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International audience; Glutathione transferases (GSTs) represent a widespread enzyme superfamily in eukaryotes and prokaryotes catalyzing different reactions with endogenous and xenobiotic substrates such as organic pollutants. The latter are often found together with metal contamination in the environment. Besides performing of essential functions, GSTs protect cells by conjugation of glutathione with various reactive electrophiles. The interference of toxic metals with this functionality of GSTs may have unpredictable toxicological consequences for the organisms. In this review results from the recent literature are summarized and discussed describing the ability of metals to inhibit intracellular detoxification processes in animals and plants.

Four new platinum(II) complexes of 3-aminocyclopentanespiro-5-hydantoin (acpsh) and 3-aminocycloheptanespiro-5-hydantoin (achpsh) were synthesized and characterized by elemental analysis, IR and 1NMR spectra. The spectral analyses indicated a cis-square planar structure of the complexes with ligands coordinated via the NH2 group. The complexes were evaluated for in vitro cytotoxicity in murine erythroleukemia (MEL) cells, clone F4N, using cell-growth and macromolecular synthesis assay. The compounds, with exception of [Pt(NH3)(achpsh)Cl2] (IV), exhibited much lower cytotoxicity than that of cisplatin (DDP). Compound IV was nearly as cytotoxic as DDP. The new complexes exerted low antibacterial activity as assessed by seven bacterial strains

The induction of HSP90 in murine erythroleukemia cells, clone F4 N, by cisplatin (DDP) was examined using indirect immunofluorescence and avidin-biotin technique, and compared with cisplatin cytotoxicity. A reverse dependence of HSP90 induction time was found on a wide range of cisplatin concentrations (0.5D10 μᴍ), which proved to be cytostatic up to 48 h of continuous treatment. Thus, the observed induction pattern of HSP90 in F4 N cells strictly correlated with their high tolerance toward DDP. This indicates that HSP90 might be responsible, at least in part, for cisplatin resistance of F4 N cells

The combination of cis-diamminedichloroplatinum (II) (DDP, cisplatin) and topoisomerase II inhibitor teniposide (VM-26) has been shown to exert a synergistic effect in the clinical treatment of cancer. In this study, the combined effect of DDP and VM-26 on the growth and induction of apoptosis in synchronized murine erythroleukemia (MEL) cells, treated at the beginning or in the middle of S-phase of cell cycle, was examined. MEL cells, clone F4 N, were synchronized by a double thymidine block leading to accumulation of 70% of cells at the G1/S boundary. The growth-inhibitory effect of DDP and VM-26 applied alone were stronger in the middle of the S-phase than at the beginning. Morphological analysis showed that the majority of the cells revealed typical signs of apoptosis: nuclei fragmentation and appearance of apoptotic bodies. The combination of both agents at low concentrations had a synergistic effect on cytotoxicity. At higher concentrations the effect was additive. The remainder of the cells were characterized by unbalanced growth, aberrant mitosis and appearance of multinucleated cells. These processes led to delayed cell death. The appearance of aberrant mitosis was more expressed after treatment in the middle of the S-phase. It is likely that as a result of the combined action of cisplatin and VM-26, cells become supersensitive to the ability of topoisomerase II inhibitor to influence mitosis, and this increased sensitivity may contribute to the observed synergism.

Glucuronidation is a major detoxification pathway in vertebrates. The reaction is catalyzed by a family of UDP-glucuronosyltransferases (UGTs) and involves conjugation of many endobiotic and xenobiotic substances with glucuronic acid, forming inactive water-soluble glucuronides. UGT prevents the accumulation of potentially toxic compounds and/or their subsequent bioactivation to more toxic intermediates, although biologically active glucuronides are also known. Impairment of UGTs may have important toxicological consequences. Substances found to inhibit or down-regulate UGT activity include endogenous compounds, a wide range of clinically used drugs, environmental contaminants, and natural toxic substances present in the diet. The development of selective, active-site-directed UGT inhibitors greatly enables the study of various UGT isoenzymes. A promising approach offers the design of transition-state analogs of the glucuronidation reaction.

