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Structural basis for the haemotoxicity of dapsone: The importance of the sulphonyl group
Abstract
The structural basis of dapsone (4,4'-diaminodiphenyl sulphone) haemotoxicity has been determined by investigation of the in vitro bioactivation of a series of 4-substituted arylamines. In the presence of rat liver microsomes, dapsone (100 microM) was the most potent former of methaemoglobin in human erythrocytes (44.8 +/- 6.7%). Substitution of the sulphone group with sulphur (11.6 +/- 1.4% methaemoglobin), oxygen (4.5 +/- 1.1%), nitrogen (0.0 +/- 3.2%), carbon (13.6 +/- 0.8%) or a keto group (34.0 +/- 6.1%) resulted in a decrease in methaemoglobin formation. Only one compound, 4,4'-diaminodiphenylamine, generated significant (P < 0.001) amounts of methaemoglobin (25.6 +/- 2.5%) in the absence of NADPH. To assess further the role of the 4-substituent in methaemoglobinaemia, the toxicity of a series of 4-substituted aniline derivatives was also studied. Of the anilines studied, 4-nitroaniline caused the most methaemoglobin (36.5 +/- 8.0%), whilst aniline caused the least (0.3 +/- 0.5%). Overall, there was a significant correlation (r2 = 0.83) between the haemotoxicity and the Hammett constant, sigma(p), suggesting that it is the electron-withdrawing properties of the substituent that influence the methaemoglobin formation. In the presence of microsomes prepared from two human livers, dapsone was the most haemotoxic bis arylamine, whereas 4-iodoaniline was the most potent methaemoglobin former (60.6 and 73.6%) and aniline the least potent (1.1 and 2.4%). As a whole, these results indicate that the sulphonyl group, which is essential for the pharmacological activity of dapsone, is also largely responsible for the haemotoxicity seen with this drug.
... For drug classes and drugs associated with the described toxicity less commonly reported, the original report has been referenced. For drug classes and drugs for which the toxicity is commonly reported and discussed, reviews or monographs have been referenced , Rowe and Welch 1994, Zhang, et al. 2009Feldmann, et al. 2005Harvey, et al. 1979Harvey, et al. 1979 Sulfa antibiotics (104) No primary Yes Yes Yes Yes -0.0421 -0.3099 Chen, et al. 2010, Dollery 1993Baldwin, et al. 1977, 1997Dollery 1993, Ward, et al. 1983Dollery 1993, Ward, et al. 1983 Aminoquinolines ( Baldwin 1976, Dearden, et al. 1995Bürgi, et al. 1974, Baldwin, et al. 1977, Feldmann, et al. 2005, Gilmore and Alabugin 2011Baldwin, et al. 1977, Gilmore and Alabugin 2011Baldwin, et al. 1977, Gilmore and Alabugin 2011 Yes primary, secondary Yes -0.0038 -0.3028 Arndt andGarratty 2005, Sanford-Driscoll andKnodel 1986 Thiazide diuretics Agrawal and Khadikar, Soglia, et al. 2004Baldwin, et al. 1977, Blair 2006 McGrath and Li, Soglia, et al. 2004 McGrath and Li, Soglia, et al. 2004 Clofazimine (123) No secondary Yes Yes Yes 0.2135 -0.3783 Svenson, et al. 1974Svenson, et al.1974Svenson, et al. 1974 (Continues) Shamovsky, et al. 2011Baldwin, et al. 1977Franz and Wilson 2013, Kovacic and Somanathan 2014, Tacke, et al. 2010Franz and Wilson 2013, Kovacic and Somanathan 2014, Tacke, et al. 2010 Nomifensine ( The drug classes refer only to the aryl amine drugs listed in Fig. 21 e.g., the COX-inhibitor naproxen which lacks the aryl amine motif is not assumed to be a member of this class in this table. ...
... In a study by Tingle, 13 substituted anilines were investigated for their ability to cause methemoglobinemia (Mahmud et al., 1997). Some structural descriptors and physiochemical properties were calculated and tested as predictors of the ability to cause methemoglobin formation in the assay. ...
