The protective role of endogenously synthesized nitric oxide in staphylococcal enterotoxin B-induced shock in mice.

Nitric oxide (NO) synthesis during experimental endotoxemia has been shown to have both deleterious and beneficial effects. In the present study, we analyzed the in vivo production and the regulatory role of NO in the shock syndrome induced by staphylococcal enterotoxin B (SEB) in mice. First, we found that intraperitoneal administration of 100 micrograms SEB in BALB/c mice induced a massive synthesis of NO as indicated by high serum levels of nitrite (NO2-) and nitrate (NO3-) peaking 16 h after SEB injection. The inhibition of NO2- and NO3- release in mice injected with anti-tumor necrosis factor (TNF) and/or anti-interferon gamma (IFN-gamma) monoclonal antibody (mAb) before SEB challenge revealed that both cytokines were involved in SEB-induced NO overproduction. In vitro experiments indicated that NO synthase (NOS) inhibition by N-nitro-L-arginine methyl ester (L-NAME) enhanced IFN-gamma and TNF production by splenocytes in response to SEB. A similar effect was observed in vivo as treatment of mice with L-NAME resulted in increased IFN-gamma and TNF serum levels 24 h after SEB challenge, together with persistent expression of corresponding cytokine mRNA in spleen. The prolonged production of inflammatory cytokines in mice receiving L-NAME and SEB was associated with a 95% mortality rate within 96 h, whereas all mice survived injections of SEB or L-NAME alone. Both TNF and INF-gamma were responsible for the lethality induced by SEB in L-NAME-treated mice as shown by the protection provided by simultaneous administration of anti-IFN-gamma and anti-TNF mAbs. We conclude the SEB induces NO synthesis in vivo and that endogenous NO has protective effects in this model of T cell-dependent shock by downregulating IFN-gamma and TNF production.

DNA alterations photosensitized by tetracycline and some of its derivatives.

Bacteriophage M13 mp10 DNA were irradiated with near-UV light in the presence of tetracycline derivatives and primed with synthetic oligonucleotide to be used for DNA synthesis using Escherichia coli DNA polymerase. Chain terminations were observed by denaturing polyacrylamide gel electrophoresis and mapped precisely. All the synthesis stops occurred before or at the level of guanine residues, showing that the photoreaction mediated by tetracycline derivatives led to a preferential alteration of guanine residues. These lesions were demonstrated to be induced in DNA through a pathway involving singlet oxygen. Tetracycline derivatives also photoinduced the breakage of the DNA sugar-phosphate backbone monitored by the conversion of supercoiled phi X174 DNA to a relaxed form. This lesion was shown to be initiated by hydroxyl radicals. The production of this free radical has been confirmed by electron paramagnetic resonance (EPR) spin trapping experiments using 5,5-dimethyl-1-pyrroline-N-oxide as spin trap. In addition to the EPR signal due to OH radicals trapping another unassigned signal has been detected.

Plasma homocysteine concentration in a Belgian school-age population.

BACKGROUND: Total plasma homocysteine (tHcy) is an independent risk factor for cardiovascular disease in adults. Data for children and adolescents are lacking. OBJECTIVE: The aim of this study was to provide a reference range for tHcy and to explore the relation between tHcy and nutritional indexes in a Belgian pediatric population. DESIGN: tHcy, folate, and vitamin B-12 were measured in 647 healthy children (353 girls and 294 boys) aged 5-19 y. RESULTS: The tHcy distribution was, as in adults, skewed to the right [geometric mean (-1 SD, +1 SD): 7.41 micromol/L (5.51, 9.96)]. Concentrations were lowest in younger children and increased with age. After the tHcy distribution was examined according to age, 3 age ranges were distinguished: 5-9 y [6.21 micromol/L (5.14, 7.50)], 10-14 y [7.09 micromol/L (5.69, 8.84)], and 15-19 y [8.84 micromol/L (6.36, 12.29)]. We observed no significant differences in tHcy values between girls and boys in children aged < 15 y; in postpubertal children, however, concentrations were higher in boys than in girls. In the 3 age groups, folate was inversely correlated with tHcy; the negative relation between tHcy and vitamin B-12 was less strong. Familial cardiovascular disease was more frequent in children who had hyperhomocysteinemia. CONCLUSIONS: These observations suggest that in children, as in adults, genetic, nutritional, and endocrine factors are determinants of the metabolism of homocysteine. The significance of tHcy values in childhood and young adulthood in terms of predicting cardiovascular risk in adulthood should be investigated.

Biomolecular photoalterations mediated by phenothiazine derivatives.

