Microbiological evaluation of ten French archives and link to occupational symptoms.

UNLABELLED: Fungi that damage documents in archives may harm workers' health, depending on which mold species are inhaled, the concentrations of fungal species inhaled, and individual factors. Our aim was to identify and quantify fungi in archives and to investigate possible links with the symptoms experienced by workers. Ten French archives were sampled using an air impactor and electrostatic dust collectors. Allergies and general symptoms felt by 144 workers were reported using a self-report questionnaire. Utilizing culture-based analysis methods along with qPCR, Penicillium chrysogenum, Cladosporium sphaerospermum, and Aspergillus versicolor were the three main fungi in air and dust in terms of quantity and frequency. Median fungal concentrations in storage areas, ranged from 30 to 465 CFU/m(3). People working in the most contaminated archives did not report more symptoms of allergy than others. However, workers in contact with moldy documents reported more headaches (odds ratio, 2.4; 95% confidence interval, 1.1-5.3), fatigue (OR, 2.9; 95% CI, 1.2-6.7), eye irritation (OR, 5.4; 95% CI, 1.9-14.9), throat irritation (OR, 2.4; 95% CI, 1.0-5.7), coughing (OR, 3.2; 95% CI, 1.2-8.4), and rhinorrhea (OR, 2.6; 95% CI, 1.0-6.4) than others. Other parameters such as dust levels and concentrations of metabolites and chemical substances should be considered as confounding factors in further investigations to isolate the role of molds. PRACTICAL IMPLICATIONS: Most studies about fungi and archives deal with the conservation of manuscripts and documents, and few discuss workers' health problems. Our study shows that archives do not represent a highly contaminated environment. Symptoms felt by workers were more often linked to direct contact with moldy documents than to high concentrations of mold in the air of archive storage areas. This study provides data on concentration levels in archives that could be used to interpret microbiological investigations in this type of environment in the future.

The INSERM expert review on glycol ethers: findings and recommendations.

The use of glycol ethers and their effects on health have recently attracted the attention of the French health authorities. At their request, INSERM, the French Institute of Health and Medical Research, conducted a collective expertise review on glycol ethers in 1999. INSERM Expertise Reviews are independent procedures performed by experts from several disciplines, to guarantee the objectivity and the relevance of the report. During several work sessions, the experts carried out a critical analysis of and reviewed studies concerning the toxicity of glycol ethers. This process resulted in a series of recommendations and conclusions. All these data have been published in the form of a report and have been used to help the public authorities to make decisions on how to prevent risks for professionals and consumers.

Bromocriptine is a strong inhibitor of brain nitric oxide synthase: possible consequences for the origin of its therapeutic effects.

The ergot alkaloid bromocriptine (BKT) was found to act as a strong inhibitor of purified neuronal nitric oxide synthase (NOS) (IC50 = 10 +/- 2 microM) whereas it was poorly active towards inducible macrophage NOS (IC50 > 100 microM). BKT affects the activation of NOS by calmodulin, as it not only inhibits L-arginine oxidation to NO and L-citrulline but also NADPH oxidation and calmodulin-dependent cytochrome c reduction catalyzed by neuronal NOS. These results suggest that BKT could exert some of its therapeutic effects by interfering with the NOS-dependent formation of nitric oxide and/or superoxide ion in various tissues.

A comparative NMR study between the macrolide antibiotic roxithromycin and erythromycin A with different biological properties.

1H nuclear Overhauser enhancement studies and 1H NMR 3J analysis establish the similarity between the major solution-state conformation of roxithromycin (1) and the erythromycin (2). A major difference between the structure of antibiotics 1 and 2 is the replacement of the 9-keto group in 2 by a 9-[O-(2,5-dioxahexyl)oxime] group. The NOE studies show that this oxime chain is oriented above the macrocyclic lactone ring and that the oxygen atoms of this chain are engaged in tight hydrogen bonding with a water molecule and with the 6- and 11-hydroxyl groups of the macrocycle. It results in a globular form of the whole roxithromycin molecule. These data explain also a relative hydrophobicity of this antibiotic. Erythromycin A (2), which presents a less rigid macrocycle with two free hydroxyl groups (6-OH and 11-OH), forms a dimer detected by FAB mass spectroscopy. 1H and 13C NMR relaxation measurements (T1) for both antibiotics show that interresidue hydrogen bonds in roxithromycin reduce the rotational freedom of the macrocyclic lactone ring and consequently the motions of desosamine and cladinose sugars. In another way, an ionization of the amino function occurs in the various media according to the nature of the antibiotic. This would allow the reactivity modification of the desosamine unit. In the biological study, the modifications of the 455-nm metabolite-cytochrome P-450 complex formation are observed.

