Selective inhibition of ribonucleotide reductase by the monofunctional alkylating agent 5(1-aziridinyl)-2,4-dinitrobenzamide (CB 1954).
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Animals↗
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Generation of semiquinone radical from carbazilquinone by NADPH-cytochrome P-450 reductase.
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Animals↗
Alteration of delta psi-dependent amino acid transports in Streptococcus pneumoniae by the antitumoral drug SOAz.
Interactions of the antitumoral drug SOAz with natural and model membranes are described. Biological studies were carried out with the bacterium Streptococcus pneumoniae taken as a model system. They reveal that SOAz is able to reduce delta psi and the delta psi-dependent amino acid transports without being cytotoxic for the bacteria. With respect to model membranes, leakage studies carried out with Na+ and K+ loaded lipid vesicles demonstrated that SOAz exhibits no ionophore activity. In contrast, the drug is shown to decrease the surface potential of monolayers of acidic phospholipids but without penetrating within the film. The possibility that SOAz might alter the delta psi part of the proton motive force by decreasing the outside surface potential of the bacterial membrane is discussed.
Amino Acids↗
The effects of inhibiting choline dehydrogenase on choline metabolism in mice.
3,3-Dimethylbutanol (Dimbunol), a competitive inhibitor of choline dehydrogenase (CDH), and ethylcholine mustard aziridinium (ECMA), an effective irreversible inhibitor of both CDH and choline transport, were investigated for their effects upon the uptake and metabolism of [3H]choline in mice. Thirty minutes after Dimbunol administration (i.p. 0.5 mmoles/kg) a reduction in the rate of choline oxidation was accompanied by an inhibition of choline phosphorylation in the kidney. Choline had accumulated to 5-fold the control level. After ECMA (i.v. 4 mumoles/kg), kidney choline was elevated 18-fold and both oxidation and phosphorylation rates were severely inhibited. In the liver Dimbunol inhibited oxidation and phosphorylation of choline and generated a 2-fold rise in tissue choline. Ethylcholine mustard aziridinium inhibited both oxidation and phosphorylation in the liver to the same extent as in the kidney but produced only a 3-fold elevation of choline. Dimbunol failed to elevate serum choline 30 min after administration and brain choline and acetylcholine levels were also unchanged. Serum choline was doubled by ECMA. These studies suggest that both transport across the renal tubules and oxidation may be important in choline regulation, that high levels of choline may accumulate in the liver and kidney which are not available for acetylcholine synthesis but that longer term studies on the effects of Dimbunol might reveal useful ways of facilitating sustained elevation of serum choline in precursor therapy.
Alcohol Oxidoreductases↗
Cellular activation of diaziquone [2,5-diaziridinyl-3,6-bis (carboethoxyamino)-1,4-benzoquinone] to its free radical species.
Human leukemic cell lines K562 and HL60, and the murine leukemic cell line L1210, reduce Diaziquone (AZQ) (NCS182986) to its free radical anion. With all cell lines, the free radical was observed immediately in both aerobic and anaerobic cell suspensions. The steady-state concentration of AZQ free radicals was approximately 1% of the total AZQ concentration. L1210 cells treated with azide reduced AZQ, but cells treated with diamide and N-ethylmaleimide did not. NADPH and L-cysteine reduced AZQ. The latter did so under anaerobic conditions; the former did so under both anaerobic and aerobic conditions.
Animals↗
Effects of ascorbic acid on biologically obtained diaziquone free radicals.
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Animals↗
A new cytotoxic, DNA interstrand crosslinking agent, 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, is formed from 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by a nitroreductase enzyme in Walker carcinoma cells.
Walker tumour cells in vivo or in vitro are exceptionally sensitive to the monofunctional alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) (Cobb LM et al., Biochem Pharmacol 18: 1519-1527, 1969). CB 1954 forms DNA interstrand crosslinks in a time-dependent manner in Walker tumour cells but not in non-toxically affected Chinese hamster V79 cells [(Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986)]. However, co-culturing Chinese hamster V79 cells with Walker cells in the presence of CB 1954 renders the hamster cells sensitive to CB 1954 and leads to the formation of interstrand crosslinks in their DNA, findings indicative of the formation by Walker cells of a diffusible toxic metabolite of CB 1954. A flavoprotein, of molecular weight 33.5 kDa as estimated by SDS-polyacrylamide gel electrophoresis, has been isolated from Walker cells and identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, EC 1.6.99.2). This enzyme, in the presence of NADH or NADPH, catalyses the aerobic reduction of CB 1954 to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. This new compound can form interstrand crosslinks in the DNA of Chinese hamster V79 cells to which it is also highly toxic.
