Reactions of nitrosochloramphenicol in blood

doi:10.1016/0006-2952(84)90670-1

Biochemical Pharmacology

Volume 33, Issue 14, 15 July 1984, Pages 2299-2308

https://doi.org/10.1016/0006-2952(84)90670-1 Get rights and content

Abstract

It has been suggested that nitrosochloramphenicol (NOCAP), a possible metabolite of chloramphenicol (CAP), may be involved in CAP-induced aplastic anemia. We found that NOCAP was rapidly eliminated from human blood in vitro (more than 90% in less than 15 sec). Analysis of the different reactions showed that 5% of NOCAP was covalently bound to plasma proteins, mainly to albumin, the remainder being metabolized in red cells. The most important reaction in red cells was the very rapid adduct formation with GSH (k = 5,500 M⁻¹s⁻¹), yielding presumably a semimercaptal which either isomerized to a sulfinamide (GSONHCAP, k = 0.05 s⁻¹) or was thiolytically cleaved by another GSH molecule with formation of the hydroxylamine (NHOHCAP) and GSSG (k = 7.1 M⁻¹s⁻¹). Another important elimination reaction was the covalent binding of NOCAP to the SH groups of hemoglobin (k = 5 M⁻¹s⁻¹), also yielding a sulfinamide. Besides these reactions with thiols, NOCAP was enzymatically reduced to NHOHCAP in the presence of NADPH (K_m NADPH = 10⁻⁵ M; K_m NOCAP = 10⁻⁴ M; V_max = 2 μmole/min per ml). This reaction was only effective at NOCAP concentrations below 10⁻⁴M, probably because of limited NADPH-regeneration. Further reduction of NHOHCAP to NH₂CAP was a slow process which did not exceed 0.5 nmole/min per ml. NH₂CAP was mainly formed from GSONHCAP, a reaction which depended on NADPH and the presence of hemolysate, indicating an enzymatic reaction. In contrast to smaller nitrosoarenes, NOCAP was a poor ligand for ferrohemoglobin (probably due to steric hindrance by its bulky molecule) and was therefore much more exposed to biotransformation. NOCAP and NHOHCAP formed ferrihemoglobin at a rate 5000 times slower than did phenylhydroxylamine. In contrast to NOCAP, NHOHCAP penetrated slowly the red cell membrane ( $t_{12}$ about 5 min), and its disposition in blood was quite ineffective. From these data, it seems likely that most of the NOCAP formed by microorganisms in the intestine or produced in the liver, will be degraded in blood before it can reach the bone marrow.

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In addition to the large volume of work on the aqueous voltammetry of nitrobenzene, there has also been a significant amount of research done on another topic relevant to the current study, the reaction of aromatic nitroso compounds with thiols in aqueous solution. This is both because of the relevance to the nitro antimicrobial drugs, but also because this is believed to provide a detoxification pathway [14,64–67] to remove mutagenic intermediates formed from enzymatic activation of both nitro and aminoaromatics. The results of these studies can be summarized by Scheme 2 [11].
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Evaluation of the clinical, immunologic, and biochemical effects of nitroso sulfamethoxazole administration to dogs: A pilot study
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Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated in humans with hypersensitivity reactions, to include fever, skin eruptions, hepatotoxicity, and blood dyscrasias. These reactions also occur in dogs, the only non-human species known to develop a similar spectrum of sulfonamide hypersensitivity. Sulfonamide hypersensitivity is not well understood, but has been hypothesized to be due to the generation of the reactive oxidative metabolite, nitroso sulfamethoxazole (SMX-NO). SMX-NO, unlike the parent sulfonamide, is cytotoxic in vitro, haptenizes tissue proteins, and is immunogenic in rodents. The purpose of this pilot study was to determine whether SMX-NO, when administered to dogs, would lead to drug-tissue adducts, anti-drug antibodies, antioxidant depletion, or clinical evidence of drug hypersensitivity. Four dogs were randomized to one of four treatments: SMX-NO 1 mg/kg; SMX-NO 3 mg/kg; SMX-NO 10 mg/kg; or vehicle control. Dosing was by the intraperitoneal route, once daily for four consecutive days per week, for 2 weeks total, followed by a third week of observation. Following this, all dogs were challenged with trimethoprim-sulfamethoxazole, 25 mg/kg for 12 h for 2 weeks. No dog developed clinical or biochemical evidence of drug hypersensitivity. Plasma cysteine and leukocyte reduced glutathione were not depleted during dosing; however, ascorbate was significantly depleted by week 2 following SMX-NO at 10 mg/kg. Anti-SMX antibodies (IgG or IgM by ELISA) were not detected in any dogs at any time points. SMX-hemoglobin adducts were detected in the spleen in SMX-NO dosed dogs; however, these adducts were not accompanied by an immunologic or systemic response. The results of this pilot study indicate that SMX-NO dosing in dogs, using a dosing protocol shown to be immunogenic in other species, produces modest ascorbate depletion and hemoglobin adduct formation, but is insufficient to produce an immunologic response or a clinical syndrome of sulfonamide hypersensitivity in this susceptible species.
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Reactions of nitrosochloramphenicol in blood

Abstract

Biochem. Pharmac.

Biochem. Pharmac.

Chem.-Biol. Interact.

Chem.-Biol. Interact.

Biochem. Pharmac.

Pharmac. Res. Commun.

J. biol. Chem.

J. biol. Chem.

Biochem. Pharmac.

J. biol. Chem.

J. biol. Chem.

Archs Biochem.

J. Pharmac. exp. ther.

Pharmacology

J. Lab. clin. Med.

J. Lab. clin. Med.