Pierre Lecointe

Ellipticines as potent inhibitors of aryl hydrocarbon hydroxylase: Their binding to microsomal cytochromes p450 and protective effect against benzo(a)pyrene mutagenicity

Abstract Ellipticine (5,11 -dimethyl-6-H-pyrido[4-3b]carbazole) and its derivatives bind strongly to the oxidized and reduced liver microsomal cytochromes P450 of differently pretreated rats, producing typical difference spectra, with peaks respectively at 428 (ox.) and 445 nm (red.) (with spectral dissociation constants around 10 −6 and 10 −5 M, respectively). The high affinity of ellipticine for microsomes containing a great proportion of cytochrome P448, explains its role of strong inhibitor of benzo(a)pyrene hydroxylase. Accordingly, we found a good correlation between the binding properties of the ellipticines and their inhibitory effect upon: (1) the microsomal formation of water soluble metabolites of benzo(a)pyrene, (2) the covalent binding of reactive metabolites of this hydrocarbon to DNA, and (3) the mutagenic activity of benzo(a)pyrene in the Salmonella typkimurium test.

Relative efficiencies of a series of square-planar platinum(II) compounds on Salmonella mutagenesis

Twelve Pt(II) compounds have been tested for mutagenicity on Salmonella typhimurium (strain TA 100). Very high mutagenic activities were found for the cis derivatives. A correlation is suggested between these results and a formerly described model of chemical reactivity towards DNA, according to which cis derivatives from intra-strand chelates with guanine. A smaller activity was found with monodentate complexes with DNA.

Comparative cytotoxic and antitumoral effects of ellipticine derivatives on mouse L 1210 leukemia.

Twelve derivatives of the antitumoral alkaloid ellipticine (E) and ellipticinium were assayed in vitro on cultured L 1210 cells. These drugs possess varying abilities to decrease the cell growth rate in a 1--1000-fold range. Some of them have a highly cytotoxic effect in the 10(-8)--10(-6) M range. Non-specific intracellular damages are produced: multilobation of nuclei, occurrence of numerous lipid granules, diminution of the size and increase in the number of mitochondrial profiles and several modifications of the internal architecture of mitochondria. 2-Methyl-9-hydroxyellipticinium (2-CH3-9-OHE) was submitted to a bioassay; it inactivates the tumorigenic potency of the cells exposed to it, when they are grafted back into mice in the same dose range which reduces in vitro the growth rate of the cells. A fairly good correlation holds between the in vitro and in vivo (antitumor effect) assays, offering a possible prescreening test for a cheaper and rapid evaluation of chemotherapeutic activity of these compounds. The results stress again the importance of the 9-hydroxy substitution in these series for improving the anticancer efficiency. The nature of the biochemical target of E and derivatives is discussed according to our data.

The hydroxylation of the antitumor agent, ellipticine, by liver microsomes from differently pretreated rats

Abstract The antitumor agent, ellipticine (5,11-dimethyl-6H-pyrido[4-3,b]carbazole) is mainly hydroxylated in position 9 by liver microsomes of differently pretreated rats, this result being in agreement with that obtained previously in vivo. A quick and reliable fluorometric assay, based on the differential fluorescent properties of ellipticine and 9-hydroxyellipticine, is described for the measurement of the 9-hydroxylase activity of different microsomes. This activity exhibits the usual features of the cytochrome-P450-dependent monooxygenases. Control rat liver microsomes exhibit a good affinity for ellipticine (Km = 3 × 10−5 M) but a low specific activity (0.1 nmole min−1 mg protein −1), perhaps related with an excess substrate inhibition. Pretreatment of rats with benzo[a]pyrene or ellipticine enhances the rate of 9-hydroxylation: pretreatment with phenobarbital does not. Metyrapone and 7,8 benzofiavone are poor inhibitors of ellipticine hydroxylation particularly in microsomes from benzo[a]pyreneor ellipticine-pretreated rats.

Ellipticines as potent inhibitors of microsomes- dependent chemical mutagenesis

9-Hydroxyellipticine (9-OHE), an inhibitor of microsomal monooxygenase activities has been shown to exert a large or even complete decrease of the mutagenicity, on the Salmonella strains of a great number of compounds (aromatic amines, polycyclic aromatic hydrocarbons, fungal toxins, azo compounds, tobacco smoke condensate). 9-OHE and 9-fluoroellipticine are more potent inhibitors than ellipticine itself. The inhibitions exerted by 9-OHE are not even equalled by 10-fold higher doses of 7,8-benzoflavone (7,8-BF). There is a good correlation between these data and the interaction properties of ellipticines with microsomal cytochromes P-450.