Twelve herbicides, representatives of two chemical groups, substituted phenoxyalkanoic acids and s-triazines, were tested for their inhibitory effect on the glucuronidation of 4-nitrophenol (4-NP), phenolphthalein (PPh) and 4-methylumbelliferone (4-MU) by rat liver microsomes. One millimole MCPA, ametryn and cyanazine significantly decreased PPh UDP-glucuronosyltransferase (UGT) activity, while propazine was found to be a most potent inhibitor of 4-NP glucuronidation. Concentrations of 0.1 mM dichlorprop and cyanazine were still inhibitory against PPh-UGT. The inhibition of 4-MU glucuronidation by the herbicides was low and not specific. As a whole, s-triazine derivatives were more inhibitory than the substituted phenoxyalkanoic acids. Kinetic studies with propazine revealed a non-competitive type of inhibition towards the acceptor substrate 4-NP, with an apparent Ki value of 0.540 mM. With ametryn, an uncompetitive type of inhibition against PPh and a mixed type of inhibition towards UDPGA were found, with apparent Ki values of 0.330 mM and 0.380 mM, respectively. © 1999 Society of Chemical Industry

In order to design potential inhibitors of UDP-glucuronosyltransferase, the synthesis of some 5′-O-oligopeptide derivatives of uridine is presented. 5′-O-(N-tert.Butyloxycarbonyl-O-benzyl-L-seryl-L-valyl)-2′,3′-O-isopropylideneuridine (1) was synthesized by the DCC/HOBt method from N-tert.butyloxycarbonyl-O-benzyl-L-serine and 5′-O-L-valyl-2′,3′-O-isopropylideneuridine in 95% yield. In a similar way, 5′-O-(N-tert.butyloxycarbonyl-L-valyl-O-benzyl-L-seryl)-2′,3′-O-isopropylideneuridine (2) was obtained from N-tert.butyloxycarbonyl-L-valine and 5′-O-(O-benzyl-L-seryl)-2′,3′-O-isopropylideneuridine in 93% yield. Treatment of 1 and 2 with HCl/EtOAc at room temperature for 30 min led to removal of both Boc and 2′,3′-O-isopropylidene groups. 5′-O-(O-Benzyl-L-seryl-L-valyl)-uridine (3) and 5′-O-(L-valyl-O-benzyl-L-seryl)-uridine (4) were obtained in 94% and 91% yields, respectively.

New platinum(II) complexes of cyclohexanecarboxylic acid hydrazide (chcah) were synthesized and characterized by elemental analysis, IR. and 1H NMR spectra. Their inhibitory effects on cell growth and macromolecular synthesis of Friend leukemia cells in culture as well as the in vivo antitumor activity towards L1210 leukemia in mice were compared with those of complexes containing differently substituted aromatic acid hydrazides. Some of the complexes exhibited antineoplastic activity. No correlation between the in vitro cytotoxicity and the in vivo antitumor activity was found. However, there was a relationship between the in vitro macromolecular synthesis inhibition profile and the in vivo antineoplastic effect, similar to that of cisplatin. On the other hand, only agents containing one ammine ligand were active in vivo. The substitution of the aromatic ring by a cycloalkane residue increased significantly the antitumor effect, with [Pt(NH3)(chcah)Cl2] being the most active compound in this study.

In an attempt to develop potential inhibitors ofUDP-glucuronosyltransferase, some 5′-O-amino acid derivatives of uridine were synthesized. N-protectedL-amino acids were coupled at the 5′-O-position of 2′,3′-O-isopropylideneuridine by esterification employing the method of symmetrical anhydrides in presence of 4-dimethylaminopyridine, 5′-O-(N-benzyloxycarbonyl-O-tert.butyl-L-threonl)-2′3′-O-isopropylideneuridine (1), 5′-O-(N-tert.butyloxycarbonyl-O-benzyl-L-seryl)-2′,3′-O-isopropylideneuridine and (2), 5′-O-(N-tert.butyloxycarbonyl-L-valyl)-2′,3′-O-isopropylideneuridine (3), and 5′-O-(N-tert.butyloxycarbonyl-L-valyl)-2′,3′-O-isopropylideneuridine (4) were obtained in good yield after column chromatography on silica gel. The treatment of2 withTFA/CH2Cl2 (6:1) at room temperature for 30 min led to a selective removal of theBoc group without deblocking of the 2′,3′-O-isopropylidene group of uridine. Treatment of2 withTFA/H2O (5:1) at room temperature for 1 h, however, released bothBoc and 2′,3′-isopropylidene groups. TheZ group of1 was deprotected by catalytic hydrogenolysis over 10% Pd/C/ammonium formate.