Conformational analysis of ketolide, conformations of RU 004 in solution and bound to bacterial ribosomes.

A new structurally distinct class of 14-membered-ring macrolides is characterized by a keto-function instead of the cladinose sugar, well-known for its fragility even in weakly acidic media. This new class called ketolides is endowed with remarkable antibacterial activity against macrolide-resistant strains. A complete assignment of the 1H and 13C NMR spectra of RU 004 in deuteriochloroform, methanol-d4 and D2O has been made using different two-dimensional (2D) chemical-shift correlation methods. The study of ketolide-ribosome interaction has been investigated using 2D transferred nuclear Overhauser effect spectroscopy (TRNOESY). A comparison of the conformations in solution and bound to ribosomes was made with those of previous macrolides. This study can highlight some of the significant differences between RU 004 and other antibiotics.

The cyclo-oxygenase-dependent regulation of rabbit vein contraction: evidence for a prostaglandin E2-mediated relaxation.

1. Arachidonic acid (0.01-1 microM) induced relaxation of precontracted rings of rabbit saphenous vein, which was counteracted by contraction at concentrations higher than 1 microM. Concentrations higher than 1 microM were required to induce dose-dependent contraction of vena cava and thoracic aorta from the same animals. 2. Pretreatment with a TP receptor antagonist (GR32191B or SQ29548, 3 microM) potentiated the relaxant effect in the saphenous vein, revealed a vasorelaxant component in the vena cava response and did not affect the response of the aorta. 3. Removal of the endothelium from the venous rings, caused a 10 fold rightward shift in the concentration-relaxation curves to arachidonic acid. Whether or not the endothelium was present, the arachidonic acid-induced relaxations were prevented by indomethacin (10 microM) pretreatment. 4. In the saphenous vein, PGE2 was respectively a 50 and 100 fold more potent relaxant prostaglandin than PGI2 and PGD2. Pretreatment with the EP4 receptor antagonist, AH23848B, shifted the concentration-relaxation curves of this tissue to arachidonic acid in a dose-dependent manner. 5. In the presence of 1 microM arachidonic acid, venous rings produced 8-10 fold more PGE2 than did aorta whereas 6keto-PGF1alpha and TXB2 productions remained comparable. 6. Intact rings of saphenous vein relaxed in response to A23187. Pretreatment with L-NAME (100 microM) or indomethacin (10 microM) reduced this response by 50% whereas concomitant pretreatment totally suppressed it. After endothelium removal, the remaining relaxing response to A23187 was prevented by indomethacin but not affected by L-NAME. 7. We conclude that stimulation of the cyclo-oxygenase pathway by arachidonic acid induced endothelium-dependent, PGE2/EP4 mediated relaxation of the rabbit saphenous vein. This process might participate in the A23187-induced relaxation of the saphenous vein and account for a relaxing component in the response of the vena cava to arachidonic acid. It was not observed in thoracic aorta because of the lack of a vasodilatory receptor and/or the poorer ability of this tissue than veins to produce PGE2.

In vivo effects of erythromycin, oleandomycin and erythralosamine derivatives on hepatic cytochrome P-450.

Rats have been treated with several derivatives of the erythromycin, erythralosamine or oleandomycin series, in order to compare their ability to induce cytochrome P-450 and to form stable 456 nm-absorbing cytochrome P-450 metabolite complexes. The data obtained confirm that the cytochromes P-450 induced in rats by various macrolides are similar to that induced by pregnenolone 16 alpha-carbonitrile: the cytochrome P-450 IIIA1 isozyme. It showed that: (i) formation of a stable inhibitory 456 nm-absorbing cytochrome P-450 complex is not a prerequisite for cytochrome P-450 induction but enhances induction by stabilization of the IIIA isozyme. Therefore, the best inducers lead also to the maximal in vivo amounts of cytochrome P-450 metabolite complex (except for 2'MBEM); (ii) affinity for cytochrome P-450 IIIA1 is not directly involved for induction; and (iii) hydrophobicity favors induction and formation of complexes. Structural factors are also involved.

