Chloramphenicol Metabolism Research Articles

Revealing the biological significance of multiple metabolic pathways of chloramphenicol by Sphingobium sp. WTD-1

Chloramphenicol (CAP) is an antibiotic that commonly pollutes the environment, and microorganisms primarily drive its degradation and transformation. Although several pathways for CAP degradation have been documented in different bacteria, multiple metabolic pathways in the same strain and their potential biological significance have not been revealed. In this study, Sphingobium WTD-1, which was isolated from activated sludge, can completely degrade 100 mg/L CAP within 60 h as the sole energy source. UPLC-HRMS and HPLC analyses showed that three different pathways, including acetylation, hydroxyl oxidation, and oxidation (C1-C2 bond cleavage), are responsible for the metabolism of CAP. Importantly, acetylation and C3 hydroxyl oxidation reduced the cytotoxicity of the substrate to strain WTD-1, and the C1-C2 bond fracture of CAP generated the metabolite p-nitrobenzoic acid (PNBA) to provide energy for its growth. This indicated that the synergistic action of three metabolic pathways caused WTD-1 to be adaptable and able to degrade high concentrations of CAP in the environment. This study deepens our understanding of the microbial degradation pathway of CAP and highlights the biological significance of the synergistic metabolism of antibiotic pollutants by multiple pathways in the same strain.

The Successional Pattern of Microbial Communities and Critical Genes of Consortia Subsisting on Chloramphenicol and Its Metabolites Through Long-Term Domestication

As a widespread emerging contaminant, chloramphenicol (CAP) adversely impacts ecological communities in the water environment. Biological treatment is widely used for aquatic pollutant removal, and the performance of functional microbes determines its outcome. Herein, a consortium with a powerful CAP-degrading capacity was domesticated from activated sludge. As the common degradation products of CAP, 4-nitrobenzoic acid (PNB) and 2,2-dichloroacetic acid (DCA) were also used as the sole substrates for long-term domestication. The successional pattern of the microbial community and critical functional genes through the 2.5-year domestication was revealed by metagenomic analysis. Sphingomonas, Caballeronia, and Cupriavidus became the most dominant populations in the CAP-, PNB-, and DCA-degrading consortia, respectively, and they were crucial degraders of PNB and DCA. Their collaboration contributed to the high mineralization rate of CAP. PNB was transformed into protocatechuic acid (PCA) and then mineralized through meta-cleavage and ortho-cleavage pathways. Crucial functional genes involved in CAP, PNB, and DCA metabolism, including CAP acetyltransferase, CAP oxidoreductase, haloacid dehalogenases, and protocatechuate dioxygenases, were significantly enriched in consortia. pH and carbon source had significant impacts on CAP biodegradation efficiency. The domesticated consortia and isolated strains are necessary microbial resources to enhance the bioremediation of CAP-, PNB-, or DCA-polluted environments.

Stereoselective metabolism of chloramphenicol by bacteria isolated from wastewater, and the importance of stereochemistry in environmental risk assessments for antibiotics

Wastewater treatment plants have been highlighted as a potential hotspot for the development and spread of antibiotic resistance. Although antibiotic resistant bacteria in wastewater present a public health threat, it is also possible that these bacteria play an important role in the bioremediation through the metabolism of antibiotics before they reach the wider environment. Here we address this possibility with a particular emphasis on stereochemistry using a combination of microbiology and analytical chemistry tools including the use of supercritical-fluid chromatography coupled with mass spectrometry for chiral analysis and high-resolution mass spectrometry to investigate metabolites. Due to the complexities around chiral analysis the antibiotic chloramphenicol was used as a proof of concept to demonstrate stereoselective metabolism due to its relatively simple chemical structure and availability over the counter in the U.K. The results presented here demonstrate the chloramphenicol can be stereoselectively transformed by the chloramphenicol acetyltransferase enzyme with the orientation around the first stereocentre being key for this process, meaning that accumulation of two isomers may occur within the environment with potential impacts on ecotoxicity and emergence of bacterial antibiotic resistance within the environment.

Metabolomic Profile of Primary Turkey and Rat Hepatocytes and Two Cell Lines after Chloramphenicol Exposure.