This survey focuses on recent developments in the field of the ultraviolet photochemistry and photobiology of phenothiazine derivatives. One of the major alterations introduced by this kind of photosensitized reaction is a covalent addition of the photosensitizer or one of its photoproducts onto the macromolecular target. This reaction has been observed with soluble and membrane proteins, lipids and DNA. In the latter case, the addition occurs at the level of guanine residues and leads to inhibition of DNA replication.

In vitro cross-linking of bovine lens proteins photosensitized by promazines.

Promazine derivatives induce cross-linking of bovine lens crystallins in vitro by irradiation with near-ultraviolet (UV) light in the presence of O2, as revealed by electrophoresis after denaturation. With the five derivatives tested (promazine [PZ], chlorpromazine [CPZ], triflupromazine [ TFPZ ], methoxypromazine [ MTPZ ], and acepromazine [ ACPZ ] ), single-hit kinetics are observed. Evidence implicating the cation radicals of the PZ derivatives as the causative agent of this in vitro effect is presented. Hydroxyl radicals do not appear to be involved in the photo-cross-linking reaction. Sodium ascorbate protects against damage induced either by PZ derivatives plus light or by PZ cation radicals in the dark. These findings are discussed with respect to development of cataracts induced by these drugs in vivo.

Termini generated at the site of the DNA breakage mediated by photoexcited promazines.

Promazine derivatives are known to be able to photoinduce, in vitro, direct single-strand breaks into DNA (Decuyper et al., Biochem. Pharmac. 33, 4025-4031 (1984]. Using [32P]end labeled DNA fragments, it is demonstrated that this DNA breakage occurs almost regardless of the nucleotide sequence of the DNA. Using 3'-[32P]end or 5'-[32P]end labeled oligonucleotide and enzymatic digestion of the fragments generated, it is demonstrated that the termini generated at the site of the breakage are 5'-phosphate, 3'-phosphate and 3'-termini which are presumed to be 3'-phosphoglycolate. This is consistent with an attack of the sugar moeity of the sugar-phosphate backbone of the DNA by the reactives species generated upon near-u.v. irradiation of promazine derivatives.

Photosensitization of SV 40 DNA mediated by promazine derivatives and 4'-hydroxymethyl-4,5',8-trimethylpsoralen. Inhibition of the in vitro transcription.

In vitro transcription by E. coli RNA polymerase was carried out on SV40 DNA photoreacted with various promazine derivatives. Inhibition of the template activity was recorded with increasing irradiation times in the presence of promazine derivatives. Promazine covalent adducts on guanine did not terminate RNA synthesis and seemed to be bypassed by the enzyme. HMT (4'-hydroxymethyl-4,5',8-trimethylpsoralen) photoreaction with DNA was carried out under two conditions: irradiation with lambda greater than 395 nm favouring monoadduction on pyrimidine residues and irradiation at 360 nm inducing a maximum of interstrand diadducts. Both adducts were able to terminate RNA synthesis on the phototreated SV40 DNA and using the O-methyl-nucleotide sequencing procedure, the termination sites were precisely mapped. Monoadducts on the coding strand and cross-links induced termination two bases away from the covalent adduct, but monoadducts on the noncoding strand did not half RNA polymerase.

Induction of breaks in deoxyribonucleic acid by photoexcited promazine derivatives.

Near-u.v. photoexcited promazine and three of its derivatives are shown to induce single-strand breaks in phi X174-DNA replicative form. The mechanisms of this DNA breakage depend upon the various photochemical properties of the promazine derivatives. Chlorpromazine is shown to act predominantly via the photodechlorination reaction both in aerobic and anaerobic conditions. The three other promazine derivatives (promazine, trifluopromazine and methoxypromazine) display two mechanisms for DNA breakage. One of them occurs through the cation radical, which is formed during near-u.v. irradiation of promazine derivatives. The second mechanism is demonstrated to act via an hydroxyl radical-dependent pathway. Acepromazine is without photoactivated action. EPR-spin-trapping studies of irradiated mixtures, containing the drugs and 5,5-dimethyl-1-pyrroline-N-oxide (as spin trap), suggest the production of superoxide radical by photoexcited promazines. When DNA is present in the irradiation mixture, this superoxide radical is converted into hydroxyl radical probably via a Haber Weiss-type reaction, catalysed by DNA-iron complexes.

Distribution of iron and iron-binding proteins in first-trimester human pregnancies.

