Desmutagenic actions of ascorbic acid and cysteine on a new pyrole mutagen formed by the reaction between food additives; Sorbic acid and sodium nitrite

Mutagenicity of 1,4-dinitro-2-methyl pyrrole, a new mutagen isolated from the reaction mixture of sorbic acid and sodium nitrite, was found to be destroyed by treatment with ascorbic acid or cysteine. Chemical studies revealed that the loss of mutagenicity was due to reduction from the C-nitro to C-amino group.     

Induction of rat liver cytochrome P450 isoenzymes CYP 1A and CYP 2B by different fungicides, nitrofurans, and quercetin

The genotoxic activity of environmental xenobiotics is manifested either in their direct interaction with cellular genetic material or in provoking secondary events, among which reactive oxygen species (ROS) production is a common phenomenon. Both pathways can be mediated by the activity of the cytochrome P450 monooxygenase system. We studied induction of the CYP 1A or CYP 2B monooxygenases in rat liver by the fungicides: thiram, captan, captafol, dodine and the drugs: nitrofurazone, furazolidone and the plant flavonoid: quercetin. A cytochrome P450 induction assay (CYPIA test) was used. S9 prepared from livers of rats treated with the test compounds were used to activate ethidium bromide (EtBr) (CYP 1A isoenzyme) or cyclophosphamide (CPA) (CYP 2B isoenzyme) in the Ames test.It was found that among the tested compounds, the most potent inducer of CYP 1A was furazolidone (3× 80 mg/kg). Less potent was thiram (1× 100 mg/kg), as well as quercetin (3× 80 mg/kg), and captafol (1× 30 mg/kg). On the other hand, thiram (1× 100 mg/kg), captafol (1× 30 mg/kg), and quercetin (3× 80 mg/kg) were most potent in the CYP 2B isoenzyme induction, while furazolidone (3× 80 mg/kg), and nitrofurazone (3× 80 mg/kg) appeared to be less potent in this respect. Captan and dodine (3× 80 mg/kg) did not affect the activity of any of the cytochrome P450 isoenzymes.     

Kinetic and structural characterisation of Escherichia coli nitroreductase mutants showing improved efficacy for the prodrug substrate CB1954

Escherichia coli nitroreductase (NTR) is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Among these substrates is the prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954) that is activated by NTR to form two products, one of which is highly cytotoxic. NTR in combination with CB1954 has entered clinical trials for virus-directed enzyme-prodrug therapy of cancer. Enhancing the catalytic efficiency of NTR for CB1954 is likely to improve the therapeutic potential of this system. We previously identified a number of mutants at six positions around the active site of NTR that showed enhanced sensitisation to CB1954 in an E. coli cell-killing assay. In this study we have purified improved mutants at each of these positions and determined their steady-state kinetic parameters for CB1954 and for the antibiotic nitrofurazone. We have also made a double mutant, combining two of the most beneficial single mutations. All the mutants show enhanced specificity constants for CB1954, and, apart from N71S, the enhancement is selective for CB1954 over nitrofurazone. One mutant, T41L, also shows an increase in selectivity for reducing the 4-nitro group of CB1954 rather than the 2-nitro group. We have determined the three-dimensional structures of selected mutants bound to the substrate analogue nicotinic acid, using X-ray crystallography. The N71S mutation affects interactions of the FMN cofactor, while mutations at T41 and F124 affect the interactions with nicotinic acid. The structure of double mutant N71S/F124K combines the effects of the two individual single mutations, but it gives a greater selective enhancement of activity with CB1954 over nitrofurazone than either of these, and the highest specificity constant for CB1954 of all the mutations studied.     

The effect of short-term dietary modification on human fecal mutagenic activity

To assess the effect of short-term modification of diet on human fecal mutagenic activity, 6 subjects consumed 2 dietary regimes hypothesized to affect risk of colorectal cancer. After a 7-day baseline period, a 'low-risk' non-meat diet was consumed for 2 weeks followed by 2 weeks on a 'higher risk' diet which emphasized beef and refined grains. Fecal samples were collected at the end of each diet period and assayed for direct-acting mutagens with the fluctuation test for weak mutagens using Salmonella typhimurium TA100 and TA98 as tester strains. Fecal mutagenic activity on TA100 was increased for all subjects during the 'higher risk' period compared to the 'low risk' period. The average mutagenicity on TA98 was also increased, but the trend was not consistent for all subjects. The baseline diet and non-meat diet resulted in approximately equal mean fecal mutagenicity levels. These findings indicate that a diet high in meat and refined grain, as characterized here, increases fecal mutagenic activity within a 2-week period.     

Metabolism of 1,8-dinitropyrene by Salmonella typhimurium

Earlier work has shown that many nitroaromatic and nitroheterocyclic compounds are directly 'activated' to their ultimate mutagenic forms through the action of bacterial nitroreductase enzymes. However, in the case of 1,8-dinitropyrene (DNP) and certain other nitroarenes the pathway of activation is more complex and neither the identity of the ultimate mutagens nor the nature of the DNA adducts formed are known. We now show that Salmonella typhimurium strains TA98 and TA1538, which are sensitive to DNP and have wild type nitroreductase complements, do metabolize DNP to 1-amino-8-nitropyrene (ANP) and 1,8- diaminopyrene (DAP) but that these compounds are much weaker mutagens than DNP. These two strains (TA98 and TA1538) contain two separable components of nitroreductase activity as determined using nitrofurazone as the substrate. The major component, at least, is capable of reducing both 1-nitropyrene (NP) and DNP although the rates are much lower than with nitrofurazone. TA98NR , a mutant of TA98 that is resistant to nitrofurazone and NP but not to DNP, lacked the major nitroreductase but retained two minor components. In contrast, a mutant ( DNP6 ) which is resistant to DNP (but not to NP) contained a full complement of nitroreductases. When the metabolism of [3H]DNP by crude extracts of TA98 was re-examined, previously undetected metabolites were found. These were more polar than DAP and ANP and were also seen when TA98NR was used as the source of enzyme. These metabolites were not formed when enzymes from TA98DNP6 or TA98NR / DNP6 were used. This work supports the notion that some enzymic activity other than (or in addition to) nitroreductase is required for the activation of DNP and that the new polar metabolites may be related to this process.