Particular ability of cytochromes P450 3A to form inhibitory P450-iron-metabolite complexes upon metabolic oxidation of aminodrugs.

The ability of 21 drugs containing an amine function to form inhibitory P450-iron-metabolite complexes absorbing around 455 nm was studied on liver microsomes from rats treated with various P450 inducers. These drugs belong to different chemical and therapeutic series and exhibit very different structures. In the case of eight compounds (diltiazem, lidocaine, imipramine, SKF 525A, fluoxetine, L-alpha-acetylmethadol, methadol and desmethyltamoxifen) whose oxidation by microsomes from rats treated with several inducers was studied, only dexamethasone (DEX)-treated rat microsomes and, to a lesser extent, phenobarbital (PB)-treated rat microsomes, were able to give significant amounts of 455 nm absorbing complexes. Ten of the 21 compounds studied gave such complexes with DEX-treated rat microsomes, while only three compounds gave complexes (in low amounts) with PB-treated rat microsomes only. For all compounds leading to complexes both with DEX- and PB-treated rat microsomes, much higher amounts of complexes were obtained with DEX-treated rat microsomes. DEX-treated rat microsomes also led to the most intense type I spectral interactions with most of the compounds studied, and very often exhibited the highest N-dealkylation activities towards the tertiary or secondary amine function of the drugs used. A few exceptions aside, there generally exists a qualitative relationship between the ability of P450 3As, induced by DEX, to bind and N-dealkylate amino compounds and their propensity to lead to 455 nm absorbing complexes. This was confirmed by in vivo experiments showing that rats treated with diltiazem, tamoxifen or imipramine accumulated large amounts of 455 nm absorbing complexes in their liver only after pretreatment with DEX and, to a lesser extent, with PB. This particular ability of P450 3As to oxidize amino drugs with formation of inhibitory P450-metabolite complexes could be of great importance for the appearance of drug interactions in man.

Dual effects of macrolide antibiotics on rat liver cytochrome P-450. Induction and formation of metabolite-complexes: a structure-activity relationship.

Previous studies have shown that the macrolide antibiotics, troleandomycin and erythromycin, are able to induce their own transformation into a metabolite forming an inactivated complex with rat liver cytochrome P-450. This paper reports the results of a study on the effects of several macrolide antibiotics including oleandomycin, erythromycin derivatives, josamycin, methymycin, tylosin, spiramycin and rifampicin, as well as antibiotics of other series, such as tetracycline and lincomycin, on rat liver cytochromes P-450 in vivo and in vitro. Only the antibiotics containing the desosamine and mycaminose amino sugars were able to give the dual effects already found with troleandomycin: induction of cytochrome p-450 and formation of an inhibitory cytochrome P-450--iron--nitrosoalkane metabolite complex in vivo or in vitro. From these studies, it appears that two structural factors are important for a macrolide antibiotic to lead to such effects: the presence of a non-hindered readily accessible N-dimethylamino group and the hydrophobic character of the molecule. These data are discussed in relation to the adverse effects observed during drug associations involving some of these macrolide antibiotics.

In vitro interaction of rat liver cytochromes P-450 with erythromycin, oleandomycin and erythralosamine derivatives. Importance of structural factors.

