Dnic May Be Used As A Biomarker For The Antimicrobial And Cytotoxic Actions Of Nitrite

The interaction of nitrite or NO with the iron-sulfur protein/enzyme is closely implicated in nitrite treatment of biological systems as described in the previous sections. Dinitro-syl dithiolato iron complex (DNIC) formed through the interaction has been shown to be relatively stable paramagnetic molecule that exhibits a characteristic electron paramagnetic resonance (EPR) signal both in solution and the frozen state [46,74-78]. In nitrite-treated cells of clostridia [7-9] and E. coli O157:H7 [79], DNIC can be detected at 77 K as an axi-ally symmetric EPR signal (g|| = 2.04, g± = 2.015). In cured meat, the DNIC signal was found to overlap with that of nitrosyl myoglobin [80], suggesting that both nitro-syl iron complexes were simultaneously formed. At the rat GEJ, the DNIC signal was found in tissues where salivary nitrite is likely to encounter highly acidic gastric juices, yielding high concentrations of NO [68], as details shown below. The presence of DNIC suggests that synchronous inactivation of iron-sulfur proteins/enzymes is involved in vital-activity as well as the release of intracellular iron after the reaction of free iron with thiol-containing proteins [47,76], further indicating that detection of DNIC in nitrite-treated specimens could be a biomarker for the anti-microbial and cytotoxic actions of nitrite. In what follows, two good examples illustrating the utility of DNIC as a biomarker will be described.

Infection with E. coli O157:H7 causes hemorrhagic diarrhea and hemolytic uremic syndrome [81,82]. This gram-negative bacterial species produces a large quantity of verotoxins during the course of the infection. Verotoxins are released from bacterial cells in the event of bacteriolysis, exerting a disastrous effect on the patient. Although some antibiotics and bacteriostatic sodium chloride can inhibit or kill E. coli O157:H7, these bactericidal agents cause bacteriolysis, releasing large quantities of verotoxins contained within these cells [78]. In contrast, nitrite treatment effectively inhibited the growth of this bacterium without triggering increased verotoxin release. EPR spectroscopy of frozen E. coli suspensions treated with nitrite in the presence or absence of ascorbate revealed that nitrite treatment abrogated the iron-sulfur protein signal at g = 1.94 and initiated the DNIC marker signals at g = 2.036 and 2.011, and that the additional treatment with ascorbate intensified the DNIC signals (Fig. 1) [78]. The addition of NO donor reagents exerts the same effect. In addition, nitrite treatment inhibited the synthesis of ATP in E. coli O157:H7 cells. These results indicate that nitrite-derived NO can inhibit bacterial growth through the inactivation of iron-sulfur enzymes in the respiratory chain. Thus, nitrite confers antibacterial activity against E. coli O157:H7 without increasing verotoxin release, because the action mechanism does not include bacteriolysis. Furthermore, DNIC acts as a valuable marker for assessing this effect.

High concentrations of NO are luminally generated at the GEJ through the enterosalivary recirculation of dietary nitrate in humans [63]. In the GEJ gastric tissues of nitrite-treated rats, the DNIC marker signal increased time- and dose-dependently, whereas no signal was observed at the distal stomach in the same rats [68]. In humans after nitrate ingestion, a low level of DNIC was detected in biopsy specimens from the cardia, but not the antrum [68]. The aconitase activity of GEJ tissues was significantly lower in nitrite-treated rats vs. control rats, while that in the distal stomach was similar between the two groups [68]. These results collectively suggest that salivary-derived NO diffuses from the stomach lumen into adjacent g = 2.036

Fig. 1. EPR spectra of cell suspensions of E. coli O157:H7 at 77 K. (Curve a) Untreated; (Curve b) treated with NaNO2 (200 mg/l); (Curve c) treated with both NaNO2 (200 mg/l) and sodium ascorbate (500 mg/l); (curve d) treated with 3-[2-hydroxy-1-(1-methylethyl) -2-nitrosohydrazino]-1-propanamine as an NO donor reagent. (Reproduced with permission from Ref. [78]. Copyright 2004 Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Fig. 1. EPR spectra of cell suspensions of E. coli O157:H7 at 77 K. (Curve a) Untreated; (Curve b) treated with NaNO2 (200 mg/l); (Curve c) treated with both NaNO2 (200 mg/l) and sodium ascorbate (500 mg/l); (curve d) treated with 3-[2-hydroxy-1-(1-methylethyl) -2-nitrosohydrazino]-1-propanamine as an NO donor reagent. (Reproduced with permission from Ref. [78]. Copyright 2004 Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

tissues, where it interacts with and disassembles vulnerable Fe-S cluster proteins, or reacts with intracellular free iron and thiol-containing proteins. This mechanism may be involved in the high prevalence of inflammation and intestinal metaplasia at GEJ in humans. Thus, it is valuable to consider the use of DNIC monitoring in future studies.

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