Nitroso-chloramphenicol Research Articles

Bioavailability, pharmacokinetics and residues of chloramphenicol in the chicken*

The pharmacokinetic properties of chloramphenicol were determined in broiler chickens after two single oral doses (30 and 50 mg/kg body weight) and after a single intravenous (i.v.) dose (30 mg/kg body weight). After oral and i.v. administration, the plasma concentration-time graph was characteristic of a two-compartment open model. After oral administration (30 and 50 mg/kg), chloramphenicol was absorbed rapidly (time to maximal concentration of 0.72 or 0.60 h) and eliminated with a mean half-life (t1/2 beta) of 6.87 or 7.41 h, respectively. The bioavailability was 29% at 30 mg/kg chloramphenicol and 38% at 50 mg/kg chloramphenicol. Concentrations greater than 5 micrograms/ml were achieved at 15 min and persisted up to 2 or 4 h post-administration, respectively. Statistically significant differences between the two routes of administration were found for the pharmacokinetic variables, half-lives of both distribution and elimination phases (t1/2 alpha, t1/2 beta) and apparent volume of distribution [Vd(area)]. The mean t1/2 beta of chloramphenicol and i.v. administration was 5.23 h. Chloramphenicol was extensively metabolized into dehydrochloramphenicol (DH-CAP), nitrophenylaminopropanedione (NPAP) and nitroso-chloramphenicol (NO-CAP) derivatives. Residues of chloramphenicol (CAP) and the three metabolites DH-CAP, NPAP and NO-CAP in kidney, liver and muscle were measured in chickens that received an oral dose of 50 mg/kg once daily for 4 days. The results indicate that CAP and DH-CAP residues were cleared slowly and were at or below the detection limit of 0.005 microgram/ml within 12 days after dosing. However, at the time of slaughter (12 days), the NPAP and NO-CAP residues were detected in the tissue.

Stability of chloramphenicol metabolites in human blood and liver as determined by high-performance liquid chromatography.

The pathogenesis of aplastic anemia from chloramphenicol (CAP) remains uncertain. Recent observations suggest that metabolites of CAP generated by intestinal bacteria may play an important role in mediating hematotoxicity from the drug. Thus, it is possible that once in circulation and after passage through the liver, some CAP metabolites may gain access to the marrow causing hematotoxicity. Based on this possibility, we have studied the stability of CAP and the three cytotoxic analogues dehydrochloramphenicol (DH-CAP), nitrophenylaminopropanedione (NPAP) and nitroso-chloramphenicol (NO-CAP) in human blood and liver. Determination of these compounds was accomplished by using a high-performance liquid chromatography system uniquely suited for their separation and detection. Several methods for deproteinization were utilized in order to ensure a full quantitative extraction of the drugs. At zero time, a 100% recovery of CAP, DH-CAP and NPAP was reached with acetonitrile (1 vol/3 vol); whereas NO-CAP was slightly or not detectable with all methods. Incubation of CAP and analogues with blood or liver at 37 degrees C for up to 30 min showed the following: CAP was stable in both tissues with full recovery; DH-CAP was stable for 5 min, then gradually decreased reaching 50 or 70% of the initial amount after 30 min of incubation with blood and liver, respectively; NPAP decreased at a faster rate than DH-CAP, and NO-CAP completely disappeared. The data suggest that if and when formed in the body, DH-CAP and NPAP may stay in the circulation long enough to reach the marrow and interact with its cellular components.

DNA damage induced by chloramphenicol and nitroso-chloramphenicol: protection by N-acetylcysteine.

We have previously demonstrated that nitroso-chloramphenicol (NO-CAP) in small concentrations causes the hydrolysis of isolated double stranded DNA in vitro and this action is blocked by sulfhydryl groups. The present study was designed to assess damage to isolated DNA as well as intact cells DNA and examine the protective effect of N-acetylcysteine (NAC). Using alkaline sucrose gradient sedimentation and the alkali elution technique of Kohn we were able to demonstrate DNA damage in Raji cells as well as phytohemagglutinin stimulated human lymphocytes after exposure to NO-CAP. Damage could be totally blocked by NAC. In preliminary studies we also observed that NAC protects bone marrow cells from the growth-inhibitory effects of chloramphenicol and thiamphenicol.

Reactions of nitrosochloramphenicol in blood

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 NH 2CAP was a slow process which did not exceed 0.5 nmole/min per ml. NH 2CAP 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 1 2 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.

