DNA strand scission by the nephrotoxin [2,2'-bipyridine]-3,3',4,4'-tetrol-1,1'-dioxide and related compounds in the presence of iron.

The capacity of non-illuminated nephrotoxin orellanine ([2,2'-bipyridine]-3,3',4,4'-tetrol-1,1'-dioxide) to induce DNA damage in the presence of ferrous iron and dioxygen has been evaluated. Maximal single-strand breaks in plasmid DNA were obtained with a metal to ligand ratio 1:3. Instantaneous oxidation of Fe2+ in presence of orellanine under air was responsible for oxy-radical production concomitant to a stable ferric complex Fe(III)Or3 formation, leading to oxidative DNA breakage at physiological pH. DNA damage was lowered in the presence of SOD and catalase or DMSO, indicating a set of reactions that leads to oxy-radical generation. Iron chelators such as DTPA and EDTA had no protecting effect, Desferal slightly protected. GSH acted as an oxy-radical scavenger, whereas cysteine induced stronger damage. Closely related bipyridine compounds were also studied in presence of Fe2+ and O2 using a combination of spin-trapping and DNA-nicking experiments, none of which were able to chelate iron and induce damage at pH 7. Both catecholic moieties and aminoxide groups are required for observing breakage at physiological pH.

Expression of the PAX2 gene in human embryos and exclusion in the CHARGE syndrome.

The CHARGE syndrome comprises ocular coloboma, heart malformation, choanal atresia, retarded growth and development, central nervous system malformations, genital hypoplasia, ear abnormalities, or deafness. The cause of the CHARGE syndrome remains unknown. In the present study, we analyzed the distribution pattern of the PAX2 gene in human embryos and found that PAX2 gene expression occurs in the primordia affected in the CHARGE syndrome. These data prompted us to consider the PAX2 gene a candidate gene in the CHARGE "association." We analyzed the PAX2 gene in 34 patients fulfilling the diagnostic criteria of the CHARGE syndrome for deletion and nucleotidic variations of the coding sequence and identified only polymorphisms. Our data suggest that mutation of the PAX2 gene is not a cause of the CHARGE association. However, the pattern of expression of PAX2 suggests that genes encoding downstream targets effectors could be candidate genes for the CHARGE syndrome.

Expression of the sonic hedgehog gene in human embryos with neural tube defects.

BACKGROUND: To estimate the rate of malformations observed during early human development, a series of 38,913 first-trimester abortions were studied. Neural tube defects (NTD) were found in 57 cases. METHODS: A histological study of serial sections performed in 25 embryos revealed a spectrum of axial structure abnormalities. Expression of the SHH gene was studied by in situ hybridization in one case of CRS and in two cases of SB. RESULTS: A cervical notochord duplication was always found in craniorachischisis (CRS, n = 8), but not in spina bifida (SB, n = 10) or diplomyelia (split cord malformation, n = 3). In the embryo with CRS, expression of SHH was found in both domains, corresponding to the duplicated part of the notochord, whereas a single signal was observed in the nonduplicated part. This expression was associated at the cervical level of the open neural tube with a broad SHH expression domain and with two or even three domains in its lumbar region, suggesting multiple functional floor plates. Similarly, in two embryos with SB, two domains of SHH expression were found in the ventral neural tube. CONCLUSIONS: Our findings suggest that notochord splitting in the cervical region might be involved in the pathogenesis of CRS. Interestingly, similar notochord abnormality and altered expression of the shh gene are observed in Lp mice with NTD. This suggests that the Lp gene could be a candidate gene for human CRS. Further studies are needed to establish the primary event responsible for the notochord splitting and for the abnormal expression of the SHH gene in the floor plate in embryos with CRS and SB.

Peroxidase-mediated oxidation, a possible pathway for activation of the fungal nephrotoxin orellanine and related compounds. ESR and spin-trapping studies.