Animals↗
The nitroreductase enzyme in Walker cells that activates 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is a form of NAD(P)H dehydrogenase (quinone) (EC 1.6.99.2).
A nitroreductase enzyme has been isolated from Walker 256 rat carcinoma cells which can convert 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to a cytotoxic DNA interstrand crosslinking agent by reduction of its 4-nitro group to the corresponding hydroxylamino species (Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986; Knox RJ et al., Biochem Pharmacol 37: 4661-4669, 1988). The enzyme has now been identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, menadione reductase (NMOR), phylloquinone reductase, quinone reductase, EC 1.6.99.2) by comparison of partial protein sequences, coenzymes, substrate and inhibitor specificities, and spectroscopic data. 2-Phenyl-5(4)-aminoimidazole-4(5)-carboxamide and 5(4)-aminoimidazole-4(5)-carboxamide were shown to be inhibitors of the isolated Walker cell enzyme. This observation could explain the reported antagonistic action of the aminoimidazole carboxamides to the antitumour effects of CB 1954.
Amino Acid Sequence↗
DNA modifications by potential antitumour bisaziridinylquinones.
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Alkylating Agents↗
The lack of correlation between toxicity and free radical formation of two diaziridinyl benzoquinones.
L1210 and K562 leukaemic cells have been used to study the relationship between cytotoxicity and free radical production by two aziridinyl benzoquinones, 2,5-bis(carboethoxyamino)3,6-diaziridinyl-1,4-benzoquinone (AZQ) and 2,5-bis(2-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ). BZQ showed a high level of toxicity in both cell lines, but no ESR signal was detectable, while AZQ readily produced an ESR signal but much lower cytotoxicity was observed, particularly in L1210 cells. The rate of superoxide formation was measured for each drug. The results demonstrate that cell killing and free radical production do not necessarily concur.
Antineoplastic Agents↗
Cellular glutathione conjugation of aziridines in isolated rat hepatocytes.
Glutathione conjugation of aziridines was found in isolated rat hepatocytes in experiments using the optical isomers of (1- and d-) aziridinecarboxylic acid (AZC) and (1- and d-) 1-methyl-2-beta-naphthylaziridine (NAZ). 1-AZC much more effectively consumed glutathione than d-AZC, and the yield of the glutathione conjugate during 2 hr of incubation exceeded 200% of the cellular glutathione detected at the initiation of the incubation. Such a high yield of 1-AZC-GSH conjugate would occur only when conjugation efficiently proceeds without interference against the GSH resynthesis route in the liver cells. The cytotoxicity of 1-AZC was very weak and did not affect cell viability of the isolated hepatocytes even after the formation of AZC-GSH conjugate. Consequently, we supposed that GSH is not essential for supporting the viability of the isolated hepatocytes. For very slow GSH conjugate formation of d-AZC, we envisaged poor membrane transport of the d-isomer resembling to the selective incorporation of d- and 1-proline observed in some plant cells. Both isomers of NAZ were markedly cytotoxic and depressed the cell viability. The yield of the glutathione conjugate from NAZ did not exceed the cellular GSH level detected at the initial stage of incubation. The highly cytotoxic compound nitrosomethane, generated in the first biotransformation step of the metabolism of NAZ, can obstruct the resynthesis route of GSH by inhibiting the ATP generation process as discussed previously (Ref. 3). Decreasing the cellular GSH by treatment with 1-AZC enhanced the susceptibility of the isolated hepatocytes to NAZ toxicity. d-AZC did not affect the viability of cells treated with NAZ.
Animals↗
The alkylation of DNA in vitro by 2,5-bis(2-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ)--I.
