Evidence is provided that the amplifiers luminol and lucigenin react with different reactive oxygen species (ROS), depending on the ROS-generating system used. H2O2 is used to produce calibration curves for luminol- and lucigenin-amplified chemiluminescence. With this chemiluminescence generator we characterized the specificity and sensitivity of luminol- and lucigenin-amplified chemiluminescence and also studied penicillin G, a known enhancer of luminol-amplified chemiluminescence. The combination of luminol and lucigenin in reciprocally changing concentrations is effective in an additive manner, but the weak amplifier penicillin increases luminol-amplified chemiluminescence distinctly more than in an additive manner in different combinations. Lucigenin-amplified chemiluminescence is increased by penicillin at about 1% of the optimum concentration of penicillin; increasing concentrations of penicillin are less and less effective. On the other hand, low lucigenin concentrations enhance penicillin-amplified chemiluminescence at optimum penicillin concentrations more than in an additive manner. Fe2+ does not alter luminol-, lucigenin- or penicillin-amplified chemiluminescence. Co2+ increases luminol-amplified chemiluminescence by a factor of 100. Lucigenin- and penicillin-amplified chemiluminescence are minimally enhanced by Co2+. Cu2+ enhances luminol-amplified chemiluminescence with increasing concentrations by a factor of 1000. Lucigenin-amplified chemiluminescence increases also by the factor of 1000, but the concentration-reaction curve is not as steep. NaOCl enhances H2O2/Fe(2+)-driven luminol-amplified chemiluminescence in a concentration-dependent manner by a factor of 10(4) (in the highest concentration of 10 mmol/L) and lucigenin amplified chemiluminescence only by a factor of about 25. Catalase (CAT) abolishes luminol-, lucigenin- and penicillin-amplified chemiluminescence completely, whereas superoxide dismutase (SOD) has no effect on luminol- or penicillin-amplified chemiluminescence, but enhances lucigenin-amplified chemiluminescence five-fold increasingly with increasing SOD activity.

Acridines↗

PubMedDate not supplied

Interaction of chlormezanone with rat liver microsomes and its degradation.

Chlormezanone binds to oxidized cytochrome P450 in rat liver microsomes with a binding curve according to type I like hexobarbital but less pronounced and with a general shift to the left. Ethylmorphine N-demethylation, ethoxycoumarin and ethoxyresorufin O-deethylation are inhibited by chlormezanone in mM concentrations only whereas pentoxyresorufin O-depentylation is inhibited by about 50% in microM concentrations. Luminol and lucigenin amplified chemiluminescence indicating the formation of reactive oxygen species was not influenced in concentration ranges between mM and microM, whereas NADPH/Fe stimulated lipid peroxidation showed a tendency of inhibition. But scavenger activity could not be demonstrated: the zymosan stimulated chemiluminescence of whole blood was not influenced significantly. The degradation process of chlormezanone was elucidated. The first step involves ring opening by chemical hydrolysis with subsequent formation of an unstable acylhalfaminal which is the source of 4-chlorobenzaldehyde. This aldehyde undergoes enzymatically controlled oxidation to 4-chlorobenzoic acid which is the parent compound of following phase II reactions. The second degradation product is 2-carboxyethane-sulfinic-acid-N-methylamide, which is hydrolyzed very quickly. Neither oxidation of the sulfinic acid or its N-methylamide derivative could be observed nor N-demethylation of chlormezanone.

Animals↗

PubMedDate not supplied

Interaction of chlormezanone enantiomers with rat liver microsomes.

Both chlormezanone enantiomers, for the first time obtained by enantiospecific HPLC with a 100% yield, bind to oxidized cytochrome P-450 in rat liver microsomes with a binding curve according to type I, similar to hexobarbital but less pronounced. There are no differences between the binding curves of the two enantiomers. Ethylmorphine N-demethylation, ethoxycoumarin and ethoxyresorufin O-deethylation are inhibited by both chlormezanone enantiomers at 0.1-1 mM concentrations: no differences could be found. Luminol and lucigenin amplified chemiluminescence indicating the formation of reactive oxygen species was not influenced by either enantiomer in concentration ranges between millimolar and micromolar, whereas hydrogen peroxide formation was inhibited. NADPH/Fe stimulated lipid peroxidation was not influenced. Scavenger activity could not be demonstrated: the zymosan stimulated whole blood chemiluminescence was not influenced significantly.

Acridines↗

PubMedDate not supplied

Interactions of melatonin with the liver microsomal cytochrome P450 system of rats and humans in vitro and effects on the P450 system and the antioxidative status in rat liver after acute treatment.

In vitro melatonin binds to human and rat liver microsomal cytochrome P-450 (P450) according to a type II substrate. The affinity is similar to that of aniline with a general left-shift. Melatonin interferes with model monooxygenase reactions indicative of different P450 forms in humans and rats (in humans according to the lower specific P450 content less pronounced): the strongest inhibition was found for ethoxyresorufin O-deethylation, indicating the binding to P450 1A, the binding to P450 2B (ethoxycoumarin O-deethylation) was less pronounced, the least inhibition was found for P450 3A (ethylmorphine N-demethylation) reaction. The oxidase function was also inhibited: luminol amplified chemiluminescence was more inhibited than the lucigenin amplified one, hydrogen peroxide formation was inhibited at concentrations higher than 10(-4) M, microsomal NADPH/Fe stimulated lipid peroxidation was inhibited at concentrations higher than 10(5) M. In vivo melatonin prolonged hexobarbital sleeping time in rats in a dose dependent manner (ip. co-administration of 1, 5 and 20 mg/kg b.w. melatonin with 100 mg/kg hexobarbital). Immediately after awakening the animals were sacrificed: a small increase in P450 concentrations cannot be explained, no changes in P450 monooxygenase or oxidase activities nor in microsomal lipid peroxidation or GSH status could be observed.

Aniline Compounds↗

PubMedDate not supplied

In vivo and in vitro inhibition of 3-methylcholanthrene-induced aryl hydrocarbon hydroxylase activity in rat liver by actinomycin D and 7,8-benzoflavone.

Using the very well investigated and sensitive microsomal monooxynogenase aryl hydrocarbon hydrolase (AHH), we investigated the dose-response relations between an inducer, 3-methylcholanthrene (3-MC), and an inhibitor of transcription, actinomycin D (ACT D), in 10 days old rats. An optimum pretreatment schedule proved to be a single dose of 1 to 40 mg/kg 3-MC ip combined with two administrations of 200 to 600 microgram/kg ACT D within 16 hr. With increasing doses of ACT D the inhibition of 3-MC-mediated AHH induction increased up to 88%. With 7,8-benzoflavone (ANF) as a relatively specific in vitro inhibitor of the cytochrome P-448 species no distinct influence on the constitutive AHH activity could be observed up to the 30th days of age. In 60 and 200 days old rats the inhibition reached a significant level of about 50%. After 3-MC induction a nearly uniform ANF-mediated inhibition by about 50% of the hepactic AHH activity was observed: in older animals the percentage became slightly higher. The investigation appears to confirm the change in cytochrome pattern,--especially as for the participation of cytochrome P-448,--according to the age and induction stimulus by 3-MC.

Age Factors↗