When reactive centers are formed in chemical conversions, intermolecular reactions tend to dominate over intramolecular alternatives whenever both alternatives are possible. Hence, when reactive metabolites are formed from xenobiotics, intramolecular quenching by moieties adjacent to a toxicophore may play an important role in reducing toxicity related to reactive intermediates. The phenomenon is likely to be particularly noticeable for toxicophores that are readily associated with a type of toxicity that is rarely caused by other structural motives. In two demonstrative investigations, it is concluded that nitrobenzenes for which the expected nitrosyl metabolite is likely to react with adjacent groups are less toxic than what is rationally expected, and that among aryl amine drugs allowing for the immediate quenching of the corresponding N-aryl hydroxylamine metabolite, the typical erythrocyte toxicity often seen with aryl amines is absent. The deliberate introduction of effective quenching groups nearby a toxicophoric moiety may present a potential strategy for reducing toxicity in the design of drugs and other man-made xenobiotics. Copyright © 2015 John Wiley & Sons, Ltd.
... [5][6][7] Other study showed that the dapsone did not oxidize hemoglobin in the absence of NADPH. 8 Among the 13 amines tested in incubations containing liver enzymes mixed with target cells, dapsone was the most potent * Author to whom correspondence should be addressed. methemoglobin former in the presence of rat liver enzymes, whilst aniline was the less potent. ...
... methemoglobin former in the presence of rat liver enzymes, whilst aniline was the less potent. 8 Nevertheless, there are no studies that explain and correlate the electronic structure-toxicity and redox mechanism of dapsone. ...
Quantum chemical calculations at the B3LYP level of theory, together with the 6-31G* basis sets, were employed to obtain electronic properties of the dapsone and related derivatives in order to study their methemoglobinemia mechanism. The electronic properties such as HOMO, LUMO, ionization potential, MEPs, and spin densities were correlated to redox properties of the compounds studied. The results show that the amine linked to the aniline moiety at the para-position is the main contributor to methemoglobin property. The sulfone moiety is responsible for the electron transition between both aniline rings. The lowest ionization potential is related with the increase of methemoglobinemia.
... For instance, structuretoxicity relationship studies on dapsone analogs indicate the parent compound as the most potent inducer of methaemoglobinemia in human erthrocytes in the presence of rat liver microsomes and that replacement of the sulfone group with S, O, NH, CH 2 , or C=O functionalities decreases the toxic effects. A significant correlation between hemotoxicity and the Hammett constant σ p suggests that the electron-withdrawing properties of the 4-substituent in dapsone influences the rate of oxidation of the primary amine nitrogen [54]. Nitrenium ion GSH = Glutathione ...
... .(54). P450 Catalyzed Epoxidation of Phenyl Rings. ...
The occurrence of idiosyncratic adverse drug reactions during late clinical trials or after a drug has been released can lead to a severe restriction in its use and even in its withdrawal. Metabolic activation of relatively inert functional groups to reactive electrophilic intermediates is considered to be an obligatory event in the etiology of many drug-induced adverse reactions. Therefore, a thorough examination of the biochemical reactivity of functional groups/structural motifs in all new drug candidates is essential from a safety standpoint. A major theme attempted in this review is the comprehensive cataloging of all of the known bioactivation pathways of functional groups or structural motifs commonly utilized in drug design efforts. Potential strategies in the detection of reactive intermediates in biochemical systems are also discussed. The intention of this review is not to "black list" functional groups or to immediately discard compounds based on their potential to form reactive metabolites, but rather to serve as a resource describing the structural diversity of these functionalities as well as experimental approaches that could be taken to evaluate whether a "structural alert" in a new drug candidate undergoes bioactivation to reactive metabolites.