Cell toxicity by BZQ could not be explained by free radical formation and thus further work has been undertaken to elucidate a possible mechanism of cell killing. By using radiolabelled BZQ, in vitro DNA-drug binding has been investigated. The effect of salt, buffer and drug concentrations was determined in the pH range 4.0 to 8.0. The influence of in situ oxidation and reduction on BZQ binding was also studied as a function of pH. In an effort to ascertain any base specificity of BZQ binding the homopolymers, Poly[dG]. Poly[dC] and Poly[dA]. Poly[dT] were treated with radiolabelled BZQ in the pH range 4.0 to 8.0. A fluorescence assay was used to demonstrate the possible involvement of DNA cross-linking in cellular activity. From this work, it was concluded that BZQ functions as a bifunctional alkylating agent by an acid-assisted aziridine ring-opening mechanism and that other factors including oxidation or reduction are much less important.
Alkylating Agents↗
Effect of nerve growth factor and thyrotropin releasing hormone on cholinergic neurones in developing rat brain reaggregate cultures lesioned with ethylcholine mustard aziridinium.
Foetal rat whole brain reaggregate cultures were prepared in a serum-supplemented (S+) or serum-free medium (S-). Ethylcholine mustard aziridinium (ECMA) was added to the cultures at 9 days in vitro (DIV) at concentrations of 12.5, 25 or 50 microM. Choline acetyltransferase (ChAT) activity was measured at +2, +48 and +96 hr following treatment. In certain experiments the neurotrophic factors, thyrotropin releasing hormone (TRH: 50 micrograms/ml, daily from 9 DIV) or nerve growth factor (NGF: 7S subunit, 5 ng/ml, 0 and +48 hr following ECMA) were added during ECMA treatment. In both types of reaggregate cultured in S+ and S- media there was a 40-80% loss of ChAT activity following ECMA exposure (final concentration = 12.5 microM), presumed to reflect cholinergic cell loss. In both S+ and S- brain reaggregates NGF produced increased ChAT activity with more marked effects in S+ (45-55% increase, +48-96 hr) than in S- medium (20-25% increase, 2-96 hr). No effect on cholinergic muscarinic receptors (specific 3H-QNB binding) was evident after treatment with NGF. TRH had no effect on ChAT activity in the S+ cultures but produced small increases in the S- culture condition (approx 20%, +2-48 hr). Despite a residual "ECMA-resistant" pool of ChAT in the cultures, neither neurotrophic agent was found to cause a reversal of the lesion. In conclusion, the cholinotoxin ECMA appears to produce a cholinergic deficit in both developing S+ and S- reaggregates. This was not reversible by NGF or TRH at the concentrations and under the conditions tested. NGF had marked effects on ChAT activity without affecting muscarinic receptors in untreated developing brain reaggregates cultured in an S+ medium.
Animals↗
AF64A, a cholinergic neurotoxin, selectively depletes acetylcholine in hippocampus and cortex, and produces long-term passive avoidance and radial-arm maze deficits in the rat.
The behavioral and biochemical effects of AF64A, a presynaptic cholinergic neurotoxin, were investigated. Bilateral administration of this compound into the lateral cerebral ventricles produced transient and dose-related effects on sensorimotor function and long-term impairments of cognitive behavior. Male Fischer-F344 rats dosed with either 15 or 30 nmol of AF64A reacted 29-62% faster than CSF-injected controls in a hot-plate test 14 (but not 1, 7, 21 or 28) days following dosing. The group administered 15 nmol of AF64A was also significantly more active (41%) than controls 28 days following dosing. The activity level of this group was comparable to that of controls at other times and hyperactivity was never observed in the 30 nmol group. Retention of a step-through passive avoidance task, assessed 35 days after dosing, was impaired in both the 15 and the 30 nmol groups. Their step-through latencies were significantly shorter than the control latencies, and they exhibited more partial entries during the 24-h retention test. Radial-arm maze performance, measured 60-80 days following treatment, was markedly impaired in the treated groups. Animals treated with AF64A made fewer correct responses in their first 8 choices, required more total selections to complete the task, and had an altered pattern of spatial responding in the maze. The neurochemical changes produced by AF64A, determined 120 days after dosing, were specific to the cholinergic system and consisted of decreases of ACh in both the hippocampus (15 and 30 nmol groups) and the frontal cortex (30 nmol group). The concentrations of catecholamines, indoleamines, their metabolites and choline in various brain regions were not affected by AF64A. Furthermore, histological analysis revealed that the doses of AF64A used in the present study did not damage the hippocampus, the fimbria-fornix, the septum or the caudate nucleus. These data support the contention that cholinergic processes in the hippocampus and/or frontal cortex play an important role in learning and memory processes. Furthermore, based upon the behavioral and biochemical data presented, it is suggested that AF64A could be a useful pharmacological tool for examining the neurobiological substrates of putative cholinergic disorders such as senile dementia of the Alzheimer's type.