Fifteen pesticides, representatives of different chemical groups, were tested for their inhibitory effect on the glucuronidation of 4-nitrophenol (4-NP) and phenolphthalein (PPh) by rat liver microsomes. Three herbicides (simazine, chlorsulfuron, tribenuron-methyl), two insecticides (dioxacarb, carbaryl) and one fungicide (zineb) significantly decreased the UDP-glucuronosyltransferase (UDPGT) activity. The carbamate insecticide dioxacarb was found to be the most potent inhibitor, at 1 mM concentration suppressing 4-NP-UDPGT activity completely, and reducing by 55% the activity associated with the conjugation of PPh. One millimole simazine and carbaryl affected only 4-NP glucuronidation, while chlorsulfuron and zineb exerted a marked inhibition of both 4-NP and PPh conversion. Concentrations of 0.1 mM carbaryl, dioxacarb and zineb were still inhibitory against 4-NP-UDPGT, with zineb producing 40% inhibition of PPh glucuronidation. As a whole, UDPGT isoforms conjugating PPh were less sensitive to the agrochemicals tested. Kinetic studies with dioxacarb, chlorsulfuron and carbaryl revealed a mixed type of inhibition with respect to the acceptor substrate 4-NP, with apparent K i values of 70 μM, 120 μM and 160 μM, respectively.

The complexes [Pt(bah)2X2], [Pt(NH3)(bah)Cl2].0.5H2O, [Pt(mbah)2X2], and [Pt(NH3)(mbah)Cl2] (bah = benzoic acid hydrazide, mbah = 3-methoxybenzoic acid hydrazide; X = Cl, Br, I) have been prepared and characterized by elemental analysis, electric conductivity, IR, 1H NMR, and electronic spectra. A cis-square planar structure with hydrazide ligands coordinated via the NH2-groups has been proposed for these complexes. The complexes have shown a growth-inhibitory effect against Friend leukemia cells in culture comparable to that of the antitumor drug cisplatin.

In this study we have extended the screening of some uninvestigated differently substituted pyrimidine bases, 2,2'-anhydropyrimidine nucleoside and pyrimidine nucleoside analogs against Uridine phosphorylase of rat liver. The most active compounds were also tested against thymidine phosphorylase of mouse liver

Induction of heat shock protein (Hsp) 70 and distinct metallothionein-like proteins (MTLPs) in response to Cd and heat treatment were studied in two strains of the aquatic hyphomycete Heliscus lugdunensis: Hl-H4, isolated from a heavy metal polluted site, and Hl-BB taken from an unpolluted area. Upon Cd-exposure, Hsp70 was actively synthesized in the strain Hl-H4, and to a much lower degree in the strain Hl-BB. The Hsp70-expression was time- and dose-dependent, reaching a maximum after 24 h incubation with 80 microM Cd. Upon heat-stress, a similar response was observed: a strong Hsp70-expression in Hl-H4, and only a marginal one in Hl-BB. The strains reacted to Cd-exposure by a specific, environmentally related induction of MTLPs, as shown by the highly sensitive bimane derivatisation method of SH-rich proteins. In Hl-H4, a strong expression of 11 kDa MTLP was registered, which followed strictly the induction pattern of Hsp70. This suggests interdependence of the induction mechanisms and roles of these stress proteins in metal resistance. On the contrary, in Hl-BB a weak expression of MTLP of about 20 kDa was observed, exhibiting completely different induction pattern. The results suggest that the specific induction of Hsp70 and/or distinct MTLPs in the range of 11 kDa in H. lugdunensis strain Hl-H4 are essential adaptive mechanisms to continuous heavy metal exposure.

The aquatic hyphomycete Heliscus lugdunensis belongs to a group of exclusively aquatic mitosporic fungi with an only scarcely explored potential to oxidatively attack xenobiotic compounds, and was used to study the biotransformation of the environmental pollutant metabolite 1-naphthol. H. lugdunensis metabolized approximately 74% of 1-naphthol within 5 days. The identification and quantification of degradation products using gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and high performance liquid chromatography revealed that approximately 12% of the parent compound was converted into 1-naphthylsulfate, 3% was transformed into 1-methoxy-naphthalene, and less than 1% was converted into 1,4-naphthoquinone. A further metabolite, most likely 4-hydroxy-1-naphthylsulfate, was also detected. In contrast to sulfate conjugate metabolites, no glucuronide and glucoside conjugates of 1-naphthol were found, and neither UDP-glucuronyltransferase nor UDP-glucosyltransferase present in H. lugdunensis showed activity towards 1-naphthol. These results support a role of fungi adapted to aquatic environments in affecting the environmental fate of pollutants in aquatic ecosystems.