Simple SummaryThe use of cell cultures can be an important alternative for animal experiments. Therefore, it seems reasonable to use in vitro models to compare the liver metabolism of a drug in different animal species. Chloramphenicol is an effective broad-spectrum antibiotic used in human and pets. The toxicity of chloramphenicol is complex and differs among animal species mainly depending on the biotransformation. The purpose of this study was to assess chloramphenicol metabolism on its cytotoxicity in primary turkey and rat hepatocyte cultures, also in human hepatoma (HepG2) cells and nonhepatic, Balb/c 3T3 fibroblasts. To the best of our knowledge, this is the first report of differences in chloramphenicol metabolism in primary turkey and rat hepatocyte cultures. The two metabolites of the drug were detected in turkey and rat hepatocyte cultures. The amount of one metabolite of chloramphenicol was closely related to the cytotoxicity of the drug. The primary turkey and rat hepatocyte cultures were more sensitive to chloramphenicol than HepG2 cells and Balb/c 3T3 fibroblasts. The primary hepatocyte cultures represent valuable tools with which to study the biotransformation of xenobiotics and determine species differences in their metabolism and toxicity.The purpose of this study was to assess the formation of chloramphenicol metabolites in primary turkey and rat hepatocyte cultures and human hepatoma (HepG2) cells and nonhepatic, Balb/c 3T3 fibroblasts. Additionally, the cytotoxicity of the drug was assessed through three biochemical endpoints: mitochondrial and lysosomal activity and cellular membrane integrity after 24 and 48 h exposure. The two metabolites of the drug, chloramphenicol glucuronide and nitroso-chloramphenicol, were detected to the greatest extent in both primary hepatocyte cultures by liquid chromatography–tandem mass spectrometry. Toxic nitroso-chloramphenicol was the main metabolite in the primary turkey hepatocyte cultures, but it was not in the primary rat hepatocyte cultures. The most affected endpoint in turkey and rat hepatocyte cultures was the disintegration of the cellular membrane, but in the cell lines, mitochondrial and lysosomal activities underwent the greatest change. The primary hepatocyte cultures represent valuable tools with which to study the species differences in the biotransformation and toxicity of drugs. To the best of our knowledge, this is the first report of differences in chloramphenicol metabolism in primary turkey and rat hepatocyte cultures.

Ensuring safe and effective medication use in pediatric patients.

This is a tremendous honor to receive peer recognition carrying the name of late Dr Sumner Yaffe. I was fortunate to have worked with him over many years through professional organizations and as a part of the pediatric pharmacology research units (PPRUs). Dr Yaffe established pediatric pharmacology programs in Buffalo and Philadelphia and made important contributions to enhance pediatric care with his research, practice, and teaching. He led the development of PPRUs and the neonatal networks, and was a force behind the framing of what became the Best Pharmaceuticals for Children Act (BPCA) and Pediatric Research Equity Act (PREA). He was indeed a visionary leader and mentor in the field of pediatric pharmacology. The objectives of my presentation today are to briefly share my background, reflect on our work, and discuss potential opportunities to advance pediatric pharmacy, pharmacology, and therapeutics.

Chloramphenicol Is a Substrate for a Novel Nitroreductase Pathway in Haemophilus influenzae

The p-nitroaromatic antibiotic chloramphenicol has been used extensively to treat life-threatening infections due to Haemophilus influenzae and Neisseria meningitidis; its mechanism of action is the inhibition of protein synthesis. We found that during incubation with H. influenzae cells and lysates, chloramphenicol is converted to a 4-aminophenyl allylic alcohol that lacks antibacterial activity. The allylic alcohol moiety undergoes facile re-addition of water to restore the 1,3-diol, as well as further dehydration driven by the aromatic amine to form the iminoquinone. Several Neisseria species and most chloramphenicol-susceptible Haemophilus species, but not Escherichia coli or other gram-negative or gram-positive bacteria we examined, were also found to metabolize chloramphenicol. The products of chloramphenicol metabolism by species other than H. influenzae have not yet been characterized. The strains reducing the antibiotic were chloramphenicol susceptible, indicating that the pathway does not appear to mediate chloramphenicol resistance. The role of this novel nitroreductase pathway in the physiology of H. influenzae and Neisseria species is unknown. Further understanding of the H. influenzae chloramphenicol reduction pathway will contribute to our knowledge of the diversity of prokaryotic nitroreductase mechanisms.

The development of drug metabolising enzymes and their influence on the susceptibility to adverse drug reactions in children.