... One possible explanation for this is that bulky groups in the ortho and meta positions can disrupt the necessary metabolic step required to produce the mutagenic nitrenium ion. A possible explanation for substitution patterns in the para position being more mutagenic than the ortho and meta positions is that certain electron withdrawing groups in the para position can stabilize the nitrenium ion (Mahmud et al., 1997). Generally, the results were consistent with the list of activating and deactivating features described in Section 1.3; however, many additional structural features have now been reported as being responsible for aromatic amine mutagenicity or lack of mutagenicity. ...
... However, a certain degree of hemolysis during DDS therapy appears inevitable, as alongside our present report, several previous studies have shown the presence of Heinz bodies during therapy with this drug [15,41]. In addition, Mahmud et al. [42] reported that the dapsone (100 microM) was the most potent former of methaemoglobin in human erythrocytes, and the substitution of the sulphone group with sulphur, oxygen, nitrogen, carbon or a keto group in drug resulted in a decrease in methaemoglobin formation. ...
This study aims to assess the oxidative stress in leprosy patients under multidrug therapy (MDT; dapsone, clofazimine and rifampicin), evaluating the nitric oxide (NO) concentration, catalase (CAT) and superoxide dismutase (SOD) activities, glutathione (GSH) levels, total antioxidant capacity, lipid peroxidation, and methemoglobin formation. For this, we analyzed 23 leprosy patients and 20 healthy individuals from the Amazon region, Brazil, aged between 20 and 45 years. Blood sampling enabled the evaluation of leprosy patients prior to starting multidrug therapy (called MDT 0) and until the third month of multidrug therapy (MDT 3). With regard to dapsone (DDS) plasma levels, we showed that there was no statistical difference in drug plasma levels between multibacillary (0.518±0.029 µg/mL) and paucibacillary (0.662±0.123 µg/mL) patients. The methemoglobin levels and numbers of Heinz bodies were significantly enhanced after the third MDT-supervised dose, but this treatment did not significantly change the lipid peroxidation and NO levels in these leprosy patients. In addition, CAT activity was significantly reduced in MDT-treated leprosy patients, while GSH content was increased in these patients. However, SOD and Trolox equivalent antioxidant capacity levels were similar in patients with and without treatment. These data suggest that MDT can reduce the activity of some antioxidant enzyme and influence ROS accumulation, which may induce hematological changes, such as methemoglobinemia in patients with leprosy. We also explored some redox mechanisms associated with DDS and its main oxidative metabolite DDS-NHOH and we explored the possible binding of DDS to the active site of CYP2C19 with the aid of molecular modeling software.
... 4 Studies concerning structure-activity relationships have demonstrated that the sulphone group is essential for the pharmacological activity of dapsone, and indeed no alternative pharmacophore has been identified to date. [5][6][7] Mahmudl et al. 8 has related that the dapsone did not oxidize haemoglobin in the absence of NADPH. Others amines tested in incubations which contained liver enzymes mixed with target cells, dapsone was the most potent methaemoglobin former in the presence of rat liver enzymes, whilst aniline was the least potent. ...
Quantum mechanical calculations at the B3LYP theory level, together with the 6-31G* basis set, were employed to obtain the energy, HOMO, LUMO, MEPs, and charge of dapsone is compared with others derivatives. Conformational analysis using density functional calculations show that symmetric conformational isomer has low energy than asymmetric conformational isomer. The symmetry level was observed by molecular charge analysis. Its redox properties by electrons transfer are dependent of amine and sulphone moieties. Our results explain the oxidation mechanism of dapsone by electron transfer.
... Thus the variation in the effect of substitution may reflect the fact that subtle changes in the molecule have altered the binding of the substrate to cytochrome P450 enzymes, which are subject to species variation in expression (Gonzalez, 1990), rather than merely a change in the intrinsic ability of the compounds to undergo oxidation. Furthermore, data for methaemoglobin formation and N-hydroxylation of the analogues in the presence of rat liver microsomes suggests that the hydroxylamines have different intrinsic toxicity towards erythrocytes compared with dapsone, as has been seen previously for procainamide hydroxylamine and phenylhydroxylamine (Tingle and Park, 1993;Mahmud et al., 1997), possibly due to the changes in the electronic properties of the molecule. ...