Several derivatives of the erythromycin, erythralosamine and oleandomycin series have been prepared. Their abilities to bind to rat liver microsomal cytochrome P-450 and to lead to the formation of stable 456 nm absorbing cytochrome P-450-metabolite complexes after their oxidative microsomal metabolism in vitro have been compared. The obtained data confirmed that cytochrome P-450 induced in rats either by macrolides or by 16 alpha-pregnenolone carbonitrile were the major isozymes involved in the binding of macrolides to liver microsomes and in metabolite-complex formation. They showed that (i) hydrophobicity was in general a beneficial factor for these two properties, (ii) the presence of a bulky substituent in position 3 of erythromycin dramatically decreased their affinity for these isozymes, and (iii) the simultaneous presence of bulky substituents in position 2' and 3 prevented iron-metabolite complex formation. These results led to the selection of two compounds, erythralosamine-2'-benzoate and erythralosamine-2',3-diacetate, which exhibited a particularly high affinity for macrolide inducible cytochrome P-450 and were very good precursors of cytochrome P-450-iron-metabolite complex formation.

Mechanism for the protective effects of silymarin against carbon tetrachloride-induced lipid peroxidation and hepatotoxicity in mice. Evidence that silymarin acts both as an inhibitor of metabolic activation and as a chain-breaking antioxidant.

Administration of silymarin (800 mg/kg i.p.) 30 min before carbon tetrachloride (18 microL/kg i.p.) did not modify total hepatic levels of CCl4 and metabolites in mice, but decreased by 40% the in vivo covalent binding of CCl4 metabolites to hepatic lipids at 2 hr. This pretreatment decreased by 60% the exhalation of ethane during the first hour after CCl4, and decreased by 50% the incidence of liver cell necrosis. In vitro, silymarin (800 micrograms/mL) decreased by 50 to 70% various monooxygenase activities, and decreased by 20% the covalent binding of CCl4 metabolites to microsomal proteins. Silymarin (800 micrograms/mL) decreased by 70% in vitro lipid peroxidation mediated by CCl4 metabolites, and decreased by 90% peroxidation mediated by NADPH alone. Silibinin, one of the three isomers composing silymarin, also decreased carbon tetrachloride-induced lipid peroxidation; this effect, however, was less than that of silymarin in vitro, and was more transient in vivo. Pretreatment with silibinin (800 mg/kg i.p.) 30 min before CCl4 (18 microL/kg i.p.) did not improve SGPT activity or liver histology at 24 hr. We conclude that silymarin prevents carbon tetrachloride-induced lipid peroxidation and hepatotoxicity in mice, firstly, by decreasing the metabolic activation of CCl4, and, secondly, by acting as a chain-breaking antioxidant.

Strong inhibition of neuronal nitric oxide synthase by the calmodulin antagonist and anti-estrogen drug tamoxifen.

The anti-estrogen drug tamoxifen (TMX) was found to act as a strong inhibitor of purified neuronal nitric oxide synthase (nNOS) (IC50 = 2 +/- 0.5 microM), whereas it was inactive toward inducible macrophage NOS (IC50 > 100 microM). TMX affected the activation of NOS by calmodulin, as it not only inhibited L-arginine oxidation to nitric oxide and L-citrulline but also NADPH oxidation and calmodulin-dependent cytochrome c reduction catalyzed by nNOS. These results suggest that TMX could exert some of its biological effects by interfering with constitutive NOS-dependent formation of nitric oxide and/or superoxide ion in various tissues.

Recognition and oxidative metabolism of cyclodipeptides by hepatic cytochrome P450.

Possible recognition of peptide derivatives by hepatic cytochrome P450 3A has been suggested by binding and metabolism of numerous pseudopeptidic compounds such as ergot derivatives and cyclosporin. Natural linear or cyclic dipeptides containing hydrophobic amino acids produced by microorganisms and present in mammals are able to interact with the P450 active site through either iron-amine interactions (Type II) or hydrophobic Type I interactions. P450 3A from dexamethasone-treated rats or yeast-expressed P450 human 3A4 are the most potent in such interactions, which are particularly strong with peptides containing a histidyl residue. Some cyclodipeptides are rapidly transformed by rat cytochrome P450 3A to mono- or dihydroxylated metabolites, with turnovers around 3 nmoles min(-1) P450(-1). Linear peptides are poorly transformed in these conditions. This metabolism of cyclodipeptides occurs in 8 species including man. Such interactions and metabolism have only minor consequences in terms of P450 3A binding and metabolism of classical P450 3A substrates. These data reinforce the concept that, in addition to their effect on the regulation of P450 neosynthesis, naturally occurring endogenous peptides are also substrates of P450 3A. The physiological activities of these peptides may be modulated by their metabolism.