Induction of Methemoglobin Formation by Nitrosochloramphenicol in Hemolyzed Blood

We studied the effects of chloramphenicol (CAP), nitroso-chloramphenicol (NO-CAP) and thiamphenicol (TAP) on methemoglobin (MeHb) formation in vitro in hemolyzed blood. At concentrations of 0.5 to 10 mM CAP and TAP did not have any effect on hemoglobin. On the other hand, NO-CAP at 0.25 to 5 mM caused both time and concentration dependent increase in MeHb formation. The oxidation of hemoglobin was reversible by potassium cyanide. Since MeHb was detected only at NO-CAP concentration 0.25 mM or higher, methemoglobin formation is not a sensitive indication of NO-CAP presence.

Reactions of the nitroso analogue of chloramphenicol with reduced glutathione

Nitroso-chloramphenicol (NOCAP) was synthetized by reduction of chloramphenicol (CAP) with zinc dust in a modification of the procedure published by Corbett and Chipko. The radioactive derivative was similarly prepared from [dichloroacetamido-1- 14C]CAP. NOCAP rapidly reacted with GSH with the formation of hydroxylamino-chloramphenicol (NHOHCAP), d -(−)-threo-1-(p- hydroxylaminophenyl)-2- dichloroacetamido-1,3- propanediol and glutathione disulfide (GSSG). In addition, a hydrophilic sulfinamide was formed (GSONHCAP), d -(−)-threo-1-(p- glutathionesulfinamidophenyl)-2- dichloroacetamido-1,3 - propanediol . Free amino-chloramphenicol (NH 2CAP), d -(−)-threo-1-(p- aminophenyl)-2- dichloroacetamido-1,3- propanediol , was not detected. The proportion of NHOHCAP formed increased with increasing GSH concn, at the expense of GSONHCAP. Analysis by stopped-flow spectroscopy revealed formation of a labile adduct in the reaction of NOCAP with GSH ( k = 5500 M −1 sec −1 at 37°, pH 7.4). This reaction was reversible because nearly all NOCAP could be extracted with ether from the labile intermediate. The equilibrium adduct/NOCAP was dependent on GSH concn ( K = 4500 M −1 at 37°, pH 7.4). The labile intermediate either isomerized to the sulfinamide, GSONHCAP (favoured by decreasing pH at constant GSH), or it was thiolytically cleaved by another GSH molecule to NHOHCAP and GSSG (favoured by increasing GSH at constant pH). At acid pH, GSONHCAP readily hydrolyzed to NH 2CAP and glutathionesulfinic acid. Thus, NOCAP reacts with thiols similar to nitrosobenzene. A scheme is presented for the proposed reaction mechanism. It is concluded that most of the NOCAP, if formed in the intestine or liver, will be rapidly disposed by reactions with GSH. Hence, toxic concns at the sensitive target, i.e. the bone marrow, may usually be prevented.

Enzymatic reduction of chloramphenicol and nitrosochloramphenicol by rat liver microsomal preparations.

Chloramphenicol (CAP) and nitrosochloramphenicol (NO-CAP) were metabolically reduced to aromatic amines by rat liver microsomes in vitro. The reduction required anaerobic conditions and was mediated by a NADPH-dependent reductase system. Both CAP and NO-CAP reduction were time and concentration dependent. Compared to CAP, NO-CAP (0.5 mM) reduction was rapid with complete conversion occurring in 90-120 min. At 2.5 mM NO-CAP reduction was depressed. In the presence of NO-CAP (0.05-2.5 mM) CAP metabolism was inhibited from 17 to 91%. These results indicate that CAP and NO-CAP are metabolized by similar microsomal reductase systems and NO-CAP is unstable, readily reduced further to the aromatic amine.

Inhibition by nitroso-chloramphenicol of the proton translocation in mitochondria