Altered drug disposition in the developing child occurs as a result of both biochemical and physiological changes. The clearance of many drugs is dependent on their biotransformation in the liver and small bowel and consequently is developmentally determined by a number of factors including both the activity and abundance of enzymes involved in Phase 1 and 2 drug metabolism. Altered drug metabolism can lead to the development of adverse effects in neonates and small infants that are not generally seen in the adult population. For instance, the altered metabolism of sodium valproate in children under 3 years of age is thought to be responsible for a higher incidence of hepatotoxicity, the impaired metabolism of chloramphenicol in neonates has resulted in the grey baby syndrome (cyanosis and respiratory failure) and metabolic acidosis following the use of propofol in the critically ill child may be due to altered drug metabolism. This paper reviews the potential contribution of the ontogeny of a number of drug metabolising enzymes including cytochrome P450 and glucuronoslytransferases to the development of adverse drug reactions in children.

Drug trials in children: problems and the way forward.

The drug-licensing system was introduced with the Medicines Act of 1968, following the thalidomide disaster, with the aim of ensuring that medicines are safe, effective and of high quality. There is increasing concern from the pharmaceutical industry, paediatricians, paediatric pharmacists and the Government that many medicines routinely used in children have not been formally evaluated by this system. Such use is therefore not supported by the assurances that the process brings. A joint report was produced in 1996 by the British Paediatric Association (now the Royal College of Paediatrics and Child Health) and the Association of the British Pharmaceutical Industry. This described in detail the current unsatisfactory situation and made specific recommendations for a way forward [1]. Since then several studies have documented the extent of unlicensed and off-label drug use in critically ill children, paediatric medical and surgical inpatients and neonates [2–4]. These studies all show that unlicensed and off-label drug use is a frequent occurrence for children in hospital with up to 90% of newborn infants in intensive care receiving either unlicensed or off-label treatment. Unlicensed medicines may be those compounded in a hospital pharmacy department or by a ‘specials’ manufacturer due to a lack of licensed alternatives. These medicines may not necessarily undergo the rigorous quality testing procedures that a licensed preparation must, and their quality may be variable. The term off-label refers to the use of a medicine outside the specifications of the product licence [5]. This covers medicines used at a different dosage to that recommended, in different age groups, by a different route or for a different indication. These off-label uses may be based on reliable published data, in which case prescribers can feel confident in using medicines in an off-label manner since this practice would be supported by peer review. In other instances they may rely more on the experience of the paediatrician or pharmacist, or drug information resources such as those of DIAL (Drug Information Advisory Line, Alder Hey Children’s Hospital, Liverpool). They are less likely to be supported by extensive clinical trial information, as would the manufacturer’s recommended uses of a licensed preparation.

Chloramphenicol oxamylethanolamine as an end product of chloramphenicol metabolism in rat and humans: evidence for the formation of a phospholipid adduct.

Chloramphenicol (CP) has been implicated as, although not proven to be, a causative agent of aplastic anemia in humans. Recent studies from our laboratory have presented evidence that CP-oxamylethanolamine was an end product of CP biotransformation in birds. Because this novel metabolic pathway has never been reported in other species, we have now expanded these investigations to rat and humans. [3H]CP was administered po (10 mg/kg) to adult male Wistar rats and to a human volunteer. Urine was collected and analyzed by HPLC and GC-MS for CP metabolite determination. In rat, the two most important metabolites in 0-24 h urine were CP-base and CP-acetylarylamine which together accounted for about 50% of the ingested radioactivity. The remainder was due to unchanged CP, CP-oxamic acid, CP-alcohol, CP-glucuronide, and CP-oxamylethanolamine. The presence of these end products was also demonstrated in man. CP-oxamylethanolamine represented 0.74% and 1.37% of the ingested radioactivity in rat and human urine samples, respectively. CP-oxamylethanolamine formation was confirmed in vitro with isolated rat hepatocytes, suggesting the involvement of liver in the production of this metabolite. The origin of CP-oxamylethanolamine has been investigated with the use of hepatic liver microsomes from phenobarbital-treated rats. The incubation of [3H]CP with this subcellular fraction led to the binding of a radiolabeled compound to the microsomal lipids, whereas no binding occurred when CP-oxamic acid was incubated with the microsomes. Enzymatic hydrolysis of the microsome lipid fraction with phospholipase D from Streptomyces chromofuscus released CP-oxamylethanolamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of chloramphenicol by glutathione S-transferase in human fetal and neonatal liver.