The effect of 2,2'-substitution with fluorine, methyl or trifluoromethyl groups on the toxicity, metabolism and pharmacological activity of dapsone has been investigated in vitro and in vivo. There was marked inter-species variation in the bioactivation (N-hydroxylation) of the compounds, as determined by methemoglobin formation. However, the inclusion of fluorine significantly (P<0.01) reduced methemoglobin formation compared with dapsone in all species studied. All three analogs resulted in significantly (P<0.001) less methemoglobinemia than dapsone when given either intraperitoneally or intravenously to the male Wistar rat. Rapid plasma clearance of the analogs through increased lipophilicity and enhanced N-glucuronidation may account for the low toxicity compared with dapsone. Although trifluoromethyl substitution resulted in a loss of activity against respiratory burst in human neutrophils in an in vitro model, all three analogs retained pharmacological activity against Plasmodium berghei malaria in an in vivo mouse model.
... The cytotoxic effects of dapsone are abolished in the presence of reducing agents suggesting that the hydroxylamine metabolite 59 undergoes a further two-electron oxidation to the highly reactive nitroso metabolite 60 that reacts with GSH to form the S-linked conjugate 64 via the unstable mercaptal 63 (see Fig. 8). Structure-toxicity relationship studies on dapsone analogs indicate the parent compound as the most potent inducer of methemoglobinemia in human erythrocytes in the presence of rat liver microsomes, and also that replacement of the sulfone group with S, O, NH, CH 2 , or C=O functionalities decreases the toxic effects [108]. A significant correlation between hemotoxicity and the Hammett constant (σ p ) suggests that the electron-withdrawing properties of the 4substituent in dapsone increases the rate of oxidation of the 1 0 amine nitrogen presumably by increasing its acidity. ...
The unexpected occurrence of idiosyncratic drug reactions during late clinical trials or after a drug has been released can lead to a severe restriction in its use or failure to release/withdrawal. This leads to considerable uncertainty in drug development and has led to attempts to try to predict a drug's potential to cause such reactions. The biotransformation of relatively inert drugs to highly reactive metabolites, commonly referred to as "bioactivation", is now recognized to be an obligatory step in several kinds of drug-induced adverse reactions. Reactive metabolites can be formed by most, if not all, of the enzymes that are involved in drug metabolism. A major theme explored in this review includes the diversity of oxidative bioactivation reactions on nitrogen-containing xenobiotics including drugs. A variety of Phase I enzymes including P450, MAO, and peroxidases bioactivate nitrogen-containing xenobiotics via direct oxidations on the nitrogen atom leading to reactive intermediates or by oxidation at an alternate site in the molecule; for the metabolite to be reactive via the latter sequence nitrogen participation in required. Examples of direct oxidations on nitrogen include the N-oxidation of aromatic amines (e.g. procainamide), single electron N-oxidation of imides (e.g. phenytoin), or alpha-carbon oxidations of arylalkyl- or alkylamines (e.g. mianserin), to reactive nitroso, nitrogen free radical and iminium species, respectively. Examples of indirect bioactivation are highlighted with aromatic amines (e.g. diclofenac) that undergo p-hydroxylation resulting in the formation of p-aminophenols, two-electron oxidation of which results in the formation of reactive quinoneimines. Potential strategies that could be utilized in the screening of novel bioactivation pathways are also discussed.
... Clinical studies with flutamide and nilutamide (Fig. 2), two structurally related androgen receptor ligands that include an A-ring nitro group, showed that most of the adverse effects (e.g., gynecomastia, breast pain, etc.) were associated with expected antiandrogenic effects of drugs (Mahler et al., 1998). It is known that intermediate metabolites of aryl amines are susceptible to extensive redox cycling leading to methemoglobinemia and hemolytic anemia (Mahmud et al., 1997). However, this is rarely a clinical problem for flutamide (Mahler et al., 1998). ...