Importance of metabolism in pharmacological studies: possible in vitro predictability.

Metabolic transformation of drug leads to the formation of a large number of secondary compounds. These metabolites may (a) participate to the elimination of the patent drug, (b) have similar or different therapeutic effects compared to the parent drug (c) exert toxic effects. Cytochromes P450 are the main enzymes involved in the biotransformation of exogenous drugs, leading to oxidized, reduced or peroxidized metabolites. Different isozymes of P450 are present in already all the organs and differ by their affinity for substrate families. P450 3A is the most abundant P450 protein in the adult human liver and is able to transform hundreds of substrates into either drugs or endogenous compounds such as testosterone. Its catalytic activities are regulated either by induction or by inhibition. Attempts to predict metabolic transformation of a given drug are based on the amount of P450 expressed in heterologous systems, induction, and inhibition experiments and by comparison to classical P450 substrates. Erythromycin metabolism and its P450 effects are used to illustrate the complexity and the consequences of metabolic transformation of a given drug.

A comparison of the protective effects of N-acetyl-cysteine and S-carboxymethylcysteine against paracetamol-induced hepatotoxicity.

The protective effect of the sulphur-containing amino acids N-acetyl-cysteine and S-carboxymethylcysteine against paracetamol-induced hepatotoxicity was evaluated in the hamster by biochemical and histological methods. Of the animals receiving paracetamol alone 25% died within 24 h following administration. All surviving animals showed acute hepatocellular injury and marked loss of cytochrome P-450 and hepatic mixed-function oxidase activities. Simultaneous administration of N-acetylcysteine decreased the mortality rate, partly prevented the paracetamol-induced liver damage and partly restored enzyme activities. Simultaneous administration of S-carboxymethylcysteine with paracetamol afforded no protection. Kidneys from all animals were histologically normal. Human liver microsomes and liver microsomes from 3-methylcholanthrene-pretreated hamsters metabolished paracetamol to intermediate(s) that bind covalently to microsomal proteins. The rate of covalent binding was inhibited markedly by N-acetylcysteine and to a lesser extent by S-carboxylmethylcysteine.

Lack of effect of phenobarbital on ex vivo leukocyte oxidative metabolism in healthy volunteers.

Oxidative metabolism in activated human polymorphonuclears catabolizes leukotriene B4 by a cytochrome P450 omega-hydroxylase and procainamide by a myeloperoxidase. The percentage of leukotriene B4 metabolized by activated human polymorphonuclears and the apparent enzymatic parameters of procainamide metabolism were studied ex vivo in six healthy volunteers before and after phenobarbital intake (100 mg/day) for 10 days and in six healthy control volunteers. No differences were found between groups for the difference in percentage of leukotriene B4 metabolized between day 11 and day 1. The apparent enzymatic parameters, Km and Vm, of procainamide oxidation did not differ significantly between the groups both on day 1 and day 11. These results do not show any evidence of inducibility of leukotriene B4 and procainamide metabolism in human polymorphonuclears. However, a positive correlation between 6 beta OH-cortisol excretion and percentage of leukotriene B4 metabolized was observed on day 11. This study suggests that human polymorphonuclears cytochrome P450 leukotriene B4 omega-hydroxylase and procainamide metabolism is not a useful method to study cytochrome P450 induction in clinical pharmacology.

Specific drug binding to rat liver cytochrome P-450 isozymes induced by pregnenolone-16 alpha-carbonitrile and macrolide antibiotics. Implications for drug interactions.

Clinical interactions of macrolides with various drugs lead to elimination impairment, increase of plasma concentration and overdose-like effects, resulting from modifications of their metabolism. Previous studies have shown that treatment of rats by the macrolide antibiotics of the oleandomycin and erythromycin series lead to the induction of an hepatic cytochrome P-450 which is implicated into their own metabolism. We have characterized PCN or macrolides induced cytochromes P-450 by their specific ability to interact with macrolide derivatives and, using the cytochrome P-450 spectral binding assays, we have shown that some compounds, implicated in drug interaction with macrolides, interact preferentially with the same cytochromes. This strongly suggests that specific blockage of cytochrome P-450 IIIA1 family by macrolides, is responsible for these drug interactions and that these interactions can be predicted easily by simple in vitro tests such as those described herein.