Acetylcholine↗
Effects of intrastriatal injections of the cholinergic neurotoxin AF64A on spontaneous nocturnal locomotor behavior in the rat.
Rats with bilateral striatal cholinergic lesions induced by the neurotoxin AF64A were found to have increased spontaneous nocturnal locomotor activity as measured in terms of the amount of time spent moving, the total distance traveled and the number of episodes and time of stereotypic behaviors. No significant differences between control and AF64A injected rats were found in the actual velocities between the two groups indicating that the rats were simply spending more time moving rather than moving with greater speed. Striatal activity of choline acetyltransferase (CAT) was significantly decreased in AF64A treated rats compared to controls, whereas the activities of glutamate decarboxylase (GAD) were not. Furthermore, there were no significant differences between groups in enzymatic activities of CAT and GAD in either the cortex or the hippocampus, indicating that the lesion was restricted to the striatum. The hyperactivity found in these rats after intrastriatal injection of AF64A supports a role for the striatal cholinergic system in locomotor behavior.
Acetylcholine↗
Non-cholinergic neurotoxic effects of AF64A in the substantia nigra.
Ethylcholine aziridinium ion (AF64A), a putative specific cholinergic neurotoxin, was unilaterally injected into the substantia nigra of male rats. One week following injection, rats that were challenged with apomorphine exhibited ipsilateral circling behavior. After two weeks, rats were sacrificed and corpus striatum and combined n. accumbens plus o. tubercle were assayed for dopamine. Striatal dopamine levels were depleted by 50%. Histological examination showed that AF64A caused extensive damage at the injection site. These data indicate that injection of AF64A into the substantia nigra can result in damage to dopaminergic neurons.
Animals↗
Selective cholinergic neurotoxin: AF64A's effects in rat striatum.
The selective neurotoxic effects of the aziridinium ion of ethylcholine (AF64A) have been examined after stereotaxic injection into the rat striatum. In a dose-response study (2-26 nmol), 8 nmol caused a 46% decrease in striatal choline acetyltransferase (CAT) activity with minimal effects on the activities of glutamate decarboxylase (GAD) and tyrosine hydroxylase (TH) at 7 days. Maximal CAT reductions of 78-82% occurred with doses of 16-26 nmol which also caused dose-related decreases in GAD and TH activities that paralleled the progressive decrements in CAT. A time course study with 8 nmol indicated a rapid 20% reduction of CAT activity by 12 h and an additional gradual fall of 20% over the next week; TH and GAD activities were not significantly reduced. The selective inhibition of CAT activity persisted for at least 3 months. Histological examination of Nissl stained sections revealed an area of nonspecific damage at the injection site with an abrupt border surrounded by apparently normal striatal neuropil; however; neuronal perikarya staining intensely for acetylcholinesterase were not reduced. These preliminary findings strongly suggest that AF64A has selective neurotoxic effects against striatal cholinergic neurons while relatively sparing striatal GABAergic intrinsic neurons or dopaminergic afferents.
Animals↗
Ultrastructural and neurochemical effects of the presumed cholinergic toxin AF64A in the rat interpeduncular nucleus.
We have studied the effect of the presumptive cholinergic neurotoxin, ethylcholine mustard aziridinium ion (compound AF64A), on ultrastructure and neurochemical markers in the rat interpeduncular nucleus (IPN). Stereotaxic injections of 1 nmol of AF64A resulted in extensive degeneration of synaptic terminals within 40 h. Ultrastructural damage to neuronal cell bodies, dendrites and axons was also sometimes observed at this stage. Five days after the injection, more severe degenerative changes were observed in a larger number of neuronal cell bodies, axons and dendrites. High affinity uptake of [3H]choline, but not [3H]GABA, was significantly decreased 24 h after toxin injection. Five days after the injection, not only choline acetyltransferase but also glutamate decarboxylase levels were significantly decreased. Our results suggest that, in addition to presynaptic cholinergic neurotoxicity, AF64A also leads to degenerative alterations of non-cholinergic neurons. Our electron microscopic observations constitute the first ultrastructural report on neuropathological damage caused by AF64A.
Animals↗