Orellanine is the tetrahydroxylated and di-N-oxidized bipyridine toxin extracted from several Cortinarius mushrooms among them C. orellanus. The pathogenic mechanism involved in the C. orellanus-poisoning by orellanine leading to kidney impairment is not yet fully understood until now. Electron spin resonance (ESR) spectroscopy has been used to study the activation of orellanine by horseradish peroxidase/H2O2 system at physiological pH. Evidence for a one-electron oxidation of the toxin by this enzymatic system to an ortho-semiquinone radical intermediate is presented. The orellanine ortho-semiquinone generated by the peroxidase/H2O2 system abstracts hydrogen from glutathione, generating the glutathionyl radical which is spin-trapped by 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and subsequently detected by ESR spectroscopy. Similarly, the ortho-semiquinone abstracts hydrogen from ascorbic acid to generate the ascorbyl radical which is detected by direct ESR. The peroxidatic oxidation of orellanine to semiquinone followed by its reduction by glutathione or ascorbic acid does not induce dioxygen uptake. The relationship between chemical structure and HRP oxidation of orellanine-related molecules, namely orelline and DHBPO2 (the parent molecule lacking of hydroxyl groups in 3 and 3' position) has been investigated in absence or in presence of reducing agents. None of the orellanine-related compounds can be oxidized by the HRP/H2O2 system, showing that both catecholic moieties and aminoxide groups are necessary for observing the formation of the ortho-semiquinone form of orellanine. As shown for the (photo)chemical oxidation of orellanine, the mechanism of toxicity could be correlated with a depletion of glutathione and ascorbate levels which are implicated in the defence against oxidative damage.

Generation of oxygen radicals from iron complex of orellanine, a mushroom nephrotoxin; preliminary ESR and spin-trapping studies.

Orellanine, [2,2'-bipyridine]-3,3',4,4'-tetrol-1,1'-dioxide, is the toxin responsible for the lethal nephrotoxicity of some Cortinarius mushrooms. Our present ESR and spin-trapping studies of the redox properties of the system of non-illuminated orellanine, ferrous iron and dioxygen contribute to understanding the molecular mechanism of its toxicity. UV-visible spectrophotometry, cyclic voltammetry and ESR in frozen medium showed the formation of a wine-red tris complex, Fe(III)Or3. This ferric complex is easily reducible (Ep = -565 mV vs Ag/AgCl/3M KCl at pH 7), involving a one-electron reversible process. Spin-trapping using DMPO is employed to detect the generation of superoxide anion and hydroxyl radicals. The instantaneous one-electron oxidation of ferrous ions in the presence of the toxin under air is concomitant with dioxygen consumption as supported by dioxygen consumption. GSH involves the toxin and ferrous ions under air in a redox cycling process resulting in the production of glutathionyl and oxygen free radicals, observed for the first time with an iron complex of a mushroom toxin. In most cases, EDTA is not able to prevent the Fe(III)Or3 and radical formation. The ortho-dihydroxylated groups borne by the di-N-oxidized bipyridine structure and not the bipyridine structure itself, are responsible for the formation of a stable ferric complex at pH 7, as they are for the generation of an apparently stable ortho-semiquinone anion radical. These one-electron mechanisms may play a major role in some of the known toxic effects of orellanine.

Novel methods for identification and quantification of the mushroom nephrotoxin orellanine. Thin-layer chromatography and electrophoresis screening of mushrooms with electron spin resonance determination of the toxin.

Orellanine, (2,2'-bipyridine)-3,3',4,4'-tetrol-1,1'-dioxide, the toxin from several Cortinariace species, induces an acute renal failure which can be very severe or even irreversible and fatal. It is therefore important to be able to quickly and simply identify orellanine in mushroom samples with classical methods, readily available in any laboratory, such as anti-poison centers. This article reports the results of three analytical methods: classical TLC on cellulose plates in n-butanol--acetic acid--water and two original methods, electrophoresis on agarose gel and direct electron spin resonance (ESR) after enzymatic oxidation. They were applied to detect orellanine in 34 Cortinariaceae and 4 other species of toadstools. Our three sets of results are convergent. TLC (detection limit: 15 ng with fluorescence densitometry), electrophoresis (25 ng) and even ESR (5 micrograms), are sensitive enough for our purpose, and a sophisticated method like HPLC (detection limit: 50 pg) is not required. As the ESR spectrum of the toxin semiquinone is highly specific, TLC or electrophoresis coupled with ESR are a convenient alternative to liquid chromatography coupled with mass spectrometry, with the same specificity, for a confirmation or with samples such as ours with high toxin contents. ESR unambiguously confirms the relatively high contents of orellanine, from 0.45% (C. henrici) to 1.1-1.4% (C. orellanus), found in five Cortinarius from the subgenus Leprocybe, section Orellani. The five species, though they are from different geographic origins, have a more or less common pattern of fluorescent compounds, among which orellinine and orelline beside orellanine. It can be useful to note that orellanine semiquinone can be easily detected by ESR directly in the fresh mushroom. The toxin is absent in the other mushrooms we tested, especially in D. cinnamomea and C. splendens, which have been claimed as toxic and suspected to contain orellanine.