S-1 [3-(4-fluorophenoxy)-2-hydroxy-2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]-propanamide] is one member of a series of potent selective androgen receptor modulators (SARMs) that are being explored and developed for androgen-dependent diseases. Recent studies showed that S-1 holds great promise as a novel therapeutic agent for benign hyperplasia [W. Gao, J. D. Kearbey, V. A. Nair, K. Chung, A. F. Parlow, D. D. Miller, and J. T. Dalton (2004) Endocrinology 145:5420-5428]. We examined the pharmacokinetics and metabolism of S-1 in rats as a component of our preclinical development of this compound and continued interest in structure-activation relationships for SARM action. Forty male Sprague-Dawley rats were randomly assigned to treatment groups and received either an i.v. or a p.o. dose of S-1 at a dose level of 0.1, 1, 10, or 30 mg/kg. S-1 demonstrated a low clearance (range, 3.6-5.2 ml/min/kg), a moderate volume of distribution (range, 1460-1560 ml/kg), and a terminal half-life ranging from 3.6 to 5.2 h after i.v. doses. The oral bioavailability of S-1 ranged from 55% to 60%. Forty phase I and phase II metabolites of S-1 were identified in the urine and feces of male Sprague-Dawley rats dosed at 50 mg/kg via the i.v. route. The two major urinary metabolites of S-1 were a carboxylic acid and a sulfate-conjugate of 4-nitro-3-trifluoromethylphenylamine. Phase I metabolites arising from A-ring nitro reduction to an aromatic amine and B-ring hydroxylation were also identified in the urinary and fecal samples of rats. Furthermore, a variety of phase II metabolites through sulfation, glucuronidation, and methylation were also found. These studies demonstrate that S-1 is rapidly absorbed, slowly cleared, moderately distributed, and extensively metabolized in rats.
... The influence of electronics on bioactivation potential is evident in structure-toxicity assessment on dapsone analogues. Replacement of the sulfone group in dapsone with sulfur, oxygen, methylene or carbonyl substituents significantly reduces methemoglobinaemia in human erythrocytes (Figure 11) [234]. Although the reason(s) for these structure-activity observations is unclear, a good correlation between haemotoxicity and the Hammett constant (σp) was observed in this analysis, which suggests the influence of the 4-substituent on the rate of oxidation of the aniline nitrogen. ...
Investigations into the role of bioactivation in the pathogenesis of xenobiotic-induced toxicity have been a major area of research since the link between reactive metabolites and carcinogenesis was first reported in the 1930s. Circumstantial evidence suggests that bioactivation of relatively inert functional groups to reactive metabolites may contribute towards certain drug-induced adverse reactions. Reactive metabolites, if not detoxified, can covalently modify essential cellular targets. The identity of the susceptible biomacromolecule(s), and the physiological consequence of its covalent modification, will dictate the resulting toxicological response (e.g., covalent modification of DNA by reactive intermediates derived from procarcinogens that potentially leads to carcinogenesis). The formation of drug-protein adducts often carries a potential risk of clinical toxicities that may not be predicted from preclinical safety studies. Animal models used to reliably predict idiosyncratic drug toxicity are unavailable at present. Furthermore, considering that the frequency of occurrence of idiosyncratic adverse drug reactions (IADRs) is fairly rare (1 in 1000 to 1 in 10,000), it is impossible to detect such phenomena in early clinical trials. Thus, the occurrence of IADRs during late clinical trials or after a drug has been released can lead to an unanticipated restriction in its use and even in its withdrawal. Major themes explored in this review include a comprehensive cataloguing of bioactivation pathways of functional groups commonly utilised in drug design efforts with appropriate strategies towards detection of corresponding reactive intermediates. Several instances wherein replacement of putative structural alerts in drugs associated with IADRs with a latent functionality eliminates the underlying liability are also presented. Examples of where bioactivation phenomenon in drug candidates can be successfully abrogated via iterative chemical interventions are also discussed. Finally, appropriate strategies that aid in potentially mitigating the risk of IADRs are explored, especially in circumstances in which the structural alert is also responsible for the primary pharmacology of the drug candidate and cannot be replaced.