The glutathione S-transferases of human fetal and neonatal liver catalyse the conjugation of glutathione with chloramphenicol at a low but measurable rate. The highest rates were 1.30 nmol/min/mg protein in a preterm neonate of 26 weeks of gestation and 1.11 nmol/min/mg in a fetus of 22 weeks of gestation, while the lowest measurable was 0.1 nmol/min/mg in a fetus of 17 weeks of gestation. The activity did not correlate with gestational age, but appeared dependent on the concentration of glutathione in the reaction mixture. The rate rose by a factor of three, from 0.39 nmol/min/mg protein with no added glutathione to 1.24 nmol/min/mg with 2 mumol/ml added to the reaction mixture. Chloramphenicol-aldehyde was detectable in the reaction mixture when a liver extract was incubated with glutathione but the proposed intermediate, glutathione-chloramphenicol, could not be demonstrated. Differences in activity with chloramphenicol or a model substrate, under varying conditions, indicate that different isoenzymes are concerned with the conjugation of glutathione to the two substrates. These data support the hypothesis that when glucuronide conjugation is depressed by immaturity, chloramphenicol is metabolised via other pathways.

A reappraisal of chloramphenicol metabolism: detection and quantification of metabolites in the sera of children

Seventy-four samples of serum from 27 children (age range: 26 weeks preterm to 12 y) treated with chloramphenicol for serious infections, were analyzed by high performance liquid chromatography (HPLC) for the presence of chloramphenicol metabolites. Five, including a previously unreported human metabolite--chloramphenicol aldehyde, were detected and identified by comparative HPLC, enzymic degradation or gas chromatography followed by mass spectrometry. The presence of these metabolites in the serum suggests that the biotransformation of chloramphenicol takes place by oxidation, reduction and conjugation and not by conjugation alone, as has been previously supposed. Sera from 84% of the patients contained one or more metabolites, however the presence of particular compounds did not correlate with toxicity in the four patients in whom this was observed.

Metabolism of Chloramphenicol: A Story of Nearly 50 Years

Metabolism of Chloramphenicol: A Story of Nearly 50 Years

Chloramphenicol: relation of structure to activity and toxicity.

The availability of an increasing number of effective broad spectrum anti­ microbials in the past decade has diminished considerably the clinical in­ dications for chloramphenicol (CAP). However, interest in this antibiotic has recently been revived both because of its antimicrobial activity and its toxic­ ity. Some factors affecting its resurgence are the emergence of ampicillin­ resistant Halmophilus influenzae, the superiority of CAP in fighting certain anaerobic infections and infections of the central nervous system, and the development of sensitive assays for the antibiotic and some of its metabolites in body fluids. Greater attention, however, has focused on hematotoxicity from CAP and its pathogenesis. The extensive use of a CAP analogue, thiamphenicol (TAP), in Europe and the Far East, without an increase in the incidence of associated aplastic anemia, has revived interest in the structure­ toxicity relationship in the CAP molecule and has focused attention on the P-N02 group as the structural feature that probably underlies the development of bone marrow aplasia in association with administration of CAP. TAP, with a similar antimicrobial spectrum, has emerged as a challenging substitute to CAP. In this chapter the comparative metabolism and toxicity of CAP and TAP are reviewed with particular emphasis on the role of the P-N02 group. For a clearer perspective, the properties of CAP, its mechanism of action, and relation of structure to activity and to bacterial resistance are briefly consid­ ered.

Disposition and metabolism of chloramphenicol in trout.

The urinary and faecal excretion, tissue distribution and metabolism of 3H-chloramphenicol (CP) were measured in rainbow trout, Salmo gairdneri R., after a single 50 mg/kg intragastric dose. The major route of excretion of 3H was faecal (64.3% of the dose), with approx. 16% in the urine in five days. Radioactivity was widely distributed in trout tissues and organs, the highest concen. being in the bile and intestine. At 48 h after dosing, the radioactivity remaining in the liver, the muscle and the perigastric adipose tissue was as CP-derived compounds bound to tissues. In addition to unchanged CP (4.3% dose after 96 h), the other metabolites excreted in the urine were CP base (5.2%), CP alcohol (4.0%) and CP glucuronide (1.8%).

Photochemical decomposition of chloramphenicol in a 0.25% eyedrop and in a therapeutic intraocular concentration

It has been postulated that in the metabolism of chloramphenicol, nitroso compounds can be formed in the “predisposed host”. This would be the cause of non-dose-related aplastic anemia. Solid proof for this metabolic formation in vivo has not been given. However, on exposure to sunlight under in vivo-related circumstances, relevant to the eye under treatment, we found beside p-nitrobenzaldehyde, p-nitrobenzoic acid and p-nitrosobenzoic acid. The latter even up to 45 mol% of the starting amount of chloramphenicol. This photoreaction is not restricted to the eye but may also occur in the skin.