OBJECTIVE: To investigate the iron distribution between the maternal and embryonic compartments in the first trimester of pregnancy. METHODS: Coelomic and amniotic fluids (AF) and maternal serum were collected from 36 apparently normal pregnancies at 7-13 weeks of gestation. Iron, transferrin, ferritin, and lactoferrin were measured in all samples. Iron concentrations were also measured in placental villi, liver, gut, and brain samples collected from two embryos. RESULTS: Significantly (median value) lower iron and transferrin levels and higher levels of ferritin were found in the coelomic fluid (iron 4.8 mumol/L; transferrin 0.22 g/L) than in maternal serum (iron 21 mumol/L; transferrin 2.5 g/L). The AF contained significantly lower levels of iron and ferritin (iron less than 1.8 mumol/L; ferritin 2.0 micrograms/L) than both coelomic fluid (iron 4.8 mumol/L; ferritin 287 micrograms/L) and maternal serum (iron 21 mumol/L; ferritin 49 micrograms/L). Transferrin was undetectable (less than 0.08 g/L) in AF samples, and lactoferrin was undetectable (less than 2 micrograms/mL) in both embryonic fluids. The iron concentration in the coelomic fluid increased significantly (P < .001) with advancing gestation (iron at 7-9 weeks 3.8 mumol/L; 9.1-11 weeks 5.9 mumol/L). There was a nonsignificant correlation between coelomic fluid and maternal serum iron and iron-binding protein levels. The highest iron levels were found in the liver (52 mmol/kg dry weight) and brain (49 mmol/kg dry weight) tissues. CONCLUSIONS: The distribution of iron and iron-binding proteins between the maternal and embryo-placental compartments in the first trimester is comparable to that found later in gestation, suggesting that placental iron transfer may occur as early as tertiary villi are formed. The exocoelomic fluid is probably the main iron reservoir in early pregnancy, and the secondary yolk sac is probably the principal route of entry of iron to the embryo.

Phototoxicity of phenothiazine derivatives. II. Photosensitized cross-linking of erythrocyte membrane proteins.

Irradiation in the presence of O2, with near-UV light of five promazine (PZ) derivatives added to erythrocyte ghost membranes, causes covalent cross-linking between proteins as revealed by a progressive decrease in the amounts of proteins separable by electrophoresis after denaturation. The induction of cross-links in the two spectrin subunits is a single-hit process as a function of the irradiation time; relatively the rate constants (in min-1) of the photoreactions were 0.060 with chlorpromazine (CPZ), 0.039 with methoxypromazine (MTPZ), 0.031 with PZ, 0.029 with triflupromazine (TFPZ) and 0.006 with acepromazine (ACPZ). A main photochemical intermediate implicated in the spectrin aggregation seems to be the cation radical of the PZ derivatives. Indeed, (i) the chemically generated cation radicals can induce the reaction in the dark; (ii) the photoaggregation is regularly reduced upon addition of increasing concentrations of NaN3; (iii) NaN3 similarly affects the amount of cross-links induced by the isolated cation radicals. Hydroxyl radicals are also involved in the photocross-linking when the reaction is initiated only by MTPZ and not by the other sensitizers. In the absence of oxygen during irradiation, PZ, MTPZ and ACPZ completely loose their cross-linking activities whereas CPZ and TFPZ remain as efficient as in the presence of oxygen.

Phototoxicity of phenothiazine derivatives. I. Inactivating and mutagenic effects on bacteriophage øX174.

Inactivation of øX174 bacteriophages as a function of the irradiation time in the near-UV and in the presence of triflupromazine (TFPZ), promazine (PZ), chlorpromazine (CPZ) or methoxypromazine (MTPZ) proceeds according to single hit kinetics. Acepromazine (ACPZ) has no significant activity. At low concentrations (0.1 mM) TFPZ and PZ are the most active compounds. Higher concentrations (up to 5 mM) result in a protective effect by these two compounds but cause increased inactivation rates in the case of MTPZ or CPZ. Photoinactivation mediated by TFPZ or CPZ increases the reversion frequency of a øXamber mutant. Neither MTPZ nor PZ sensitization induces mutagenesis. The effect of NaN3 on the phage inactivation rate varies depending upon both the sensitizer and the concentration of the quencher. Phage inactivation in an N2 atmosphere is measurable only in the presence of high concentrations of CPZ and MTPZ. The drugs do not show any selectivity for calf thymus DNA or bovine serum albumin, at least as measured by dialysis equilibrium experiments.

[The medical chemistry department]. / Le Service de Chimie Médicale.