Aromatic nitro and amino compounds have various commercial, governmental, and public applications. Their uses include manufacturing of dyes, pesticides, drugs, and explosives. These substances are typically considered high‐nitrogen compounds and as such share many of the same chemical and biological properties. Many have amines and/or nitrate groups on the ring and because of this exhibit properties where they are not highly water or lipid soluble and many tend to form crystals. When released to the environment, nitrate‐containing moieties are often reduced to amines by bacteria in wet soils and sediments. Acute effects typically target the nervous system; however, sublethal effects from repetitive exposures include anemia, methemoglobinemia, and often male reproductive effects. Cyanosis has been described from relatively high repetitive exposures. Oral exposures can affect the liver. Many are absorbed through the skin where dermal exposures can significantly contribute to systemic dose. Skin sensitization and irritation have been observed from exposure to some compounds. Many are eliminated via the urine; however, conjugated metabolites can be eliminated via the feces. Nitroso metabolites are suspected as having influence in observation of cancer, particularly in the bladder. Other similar nitramine compounds are also briefly described.
Toxicological Effects of Veterinary Medicinal Products in Humans is the first definitive guide to discuss the adverse effects of veterinary medicinal products in humans. The chapters focus on occupational safety and consumer issues and examine the circumstances under which exposure is likely to occur. To be in context, it reviews this against the background of adverse health effects from other sources in the veterinary and farming professions. The book examines adverse drug effects reported to regulatory agencies (mainly the FDAÆs Center for Veterinary Medicine) and then considers a series of individual drugs, including antibiotics, anaesthetics and organophosphorus compounds. The chapters also discuss the fundamental aspects of regulatory issues relating to safety assessment, and examine the manner in which user safety is assessed prior to authorisation/approval and what measures can be taken after authorisation/approval in the light of findings from pharmacovigilance activities. There is growing concern over the issue of antimicrobial resistance and the contribution made by veterinary medicinal products. This too is addressed along with the significance to human health and measures that can be taken to mitigate the effects (if any) of the use of antibiotics in animals e.g. prudent use measures. The book will be an essential resource for medical practitioners in hospitals and general practice, pharmaceutical industry scientists, analysts, regulators and risk managers.
The cytotoxic effect of methylgerambullin, a sulphone isolated from Glycosmis calcicola was screened against various cancer cell lines such as CEM-SS (T-lymphoblastic leukemia), KU812F (chronic myelogeneous leukemia), UACC-62 (melanoma) and HT29 (colon cancer) using a colorimetric tetrazolium (MTT) assay. T-lymphoblastic cell line was the most sensitive towards the compound, with CD50 value of 0.25 μg/ml, whereas for other cell lines the CD50 was from 1.3 to 1.7 μg/ml except for the peripheral blood mononuclear cells and normal mouse fibroblast cell line (3T3) the CD50 was 30 and 10 μg/ml, respectively. Examination by acridine-orange/porpidium iodide staining and electron microscopy showed that the compound caused both apoptotic and necrotic cell death.
Carbocyclic sixmembered aromatic rings are widely present in natural products and endogenous ligands and have therefore become the backbone of new chemical entities designed and synthesized by the medicinal chemists. Addition of a phenyl ring into a molecule certainly influences its lipophilicity. Incorporation of a substituent or a functional group in an aromatic ring in place of hydrogen atom can significantly perturb the electronic, steric, hydrophobic, hydrophilic, and hydrogenbonding parameters of the ring depending on the characteristics of that group. Since absorption, distribution, and clearance are all affected by physicochemical properties, incorporation of these functional groups can change ADME characterisitics of a compound. The objective of the current treatise is to review the influence of phenyl and substituted phenyl rings on the ADME properties of the molecules.
