The mechanism of the trichomonicidal activity of metronidazole and other 5-nitroimidazoles appears to depend on the ferredoxin-mediated reduction of their nitro group, with generation of a reactive metabolite or metabolites which interact with DNA leading to a subsequent inhibition of nucleic acid and protein synthesis. Redox cycling of these compounds under aerobic conditions appears to be a detoxification reaction by inhibiting net reduction of the drugs, thereby inhibiting their uptake. On the other hand, redox cycling of nitrofurans or other compounds with more positive reduction potential results in formation of high steady-state concentrations of oxygen-derived metabolites that might be of toxicological significance. It seems likely that reduced metabolites of nitroimidazoles (perhaps through covalent binding to tissue macromolecules and/or thiols depletion) are also involved in the nitroimidazoles' toxic effects to animal tissues and in their mutagenic and carcinogenic action.

Mechanism of toxicity of nitro compounds used in the chemotherapy of trichomoniasis.

Glucose and lipid metabolism in the brain, liver and in a transplanted tumour were found to be variously altered within 2 to 3 h of administering single doses of the radiosensitizer Ro-03-8799 to normal and tumour-bearing mice. Hepatic lactate and glycerol-3-phosphate (G3P) levels were decreased but those of the ketone body beta-hydroxybutyrate (beta-HOBu) were raised. However, in the tumour, these levels were all enhanced. The lactate levels in brain remained relatively constant but both beta-HOBu and G3P levels were altered in a manner similar to that in the liver. The levels of glucose were approximately doubled in blood, brain and tumour, but whereas tumour G6P levels increased, those in the brain were lowered to below the limits of detection. Hepatic glucose levels were significantly decreased after 1 h but G6P levels were not affected. These changes could neither be related to inhibitory effects on hepatic glucokinase or brain hexokinase activity nor to limiting amounts of ATP in both tissues. However, the activity of glucose-6-phosphatase (G6P'ase) was distinctly raised in the liver and the hepatic glycogen stores were also rapidly lowered. Overall, the results suggest that Ro-03-8799 exerts a stimulatory effect on glucose production in the liver. In both liver and brain the levels of free fatty acids and phospholipids were increased whereas those of esterified fatty acids were lowered. Most importantly, the changes in metabolite levels affect the cellular redox couples; those of the cytosol (lactate/pyruvate; G3P/dihydroxyacetone phosphate (DAP] are directed towards the oxidised state in the liver but to a more reduced state in the tumour. The mitochondrial couple (beta-HOBu/acetoacetate (AcAc)) in both tissues is shifted towards the reduced state. These metabolic changes may result in an increase in the degree of hypoxia in the tumour and may well play an important role in the development of neuropathies.

Effects on intermediary metabolism in mouse tissues by Ro-03-8799

Indiana University-Purdue University Indianapolis (IUPUI)%%%%Plants are an essential resource providing the world with food, oxygen to breath, and animal habitats. Within each plant, there is an integrated network of pathways that produces phytochemicals either metabolism [1, 2]. Following the discovery of the first specialized metabolite in 1806, the common view has shifted from describing a collection of “by-products” to primary metabolism [3, 4] to the products of a value-added expansion of primary metabolic processes that provides an adaptive advantage to the producing organism. Herein, we report the identification of metabolites for three lipid-related pathways and insights into their origins. The first study was initiated by the serendipitous discovery of unusual fatty acids in the seed oil of Orychophragmus violaceus. Structural analyses together with targeted syntheses of derivatives and molecular fragments revealed two C24 dihydroxy fatty acids identified as 7,18-(OH)2-24:1∆15 and 7,18-(OH)2-24:2∆15,21 that account for ~50% of the seed oil. Evidence indicates that these hydroxylated fatty acids are made via discontinuous elongation, a novel process intermediate between fatty acid and polyketide syntheses. Second, 13C and 2H labeled precursors were used to gain insight into the biosynthetic pathway of alkamides produced by Echinacea. It was determined that the alkamide’s amide moiety is derived from valine and isoleucine, which led to the discovery of the first plant branched-chained amino acid decarboxylase. Separately, the involvement of these atypical acyl chains in alkamide biosynthesis was tested with [2H3] 2E-dodec-, tridec-, and tetradecenoic acids, and their isobutylamides. Analogous CoA derivatives were synthesized to characterize a ligase potentially responsible for the amide bond formation. Finally, to gain insight into the mechanism of a divergent CYP51 enzyme involved in avenacin biosynthesis, epoxy and hydroxylated triterpenes formed by mutant enzymes were identified. Collectively, these studies further the knowledge of the biosynthesis of specialized plant natural products.