Structure-activity relationships in the inhibitory effects of ellipticines on benzo(a)pyrene hydroxylase activity and 3-methylcholanthrene mutagenicity.

Abstract The structural features which determine the ability of ellipticine (5,11dimethyl6Hpyrido[4-3b] carbazole) and its derivatives to interact with cytochrome P-450 and to inhibit rat liver microsomal benzo(a)pyrene hydroxylase as well as to inhibit the mutagenicity of 3-methylcholanthrene have been studied. Spectral interactions studies were carried out with either Aroclor 1254-, 3-methylcholanthrene-or phenobarbital-induced microsomes. Inhibitory activities towards benzo(a)pyrene hydroxylase and 3-methylcholanthrene mutagenicity (Ames test), were determined using Aroclor 1254-induced microsomes. It appears that every ellipticine derivative having significant inhibitory effects on hydroxylation of benzo(a)pyrene or mutagenicity of 3-methylcholanthrene also exhibits a very good affinity for microsomal cytochromes P-450. The accessibility of the pyridinic nitrogen of ellipticine derivatives appears as the most important factor for their binding to cytochromes P-450 and the presence of methyl groups in 5 and 11 positions of ellipticine derivatives is an essential condition for the expression of the inhibitory power. Various substitutions in the A ring of ellipticine appear to be of secondary importance. On the other hand the location of the pyridinic ring and consequently the arrangement of the molecule within the hydrophobic pocket of cytochrome P-450 seems also to play an important role in the inhibitory power since isoellipticines are devoid of such properties. These results should help in the design of particularly efficient inhibitors of drug and carcinogen metabolism.

Correlation between the toxicity of platinum drugs to l1210 leukaemia cells and their mutagenic properties.

Abstract There is a good correlation between the growth inhibitory properties of a series of Pt(II) derivatives on cultured L1210 mouse leukaemia cells and their efficiency of reversion to his + of S. typhimurium TA100, or with their forward mutagenic effects on prophage λ. Reactivity towards DNA may thus explain the antitumor properties of these drugs.

The hepatic metabolism of ethidium bromide to reactive mutagenic species: biochemical and structural requirements

Abstract Ethidium bromide, which was known to undergo N -acetylation in rats, is shown to be metabolized by rat liver cytochrome P448 and a soluble protein to one or several reactive species which are detected in the Salmonella assay. This biotransformation depends on the presence of a free aromatic amino group at position-3, on the basis of a structure-mutagenic activity study of eleven phenanthridine derivatives. Using the Salmonella assay as a biological test for the production of reactive intermediates, we have attempted to purify this enzyme and demonstrated its lack of N -acetyl- or sulfotransferase activity. Ethidium bromide thus behaves as a mutagenic aromatic amine, the bioactivation of which differs from previously described mechanisms.

Ellipticines as potent inhibitors of drug metabolism. Protective effect against chemical mutagenesis and carcinogenesis

Summary Ellipticine and its derivatives (ellipticinium salt excluded) bind strongly to the oxidized and reduced liver microsomal cytochromes P450 of differently pretreated rats, producing typical difference spectra, with peaks respectively at 428 nm for the oxidized form and 445 nm for the reduced form. The high affinity of ellipticines for cytochromes P450 (spectral dissociation constants around 10−6 M (ox.) and 10−5 M (red)) explains their role of strong inhibitors of various liver monooxygenase activities. Accordingly, it has been shown that 9-hydroxy-ellipticine exerts a large or even complete decrease of the mutagenicity, on the Salmonella strain, of a great number of compounds such as aromatic amines, polycyclic aromatic hydrocarbons, fungal toxins, azo compounds and tobacco smoke condensate.

Correlation between the in vivo effects of some griseofulvin derivatives and their in vitro interactions with mammalian microtubules

Griseofulvin, an antifungic metabolite produced by several species of Penicillium [ 1 ] , is widely used in human and animal therapy [2]. Its mechanism of action remained puzzling for along time [3]. Recently it has been shown that griseofulvin inhibits the polymerization of purified brain tubulin [4,5]. Although this property may account for its in vivo effects, this result does not allow to rule out the hypothesis that microtubule proteins may not be the main target of this drug [6]. In order to check this possibility, several griseofulvin derivatives exhibiting a lower or a higher activity than griseofulvin, were synthesized. Their in vivo effects were studied on a mouse leukemia cell line (L1210) and on a Myxomycete (Physarum). Their in vitro action was studied by monitoring the induced aggregation and the inhibition of polymerization of purified brain mammalian microtubule proteins [4,5]. It was found that the in vitro activities of the various griseofulvin derivatives were in correlation with their in vivo activities suggesting that microtubule proteins do represent the pharmacological target ofgriseofulvin.