The laboratory of clinical chemistry performs more than 300 different tests in biochemistry, hormone and tumor markers analysis, therapeutic drug monitoring and toxicology. For the most basic tests it has followed the trend of clinical chemistry towards automation and since 2001 the heart of the laboratory is a modular automated system (MODULAR) including a preanalytical platform, unique in Belgium. For more sophisticated tests, the most recent techniques have been implemented, in particular capillary electrophoresis and ICP-MS ("inductively coupled plasma-mass spectrometry). Since 1994, the laboratory has become a reference center in the field of erythrocyte hereditary diseases, combining screening, diagnosis and research. The other research themes are the physiopathology of first trimester pregnancy and the P2Y receptors of extracellular nucleotides.

Breath alkanes as an index of lipid peroxidation.

Formation of free radicals and lipid peroxidation are mechanisms that are involved in many conditions including cellular damage. In a human body, there are many antioxidant systems likely to limit the production of free radicals and to reduce their deleterious effect. The peroxidation of polyunsaturated fatty acids, such as linoleic acid and linolenic acid which are cell membrane components, induces the formation of volatile alkanes that are excreted in the breath. The determination of breath alkanes (pentane and ethane) is considered to be a valuable and elegant method to assess lipid peroxidation. The method for collecting a breath sample and the chromatographic analysis of the collected gas require a strict methodology. Moreover, we must take into consideration some factors, such as the hepatic function, which may influence the pentane metabolism itself. We critically describe the results of breath alkane determination obtained in humans in different clinical conditions.

Activated oxygen species produced by photoexcited furocoumarin derivatives.

Furocoumarin derivatives (FCD) are investigated in order to determine their ability to photosensitize the production of activated oxygen species. Using the method based on the specific singlet oxygen (1O2) oxydation of cholesterol, all FCD except bergaten appeared to be able to produce 1O2 with various efficiencies. EPR spin trapping experiments show that photoexcited FCD produce hydroxyl radicals as detected by the formation of a DMPO-OH signal which can be abolished when the photosensitization reaction is carried out in the presence of specific OH scavengers. Moreover, the photo-ejection of hydrated electron (e-) by FCD is demonstrated by the loss of paramagnetic absorption of nitroxide free radicals as e- trap.

Termination sites of the in vitro DNA synthesis on single-stranded DNA photosensitized by promazines.

Bacteriophage phi X174 and M13 mp9 single-stranded DNA molecules were primed either with restriction fragments or synthetic primers and irradiated with near UV light in the presence of promazine derivatives. These DNAs were used as template for in vitro complementary chain synthesis by Escherichia coli DNA polymerase I large fragment. Chain terminations were observed by denaturing polyacrylamide gel electrophoresis of the synthesis products and localized by comparison with a standard dideoxy sequencing pattern. More than 90% of the chain terminations were mapped exactly one nucleotide before a guanine residue. In addition, photoreaction was shown to occur more predominantly with guanine residues localized in single-stranded parts of the genome. The same guanine residues could also be damaged when the reaction was performed, in the dark, in the presence of the artificially generated promazine cation radicals. Using the BamHI-SmaI adaptor (5'GATCCCCGGG-3'), it was shown that the guanine alteration was a covalent addition of the promazine, or of a cation radical photodegradation product, on the guanine moiety. Kinetics of chlorpromazine photoaddition on single-stranded and double-stranded DNAs were determined.

Lack of singlet oxygen formation by photoexcited promazine derivatives in aqueous and ethanolic solutions.

The EPR detection of nitroxide formation and the observation by thin layer chromatography of the specific 1O2 oxidation product of cholesterol, have been used to appreciate singlet oxygen production by promazine and four of its derivatives during irradiation with near-UV light of ethanolic and aqueous solutions. Within the range of sensitivity of the methods, no 1O2 had been detected.

Visible-light-induced OH radicals in DNA-proflavine complexes: an e.p.r. and spin trapping study.

Using the spin-trapping technique, irradiation with visible light of complexes between DNA and proflavine was shown to generate OH radicals. The characteristic spectra were not obtained when proflavine or DNA were irradiated alone, nor when oxygen was absent. Using DMPO as a spin trap we found that the intensity of the DMPO-OH e.p.r. signal was enhanced when the molar ratio between bound proflavine and the DNA phosphorus increased up to a value of 0.15 demonstrating the efficiency of the intercalated dye molecules. A strong decrease of the e.p.r. signal was observed in the presence of various OH. scavengers like t-butanol, isopropanol or sodium benzoate. The OH. production was also decreased when the irradiation was made in the presence of SOD, ceruloplasmin or catalase and after addition of Chelex 100 resin.