Chloramphenicol dosage and pharmacokinetics in infants and children.

Twenty infants and children receiving intravenous chloramphenicol were studied to examine the pharmacokinetics of the parent compound and its precursor, the succinate ester (CAP-S). Plasma samples were obtained just prior to a 30-minute infusion of chloramphenicol succinate, immediately after or 30 minutes after infusion, and 90, 210, and 330 minutes after infusion. Complete 6-hour urine collections were obtained during 11 studies. Plasma and urine were assayed for chloramphenicol and its succinate ester by high-performance liquid chromatography. Peak plasma concentrations ranged from 11.0 to 51.1 micrograms/ml on doses of 50 to 100 mg/kg/day and were higher in the youngest age group. The elimination half-life of chloramphenicol averaged 4.0 hours. Multilinear regression analysis demonstrated an excellent relationship between body surface area, trough plasma chloramphenicol concentration, and total body chloramphenicol clearance. The hydrolysis of succinate ester to free chloramphenicol may delay the peak free concentration, and its renal elimination (average 21 per cent of the dose administered) significantly affects chloramphenicol pharmacokinetics. The clearance of chloramphenicol exhibited enzyme saturation kinetics in one patient studied at two different doses. Dosage adjustments of intravenous chloramphenicol in children must be made in relation to the trough chloramphenicol plasma concentration, renal elimination of CAP-S, and possible saturation of chloramphenicol metabolism.

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.

Interactions among chloramphenicol, phenytoin, and phenobarbital in a pediatric patient

O N E of the important factors determining serum concentration of chloramphenicol is the rate of hepatic metabolism to inactive metabolites by the microsomal enzyme glucuronyl transferase. 1 Since both phenytoin and phenobarbital can induce hepatic microsomal enzymes, their use may increase the rate of chloramphenicol metabolism. 2 Because these drugs may be used with chloramphenicol in treating pediatric infections of the central nervous system, it is important to know how these drugs interact. We report a child with hydrocephalus who was treated with chloramphenicol for a ventriculojugular infection. Concomitant with the sequential addition of phenytoin and phenobarbital, there was a significantly lowered chloramphenicol serum concentration and an increased total body clearance of chloramphenicol relative to values reported in pediatric patients.

Comparative effects of antacids, enteric coating, and bile salts on the efficacy of oral pancreatic enzyme therapy in cystic fibrosis

70% of the areas obtained when chloramphenicol was administered orally. Our experience confirms that of others 3. ' that the wide variation in chloramphenicol metabolism and excretion requires monitoring of serum concentrations periodically during therapy to ensure therapeutic concentrations and at the same time minimize the risk of dose-related toxicity. Dosage requirements may vary threefold in children of the same age. This variation is even greater in newborn and young infants, making monitoring of serum concentration even more imperative in this age group. We gratefully acknowledge the collaboration of Dr. Ronald Poland, Chief of Neonatology, Children's Hospital of Michigan, in collecting data on some of the infants in this study.

A new pathway of metabolism of chloramphenicol which influences the interpretation of its irreversible binding to protein in vivo

The mechanism of the metabolic activation of chloramphenicol (CAP) in vivo in the rat has been studied by measuring the irreversible binding to protein of specifically labeled [1, 2- 14C] dichloroacetamido and 1- 3H-benzylic derivatives of CAP. In every tissue examined, the 14C-label of CAP bound significantly more than the 3H-label, indicating that only part of the structure of CAP was bound. When 14C-labeled plasma protein, from rats given [ 14C]CAP, was hydrolyzed chemically or enzymatically, glycine and serine were identified by thin-layer chromatography and reverse isotope dilution analysis as the predominant radioactive products. These results indicate that the majority of the irreversible binding of CAP to protein in vivo is not due to its metabolic activation into a reactive metabolite as was found in vitro in liver microsomes, but instead is due to its metabolism into small molecular fragments, which are incorporated into pools of normal tissue constituents. These small molecules are subsequently biosynthesized into glycine and serine, and these amino acids are then incorporated into the structure of tissue protein. The general importance of these results and possible pathways for the metabolism of CAP into glycine and serine are discussed.