A survey of nitrate contents in Indonesian milk by enzymic analysis.

In this paper, a rapid and simple enzymic method is described for the determination of nitrate in 32 fresh and five dry Indonesian milk samples, deproteinized by Carrez reagents. Interference from albumin, casein, lactose and chloride ions was controlled. The calibration graph was linear over the range l-12.5 micrograms/ml NO3-; r = 0.9998. The limits of detection and quantification were found to be 0.45 micrograms/ml NO3- and 1 microgram/ml NO3- respectively. Standard nitrate solutions (10 micrograms/ml NO3-) were used to evaluate the precision. The results showed an average of 10.1 micrograms/ml, a standard deviation of 0.3 and a relative standard deviation of 3.4%. Adequate agreement was found between results obtained by the enzymic method and those of the French official reduction/photometric reference method (AFNor). Good recoveries (100% +/- 5%) were found for nitrate added to milk. The nitrate levels were in the range 1-2.6 mg/kg NO3- for fresh milk and 1.1-18 mg/kg NO3- for dry milk. All the results are in good agreement with those previously published for UK and American milk.

Validation of a modified spectrophotometric method for the determination of nitrate in dry milk using 2-sec-butylphenol.

A rapid, simple and direct visible spectrophotometric method, based on the quantitative nitration of 2-sec-butylphenol in concentrated sulfuric acid, is described for the determination of nitrate in milk. The nitration product was extracted into toluene and the yellow nitrophenoxide compound formed in alkaline aqueous solution was determined at 418 nm. Carrez reagents were used for protein precipitation. Interference from lactose, chloride and nitrite ions and the effect of reagent concentration was controlled. The calibration graph was linear over the range 0.5-5 micrograms ml-1 NO3-; y = -0.0019 + 0.0681 x; r = 0.9997. The limits of detection and quantification were found to be 0.18 and 0.5 microgram ml-1 NO3-, respectively. The accuracy of the method did not depend on nitrate content in spiked milk. The mean recovery of the spiked milk was found to be 99.81% with the confidence limits of 0.67%, a relative standard deviation (RSDr) of 1.79% for repeatability and an RSDR value of 1.90% for reproducibility. Adequate agreement was found between results obtained by the nitration method and those of the French official reduction spectrophotometric reference method (AFNor). For the Indonesian dry (powdered) milk sample studied the nitrate levels were in the range 10.7-29.5 mg kg-1 NO3-.

First electron spin resonance evidence for the production of semiquinone and oxygen free radicals from orellanine, a mushroom nephrotoxin.

Orellanine is the tetrahydroxylated and di-N-oxidized bipyridine toxin from several Cortinarius mushrooms. The mechanism responsible for its lethal nephrotoxicity was unknown until now. Our present ESR spectroscopic study of the redox properties of the toxin is an original contribution to the knowledge of its toxicity. It was achieved in particular by comparison of the properties of orellanine to that of other bipyridine compounds. After a one-electron oxidation (e.g., photochemical oxidation upon visible light), a radical form of orellanine is observed at physiological pH under aerobic or anaerobic conditions. This radical, identified as ortho-semiquinone anion radical, can also be generated by oxidation with biological oxidizing agents or enzymatic systems. Production of superoxide and hydroxyl radicals is shown by the spin-trapping method using DMPO as a spin trap. Bioreducing agents like GSH and cysteine involve in vitro the semiquinone radical and orellanine in a redox cycling process resulting in the production of glutathionyl and oxygen free radicals. This process leads in vitro to a large oxygen consumption and to a dramatic depletion of glutathione level. The formation of an apparently stable ortho-semiquinone anion radical and of reactive oxygen radical species is observed for the first time with a mushroom toxin. It is due to the catechol-like functions borne by the di-N-oxidized bipyridine structure of the toxin and may at least partly explain the toxicity of orellanine.

Prevalence of hepatitis A antibodies in French recruits in 1985.

The epidemiological study of hepatitis A antibodies prevalence in 1000 french recruits shows a 20% fall in people of 18-20 years old between 1979 and 1985, and identifies variables such as residence in coasting area, stay overseas, study level, as the most important social and geographical risk factors. These results are in agreement with the evolution observed in different other european countries.