The canavanine derivatives L-canavanine hydrazide (CH), L-canavanine-bis-(2-chloroethyl)hydrazide (CBCH) and L-canavanine phenylhydrazide (CPH) were synthesized and evaluated for biological activity in microorganisms, plants and tumor cells using canavanine as a positive control. (1) In microbial systems, the compounds exerted activity, as assessed in 14 bacterial strains. The effect of canavanine was easily removed by equimolar concentrations of arginine or ornithine, while the effect of CBCH or CPH was abolished by 10-fold excess of arginine or 10- to 100-fold excess of ornithine. (2) In plants, the activity of CH and CBCH were relatively low, whereas the inhibitory potential of CPH was comparable or even superior to that of canavanine, resulting at 1 mM concentration in a nearly complete block of tomato cell growth, and reducing by up to 80% the length of radicles of cress, amaranth, cabbage and pumpkin. (3) In pumpkin seeds, CPH or canavanine induced the synthesis of four small heat shock proteins of hsp-17 family in the pH range of 6 to 7.5. The proteins exhibited in both cases a similar profile, but differed in the timing of their expression and/or accumulation. With canavanine, the highest hsp-17 expression was found after 48 h of drug treatment, while with CPH this maximum was shifted to 24 h. (4) CPH proved to be highly cytotoxic against Friend leukemia cells in culture, exceeding by one order of magnitude the cytotoxicity of canavanine. The effect of canavanine was completely removed in the presence of equimolar amounts of arginine, while a 20-fold excess of arginine failed to abolish the cytotoxicity of CPH. Thus, a proper hydrazide modification of canavanine may lead to a significant increase in its growth-inhibitory activity and to a change in the mode of action of the parent compound.

New platinum(II) complexes of cyclopentanecarboxylic acid hydrazide (cpcah) were prepared, characterized by elemental analysis, IR and 1H NMR spectra, and evaluated for in vitro cytotoxicity in Friend leukemia (FL) and A2780 ovarian tumor cells, induction of apoptosis in FL cells, as well as for in vivo antitumor activity toward murine L1210 leukemia and Lewis lung carcinoma. The spectral analyses indicated a cissquare planar structure of the complexes with hydrazide ligand coordinated via the NH2 group. The compounds exerted significantly lower in vitro and in vivo toxicities as compared with those of cisplatin (cis-diamminedichloroplatinum(II), DDP). On the other hand, the complex [Pt(NH3)(cpcah)Cl2] exhibited antitumor activity against L1210 leukemia in mice comparable to that of cisplatin, resulting at a dose of 42 mg/kg (administered 3 times) in a T/C (mean survival time) of 280%. This compound displayed an in vitro macromolecular synthesis inhibition pattern similar to that of DDP. At concentrations close to the cytostatic ones (10–20 μM) this complex, as well as DDP, was able to induce apoptosis in FL cells as shown by neutral comet assay and morphological analysis. We concluded that there is a correlation between the ability of platinum complexes to induce apoptosis and their antitumor activity.

The inhibitory effect of a series of 5′-O-amino acid and oligopeptide derivatives of uridine on rat liver UDP-glucuronosyltransferase (UGT) activities was investigated using two assay systems. A quantitative structure-activity relationship (QSAR) study was performed. The compounds include a lipophilic residue linked to the nucleoside by a variable spacer. More­ over, half of the derivatives have two spacers linked to the uridine moiety. Compound 1, a serine derivative of isopropylideneuridine, was found to be the most potent inhibitor of both 4-nitrophenol (4-NP) and phenolphthalein (PPh) glucuronidation, with an I50 of 0.45 mᴍ and 0.22 mᴍ , respectively. Kinetic studies with this substance revealed a mixed type of inhibition towards 4-NP and UDP-glucuronic acid, with apparent Ki values of 150 μᴍ and 120 μᴍ , respectively. The dipeptide derivatives 11-14 exhibited a low activity against 4-NP conjuga­ tion. However, a marked suppression of PPh glucuronidation was found with compounds 11 and 13. Generally, compounds with two spacers are more inhibitory against the UGT activi­ties studied. The QSAR analysis outlined the significance of the spacers with a minimum length of 5 atoms and lipophilic residues linked to them for the inhibitory effect of the compounds. The most significant contribution to this effect is given by the six-atom spacer for both, 4-NP and PPh substrates. 4-NP converting UGT isoforms seem to respond more specifically to the inhibitors: a five-atom for the first and a six-atom for the second spacer enhance binding to both 4-NP and PPh conjugating isoenzymes, while a long second spacer contributes to inhibitor binding to UGT isoforms only converting PPh.

Cis-diamminedichloroplatinum(II) (cis-DDP) is known as an effective anticancer drug. Its therapeutic effect is supposed to be a consequence of the covalent binding to DNA. A number of cellular proteins were found to bind selectively to DNA modified by cis-DDP (but not by its isomer frans-DDP). Here we present our observations on interaction of the linker histone H1 with cis- and trans-DDP modified DNA fragments. The results afford new experimental information about the preferential binding of histone H1 to cis-DDP-distorted DNAs versus trans-DDP modified ones.