We have found recently that, unlike chloramphenicol (CAP), its nitroreduction derivative nitroso-chloramphenicol (NO-CAP) behaved as a potent inhibitor of the energy-conserving mechanism in mitochondria [Abou-Khalil et al. Biochem. Pharmac. 29, 2605 (1980)]. Concentrations of 75 and 250 μM NO-CAP were required to inhibit ATP formation with glutamate and succinate, respectively, whereas similar CAP concentrations were without effect. Testing several key reactions associated with the biosynthesis of ATP, inhibitory concentrations of NO-CAP were found to interfere as follows: (a) the transport of an NAD-linked substrate (e.g. glutamate) into mitochondria was only partially inhibited, whereas that of an FAD-linked substrate (e.g. succinate) was not inhibited but was rather slightly activated; (b) the transport of P i was only inhibited at about 50%; (c) mitochondrial ADP transport was not affected at all; (d) the ATPase activity, measured either by Pi release in the presence of an uncoupler or by H + ejection, was only slightly affected; and (e) under either phosphorylation or no phosphorylation conditions and in the absence of P i, NO-CAP was found to completely block mitochondrial H + extrusion resulting from the oxidation of either succinate or glutamate; however, under similar conditions the oxidation of the two substrates was not totally inhibited. The possibility of interference by NO-CAP with reactive mitochondrial thiols groups is discussed in the light of previous data and current experiments showing protection by P i against NO-CAP effects on respiration. Moreover, NO-CAP as compared to conventional inhibitors of oxidative phosphorylation (e.g. rotenone, antimycin A, oligomycin, mersalyl and others) appeared to have a distinct mode of action on that process. The results demonstrate that the inhibitory effect of NO-CAP is primarily located at the respiratory chain level where the proton translocation activity is fully blocked.

Degradation of isolated deoxyribonucleic acid mediated by nitroso-chloramphenicol: Possible role in chloramphenicol-induced aplastic anemia

Reduction of the nitro group of chloramphenicol (CAP) gives rise to more highly reactive intermediates which may be involved in the aplastic anemia associated with CAP use. One such intermediate, nitroso-chloramphenicol (NO-CAP), has been found to be a potent agent for mediating degradation of isolated DNA. In a reaction mixture containing 100 μM NO-CAP, 100 μM CuCl 2, and 5 mM NADH, 7 μg of Escherichia coli [ 3H]DNA was completely degraded to acid-soluble fragments in 30 min. Damage to DNA was in the form of single-stranded scissions. The requirement for copper was specific, and copper chelating reagents blocked the degradation. The need for a reducing agent could be met equally well by NADH or NADPH, but not by sulfhydryl reagents such as glutathione, dithiothreitol and 2-mercaptoethanol. Oxygen was also necessary for the NO-CAP-mediated DNA damage, with reduced forms of oxygen participating in the reaction. A role for H 2O 2 was indicated by the inhibition of the degradation seen when catalase was included in the mixture. Hydroxyl radicals are known to be produced in the reaction of H 2O 2 with certain transition metals. Scavangers of hydroxyl radicals also inhibited strand-scission, suggesting that the radicals may be the primary agents in DNA degradation. The importance of the nitroso moiety of NO-CAP was evidenced by the lack of DNA damage seen when NO-CAP was replaced by CAP under the conditions tested.

Differential effects of chloramphenicol and its nitrosoanalogue on protein synthesis and oxidative phosphorylation in rat liver mitochondria

Protein synthesis and oxidative phosphorylation were chosen as measures to study the differences between the effects of chloramphenicol (CAP) and its derivative nitroso-chloramphenicol (NO-CAP) on rat liver mitochondria. [ 14C]Leucine incorporation into mitochondrial protein was inhibited 83 per cent by 30 μM CAP and was equally inhibited by a similar concentration of thiamphenicol; 30μM NO-CAP, however, inhibited [ 14C]leucine incorporation only 34 per cent and 30μM nitrosobenzene had no effect. A millimolar concentration of CAP was required to inhibit oxidative phosphorylation, whereas 75 μM NO-CAP was inhibitory. Unlike CAP, NO-CAP at 100 μM slightly inhibited state 4 respiration with glutamate as substrate, but slightly activated it with succinate. Respiratory state 3 with glutamate was completely inhibited by 75 μM NO-CAP, whereas the same concentration of CAP was only 10 per cent inhibitory. With succinate, 250 μM NO-CAP was required to inhibit state 3, whereas 600 μM CAP had no effect. The uncoupled state triggered by 2,4-dinitrophenol in the presence of either glutamate or succinate was inhibited totally by NO-CAP, but not by CAP. The inhibition by NO-CAP was mitochondrial protein dependent, for more NO-CAP was required for inhibition with a larger amount of protein. NO-CAP effects could be prevented or released by cysteine, but not by washing. Oxidative phosphorylation was also inhibited by another nitroso compound, nitrosobenzene, which, however, did not affect mitochondrial protein synthesis. The results indicate that, unlike CAP, NO-CAP is a potent inhibitor of the energy conserving mechanism.