal by 5-aminosalicylic acid and glucocorticoids

Studies in animal models and humans suggest anti-inflammatory roles on the N-acylethanolamide (NAE)-peroxisome proliferators activated receptor alpha (PPARα) system in inflammatory bowel diseases. However, the presence and function of NAE-PPARα signaling system in the ulcerative colitis (UC) of humans remain unknown as well as its response to active anti-inflammatory therapies such as 5-aminosalicylic acid (5-ASA) and glucocorticoids. Expression of PPARα receptor and PPARα ligands-biosynthetic (NAPE-PLD) and -degrading (FAAH and NAAA) enzymes were analyzed in untreated active and 5-ASA/glucocorticoids/immunomodulators-treated quiescent UC patients compared to healthy human colonic tissue by RT-PCR and immunohistochemical analyses. PPARα, NAAA, NAPE-PLD and FAAH showed differential distributions in the colonic epithelium, lamina propria, smooth muscle and enteric plexus. Gene expression analysis indicated a decrease of PPARα, PPARγ and NAAA, and an increase of FAAH and iNOS in the active colitis mucosa. Immunohistochemical expression in active colitis epithelium confirmed a PPARα decrease, but showed a sharp NAAA increase and a NAPE-PLD decrease, which were partially restored to control levels after treatment. We also characterized the immune cells of the UC mucosa infiltrate. We detected a decreased number of NAAA-positive and an increased number of FAAH-positive immune cells in active UC, which were partially restored to control levels after treatment. NAE-PPARα signaling system is impaired during active UC and 5-ASA/glucocorticoids treatment restored its normal expression. Since 5-ASA actions may work through PPARα and glucocorticoids through NAE-producing/degrading enzymes, the use of PPARα agonists or FAAH/NAAA blockers that increases endogenous PPARα ligands may yield similar therapeutics advantages.     

Preferential radioprotection to DNA of normal tissues by ferulic acid under ex vivo and in vivo conditions in tumor bearing mice

Our previous study showed that ferulic acid (FA) offered good radioprotection under in vitro and in vivo conditions to DNA and enhanced the DNA repair process in the peripheral blood leucocytes of mice in vivo. This study concerns radioprotection of normal versus tumor cells. Administration of FA (50 mg/kg body weight) to mice bearing fibrosarcoma tumor, 1 h prior to/ or immediately after radiation exposure (4 Gy) showed preferential radioprotection to normal cells i.e. peripheral blood leucocytes and bone marrow cells in comparison to tumor cells. This preferential protection under in vivo conditions could be attributed to poor vasculature in the tumor or peculiar characteristics of the tumor cells either to restrict its entry inside the cells or metabolize or inactivate the drug. To resolve these ex vivo study was carried out using bone marrow and tumor cells. It was found that under ex vivo condition also only bone marrow cells were protected by FA. Thus the studies revealed that FA showed preferential protection to normal cells under both in vivo and ex vivo conditions. (Mol Cell Biochem xxx: 1–10, 2005)     

Development and validation of a HPLC-ESI-MS/MS method for the determination of 5-aminosalicylic acid and its major metabolite N-acetyl-5-aminosalicylic acid in human plasma

A new HPLC method for the determination of 5-aminosalicylic acid (5-ASA) and N-acetyl-5-aminosalicylic acid (N-Ac-5-ASA) in human plasma was developed and validated. Plasma samples were analyzed after protein precipitation with methanol and the two analytes were separated using a C18 column with a mobile phase composed of 17.5mmol/L acetic acid (pH 3.3):acetonitrile=85:15 (v/v) at 0.2mL/min flow rate. 4-ASA and N-Ac-4-ASA were used as internal standards. Selective detection was performed by tandem mass spectrometry with electrospray source, operating in negative ionization mode and in multiple reaction monitoring acquisition (m/z 152-->108 for 5-ASA; m/z 194-->150 and 194-->107 for N-Ac-5-ASA). The limit of quantification (LOQ) was 50ng/mL for both analytes (0.2ng injected) and matrix-matched standard curves showed linearity up to 4000ng/mL. In the entire analytical range the within- and between-batch precision (R.S.D.%) values were respectively 90% for 5-ASA and >95% for N-Ac-5-ASA (R.S.D.%相似文献   

In vivo measurements by differential pulse voltammetry of extra-cellular 5-hydroxyindoleacetic acid in the rat brain

The use of differential pulse voltammetry, performed with electrochemically treated carbon fiber electrodes, enables us to detect in vitro or in vivo, in the striatum of anaesthetized rats, an oxidation peak (3) at a potential of + 300 mV. Electrolytic or 5-7-dihydroxytryptamine lesions of the medial forebrain bundle are followed by a decrease of 59 and 62% respectively of this peak. Biochemical measurements are significantly correlated with the measured peak (3) and decreases. Thus, peak (3) increases obtained after injection of L-tryptophan and/or Reserpine, as well as the decreases observed after injection of Clorgyline or 3-hydroxybenzylhydrazine, confirm that peak (3) is dependent upon 5-hydroxyindoleacetic acid concentration. The detection of a peak (3) in the cerebrospinal fluid and its increase after injection of Probenecid reinforce this conclusion.     