Structure Elucidation and Isotopic Labeling Experiments Provide Insights into the Biosynthesis of Three Phytochemicals: Alkamides, Avenacin, and Hydroxylated Fatty Acids

Hypoxic BP-8 murine sarcoma cells were exposed to misonidazole and/or radiation and the kinetics and extent of cell death were evaluated with the [125I]iododeoxyuridine-prelabeling assay. Cell death after treatment with lethal doses of misonidazole was rapid and essentially complete within 2 or 3 days after drug exposure. In contrast, radiation death became apparent only after a delay period of 4 days and was complete by Day 10 after irradiation. Radiosensitization by short exposures to sublethal doses of misonidazole affected only the delayed component of cell death, that is, the radiation component of death. In experiments involving sequential radiation and drug treatment, prior irradiation of cells did not enhance the direct cytocidal effects of misonidazole, as evidenced by the fact that the early component of cell death was equal in control and preirradiated cells. However, postirradiation treatment with misonidazole did enhance the delayed radiation component of cell death. These results suggest that radiosensitization and direct killing by misonidazole are two distinct phenomena mediated by different cellular mechanisms, and radiosensitization by misonidazole represents a two-component effect composed of true dose modification and dose additive damage interactions, but these additive effects must occur at a site different from the cellular structure responsible for direct drug-induced cell death.

Radiosensitizing and Cytocidal Effects of Misonidazole: Evidence for Separate Modes of Action

The three characteristic lesions of Alzheimer’s disease are composed of B-pleated sheet fibrils called amyloid. Two of these, the cerebrovascular amyloid deposits and the senile plaques have as their major component a unique protein designated s protein (sP)1 and the fibers denoted as As. This unique 28–42 mer polypeptide has also been isolated from the amyloid-laden plaques and vessels in Down’s syndrome (trisomy 21).2 This suggested that sP was encoded by a gene on chromosome 21.2 Subsequently the gene for the sP was identified on chromosome 21 and the deduced amino acid sequence consisted of 695 amino acids, the s protein precursor (sPP), and to have characteristics of a membrane associated protein.3 Kunitz protease inhibitor sequences were identified in 751 and 770 mer isoforms of the sPP. Gene structure provides evidence that the sP is derived from two exons and thereby arises by proteolytic cleavage of sPP. Proteolysis has been previously implicated in the creation of amyloid fibrils from the AL and AA types of amyloid disease.4

A Leptomeningeal Protease Releasing the β Protein from the β Protein Precursor of Alzheimer’s Disease

1. The effect of the addition of a number of nitroimidazoles was tested on fatty acid synthesis by germinating pea seeds, isolated lettuce chloroplasts and a soluble fraction from pea seeds. 2. All the compounds tested had a marked inhibition on stearate desaturation by lettuce chloroplasts and on the synthesis of very-long-chain fatty acids by pea seeds. 3. In contrast, the effect of the drugs on total fatty acid synthesis from [14C]acetate in chloroplasts was related to the compound's electron reduction potentials. 4. Of the compounds used, only metronidazole had a marked inhibition on palmitate elongation in the systems tested. 5. The mechanism of inhibition of plant fatty acid synthesis by nitroimidazoles is discussed and the possible relevance of these findings to their neurotoxicity is suggested.

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M R Stratford

Papers

Inhibition of plant fatty acid synthesis by nitroimidazoles.