In vivo and in vitro effects of a new macrolide antibiotic roxithromycin on rat liver cytochrome P-450: comparison with troleandomycin and erythromycin.

The effects of a new macrolide antibiotic (Roxithromycin) and one of its major metabolite (RU 39001) on rat hepatic drug metabolizing enzymes were compared to those of erythromycin, erythralosamine and troleandomycin (TAO) both in vitro and in vivo. In contrast to erythromycin, erythralosamine and TAO, roxithromycin and its metabolite RU 39001 exhibit: (i) a very poor affinity for rat liver cytochrome P-450, (ii) an unability to be metabolized into a stable inhibitory metabolite-cytochrome P-450 complex and (iii) a decreased ability to induce liver cytochrome P-450 PCNE, an isozyme implicated in drug associations involving some macrolide antibiotics.

Selective inhibition of the safrole-induced mixed-function oxidase activities by 9-hydroxyellipticine.

Intraperitoneal administration of 9-hydroxyellipticine, a specific cytochrome P-448 inhibitor, inhibited 3-methylcholanthrene-induced cytochrome P-448 activity (ethoxyresorufin O-deethylase, biphenyl 2-hydroxylase) and formation of the safrole carbene ligand complex with this cytochrome, but did not inhibit phenobarbital-induced cytochrome P-450 activity (ethyl-morphine N-demethylase) or formation of the safrole carbene ligand complex with this cytochrome. Biphenyl displaced the 9-hydroxyellipticine ligand from cytochrome P-448 leading to increased free cytochrome, but with no corresponding increase in mixed-function oxidase activity when biphenyl was used as substrate. It is concluded that following dissociation of the ligand complex, 9-hydroxyellipticine, which also exhibits type I binding, competes with biphenyl for the substrate binding site. Administration of 9-hydroxyellipticine to safrole-pretreated rats inhibited the cytochrome P-448-catalysed activity, but had no effect on the cytochrome P-450-catalysed activity. These results indicate that safrole induces a mixture of cytochromes P-450 and P-448 rather than a single novel haemo-protein. The type I substrate biphenyl displaced both the safrole carbene and the 9-hydroxyellipticine ligands from cytochrome P-448 resulting in increased free cytochrome. Displacement of the safrole carbene ligand was accompanied by increased mixed-function oxidase activity but, in contrast, displacement of the 9-hydroxyellipticine ligand resulted in no increase in mixed-function oxidase activity.

Inhibition of cytochrome P-448 mixed function oxidase activity following administration of 9-hydroxyellipticine to rats.

The in vitro inhibitor of mixed-function oxidation, 9-hydroxyellipticine, non-competitively inhibited the binding of the type II substrate, aniline, to cytochrome P-448 of hepatic microsomal preparations from rats pretreated with 3-methylcholanthrene. In contrast, 9-hydroxyellipticine did not inhibit the binding of aniline to cytochrome P-450 of hepatic microsomal preparations from rats pretreated with phenobarbitone, nor did it inhibit the binding of the type I substrate, hexobarbitone to either cytochrome P-450 or cytochrome P-448. Following the pretreatment of rats intraperitoneally with 9-hydroxyellipticine and phenobarbitone, the cytochrome P-448-specific enzyme activity, ethoxyresorufin O-deethylase, was 50% inhibited in vitro but cytochrome P-450, cytochrome P-450 reductase, and other mixed function oxidase activities were unaffected. With rats pretreated with 9-hydroxyellipticine and 3-methylcholanthrene, inhibition of ethoxyresorufin O-deethylase was 90%, and cytochrome P-450/P-448, cytochrome P-450 reductase, biphenyl 2- and 4-hydroxylase were inhibited by 30, 15, 50 and 40% respectively. It is concluded that 9-hydroxyellipticine administered in vivo markedly inhibits mixed-function oxidations which are specific to cytochrome P-448, but has no effect on cytochrome P-450-catalysed microsomal oxidation.