Dextran and 5-aminosalicylic acid (5-ASA) conjugates: synthesis, characterisation and enzymic hydrolysis

Mesalamine (5-aminosalicylic acid) is the drug of choice for the treatment of Crohn's disease. A scheme for the synthesis of 5-aminosalicylic acid (5-ASA) conjugates of dextrans was developed with a focus on Crohn's disease applications. Dextrans were oxidised using sodium periodate (NaIO(4)), where the aldehyde groups formed were coupled with the alpha-amino (-NH(2)) group of 5-ASA. The resulting imine bonds were unstable in water and were consequently reduced to secondary amine groups. The effects of different aspects of the conjugation reaction were studied. These included the following: the molecular weight of the dextrans, the molar proportion of NaIO(4) to the dextrans (for periodate oxidation), the pH of the conjugation solutions, the ratio 5-ASA to oxidised polysaccharide and the relationship between the degree of conjugation and the amount of enzyme hydrolysis. Conjugates incubated in HCl were stable in 0.5 and 1.0M HCl, but they underwent degradation in 2.0 and 4.0M HCl. Dextrans (MW 20,000) with various degrees of oxidation (12%, 26%, 46%, 65%, 90% and 93%) were also prepared. Each oxidised dextran sample was conjugated with 5-ASA, and the product was quantified by high-performance liquid chromatography (HPLC). Dextrans with a maximum degree of oxidation (93%) unsurprisingly gave maximum conjugation of 5-ASA (49.1mg per 100mg of product) but were resistant to dextranase hydrolysis. Less oxidised dextrans (12%) conjugated proportionally less 5-ASA (15.1mg per 100mg of product) but were successfully hydrolysed by dextranase, suggesting their potential applications for the treatment of Crohn's disease in the distal ileum and proximal colon.     

In vivo conversion of 5-oxoproline to glutamate by higher plants

l-(U-(14)C)-5-oxoproline (pyrollidone carboxylic acid or pyroglutamic acid) was infiltrated into detached leaves of a number of species and incubated for 1 to 6 hours. In every case, conversion to labeled glutamate and glutamine was observed. The amount converted varied from 1 to 64% of the total label fed depending on the species. The ratio of glutamate-(14)C to glutamine-(14)C ranged from 5 in Vicia faba to 1 in sugar beet. This ratio could be affected by preinfiltrating various compounds before allowing the uptake of the 5-oxoproline. When l-methionine-dl-sulfoximine was prefed to sugar beet leaves, the glutamate-glutamine ratio increased from 1 to 10. Prior treatment of V. faba leaves with azaserine resulted in essentially only labeled glutamine being recovered. Preinfiltration with NaF or ATP gave similar results in that the glutamate-glutamine ratio was greatly decreased. The results are consistent with glutamate being produced from the 5-oxoproline and then being converted to glutamine.     

In vivo formation of leukotriene E5 by murine peritoneal cells

Resident mouse peritoneal cells, stimulated in vivo with opsonized zymosan, produced leukotriene C4 and E4, with LTE4 being the major (80-90%) product. When mice were placed on diets containing increasing amounts of fish oil, four additional sulfidopeptide leukotrienes (SP-LT), LTC5, LTE5, 11-trans LTC5 and 11-trans LTE5, were identified. The identity of LTE5 was confirmed by spectrophotometric, chromatographic and enzymatic methods. When equivalent amounts of n-6 and n-3 polyunsaturated fatty acids (PUFA) were included in the diet, the stimulated peritoneal cells (in vivo) produced higher quantities of LTE5 (30.2 +/- 5.4 ng/10(6) cells) than LTE4 (22.8 +/- 7.3 ng/10(6) cells). In addition, in vitro studies demonstrated a 60% reduction in LTC4 (42.0 +/- 10.8 ng/10(6) cells to 16.7 +/- 6.2 ng/10(6) cells) and the appearance of LTC5 (2.1 +/- 0.9 ng/10(6) cells) in resident macrophages (stimulated with A23187) from mice maintained on a fish oil diet compared to mice fed the control diet. This study demonstrated that formation of the pentaenyl SP-LT in vivo, in particular LTE5, by peritoneal cells can significantly contribute to the endogenous SP-LT pool in response to an inflammatory stimulus following a dietary regimen containing fish oil.     

Scavenging of reactive oxygen and nitrogen species by the prodrug sulfasalazine and its metabolites 5-aminosalicylic acid and sulfapyridine

Sulfasalazine is a prodrug composed by a molecule of 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP), linked by an azo bond, which has been shown to be effective in the therapy of inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease, as well as of rheumatic diseases, such as rheumatoid arthritis and ankylosing spondylitis. The precise mechanism of action of sulfasalazine and/or its metabolites has not been completely elucidated, though its antioxidant effects are well established and are probably due to its scavenging effects against reactive oxygen and nitrogen species (ROS and RNS), as well as metal chelating properties, in association to its inhibitory effects over neutrophil oxidative burst. The present work was focused on screening and comparing the potential scavenging activity for an array of ROS (O(2)(?-), H(2)O(2), (1)O(2), ROO(?) and HOCl) and RNS ((?)NO and ONOO(-)), mediated by sulfasalazine and its metabolites 5-ASA and SP, using validated in vitro screening systems. The results showed that both 5-ASA and sulfasalazine were able to scavenge all the tested ROS while SP was practically ineffective in all the assays. For HOCl, (1)O(2), and ROO(?), 5-ASA showed the best scavenging effects. A new and important finding of the present study was the strong scavenging effect of 5-ASA against (1)O(2). 5-ASA was shown to be a strong scavenger of (?)NO and ONOO(-). Sulfasalazine was also able to scavenge these RNS, although with a much lower potency than 5-ASA. SP was unable to scavenge (?)NO in the tested concentrations but was shown to scavenge ONOO(-), with a higher strength when the assay was performed in the presence of 25 mM bicarbonate, suggesting further scavenging of oxidizing carbonate radical. In conclusion, the ROS- and RNS-scavenging effects of sulfasalazine and its metabolites shown in this study may contribute to the anti-inflammatory effects mediated by sulfasalazine through the prevention of the oxidative/nitrative/nitrosative damages caused by these species.