The complexes [Pt(NH3)(pmbah)Cl2], [Pt(NH3)(pcbah)Cl2], [Pt(pmbah)2X2] and [Pt(pcbah)2X2] (pmbah = 4-methoxybenzoic acid hydrazide, pcbah = 4-chlorobenzoic acid hydrazide; X = Cl, Br, I) have been synthesized and characterized by elemental analysis, electric conductivity, 1H NMR, IR, and electronic spectra. A cis-square planar structure with hydrazide ligands coordinated via the NH2 groups has been proposed for these compounds. The complexes, but not the free ligands, have shown a strong growth inhibitory effect in Friend leukemia cells in vitro, most of which are more active than cisplatin.

The inhibition of [14C]thymidine incorporation into DNA of tumor and normal tissues of L1210-leukemia-bearing mice by single doses of cis-diamminedichloroplatinum (II) (cisplatin, cis-DDP) and two newly synthesized platinum (II) complexes containing as ligands dimethyl aminomethylphosphine oxide (complex I) and methyl bis(aminomethyl)phosphine oxide (complex II) was studied and used as an indication of drug toxicity. All three complexes caused selective inhibition of precursor incorporation in L1210 cells as compared to host tissue cells. cis-DDP caused a complete block of incorporated thymidine in tumor cells during more than 48 h, whereas in intestinal mucosa and bone marrow reverse inhibition was observed. In spleen, liver and kidney the inhibition was about 50% and endured up to 96 h without reversal. Complex I treatment of L1210 cells resulted in an earlier recovery of thymidine incorporation into DNA in comparison with cis-DDP. Towards all other normal tissues compound I was less toxic than cis-DDP. Unlike cis-DDP and complex I, complex II was less active against L1210 cells and most toxic against bone marrow and kidney.

1. 1. The in vitro inhibition of purine biosynthesis de novo by a series of cyanopyrazoles was studied. 2. 2. At concentration 1 mM trichloromethyl analogs (3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole and N-hydroxyethyl-3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole) were found to inhibit IMP synthesis 80 and 30% respectively. 3. 3. GAR synthesis was inhibited at a lower degree at the same range of concentrations. 4. 4. The compounds demonstrated a similar pattern of inhibition of the last steps, e.g. AICAR formylation and cyclization as found on the whole pathway.

The pyruvic acid thiosemicarbazone was found to inhibitin vitro the soluble ornithine carbamoyl transferase as well as the enzyme in a mitochondria-rich cell fraction. For proving the inhibition of ornithine carbamoyl transferase in mitochondria-rich fraction a new method for DEAE-thin-layer chromatography of radiocarbon labelled citrulline and ornithine was used. The mitochondria prepared by discontinuous sucrose density gradient centrifugation are physiologically active and oxidized succinate under state 3 conditions. The respiratory control ratio was determined to 3.3. The results obtained show that this analogue of pyruvic acid is an inhibitor of ornithine carbamoyl transferase.

RNA, DNA and ribonucleoprotein particles can be recovered from solutions containing less than 0.1 A 260 -units/ml by co-precipitation with magnesium phosphate.

The inhibitory effect of some pyrazole derivatives on GAR synthesis was studied. It was found that nonalkylating analogues and chloroethyltriazeno analogues inhibited GAR synthesis. A possible mechanism of action of triazeno derivatives on purine biosynthesis de novo was shown.

Rat liver polysomes, single ribosomes, EDTA-produced subparticles and free ribosomal RNA were treated with formaldehyde and their properties were studied. The following results were obtained. 1 RNA can be isolated from fixed polysomes or subparticles by exhaustive digestion with pronase. Such RNA shows a normal electrophoretic behaviour in agar gel. 2 In free RNA treated with formaldehyde the mobility of 18-S and 28-S RNA fractions is reduced; latent breaks are not revealed by heating; sensitivity towards ribonuclease is increased. No such effects are observed with RNA treated with formaldehyde while in the particles. 3 In fixed polysomes or single ribosomes the two subparticles appear to be covalently bound through their proteins. No intermolecular bonding occurs between 18-S and 28-S RNA either in fixed polysomes or in free RNA. The fixed ribosomes and subparticles can be fractionated by agar gel electrophoresis and give profiles similar to those of untreated particles. These results may prove useful in combining CsCl density gradient fractionation with gel electrophoretic methods.