Sulfoximine- and sulfilimine-based diamino-diphenyl
sulfone (DAPSON) analogues have been prepared and their COX-
1 and COX-2 inhibition potencies as well as LTB4 and TNF binding
properties were studied. Furthermore, their antiproliferative activities
on cancer cell growth were investigated. Neither compounds
showed significant bioactivities
We have developed a green and practical method for the Huisgen cycloaddition of water-insoluble sulfonyl azides with alkynes ‘on water’ at room temperature under catalysis of the inexpensive catalyst system CuX/PhSMe, and addition of the ligand (thioanisole) greatly inhibited cleavage of the N1N2 bond in the 5-cuprated N-sulfonyltriazole intermediates to amides and improved the yields of N-sulfonyltriazoles.
This chapter summarizes structural alerts where there is a significant weight of evidence that their incorporation into a drug candidate molecule is likely to result in a higher than average incidence of attrition due to adverse safety/or toxicity findings. The mechanism of formation of reactive metabolites from each structural alert is summarized. The structure–toxicity relationships and the boundary of definition for each structural alert are discussed. The benefits of low exposure and alternative routes of metabolism are highlighted along with screening methods to manage the risks associated with structural alerts.
Keywords:
CYP inhibition;
idiosyncratic toxicity;
reactive metabolite;
structural alert;
toxicophore
The invasive growth of the introduced green alga Caulerpa taxifolia, already affecting the richness and diversity of the littoral ecosystems, has become a major ecological problem in the Mediterranean Sea. Previously, we demonstrated that the water pollutant tributyltin induces apoptosis in tissue of the marine sponge Geodia cydonium at concentrations of 3 μM and higher. Here we show that exposure of G. cydonium to low (non-toxic) concentrations of Caulerpa extract or purified caulerpin (10 μg/ml) together with low doses of tributyltin (1 μM; non-toxic), results in a strong apoptotic effect. Evidence is presented that the enhancement of toxicity of tributyltin by Caulerpa extract is at least partially caused by inhibition of the multixenobiotic resistance (MXR) pump by the algal toxin. Caulerpa extract, as well as caulerpin, strongly enhance the accumulation of the test substrate of MXR, rhodamine B, in the gills of the mussel Dreissena polymorpha, used as a model system for testing MXR-inhibiting potential.
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.
Eleven novel sialidase inhibitors 9 and 10 with an N-sulfonylamidino group at the C-4 position of Neu5Ac2en 1 against human parainfluenza virus type 1 (hPIV-1) were synthesized using copper-catalyzed three-component coupling reactions, and their inhibitory activities against hPIV-1 sialidase were studied.
Aromatische Amine stellen aufgrund ihrer weiten Verbreitung und hohen Toxizität eine wichtige Gruppe von Umweltchemikalien dar. Ihre Analytik gestaltet sich schwierig, da sie als polare Verbindungen bei der erforderlichen Anreicherung Probleme bereiten. Im Rahmen dieser Arbeit wurde deshalb ein Derivatisierungsverfahren entwickelt, bei dem die Verbindungen direkt in wässriger Lösung diazotiert und durch Zugabe von Iodid zu aromatischen Iod-Verbindungen umgesetzt werden. Die Polarität der Analyten wird dadurch gemessen an den Oktanol-Wasser Verteilungskoeffizienten um durchschnittlich 2 Größenordnungen je Aminogruppe vermindert, so dass selbst polare aromatische Amine sehr gut mit Festphasenmikroextraktion (SPME) angereichert werden können. Das Verfahren ist auf ein breites Spektrum aromatischer Amine anwendbar, die sich zusammen derivatisieren und analysieren lassen. Neben alkylierten und halogenierten aromatischen Aminen ist die Derivatisierung gleichermaßen für desaktivierte nitrierte und dinitrierte sowie Diamino-Verbindungen geeignet. Die Derivate wurden mit Flüssig- wie Gaschromatographie getrennt. Mit GC-MS wurden für die untersuchten aromatischen Amine Nachweisgrenzen im unteren ng/L-Bereich erzielt. Durch die Detektion im fullscan-Modus war neben einer sicheren Identifizierung gleichzeitig der Nachweis unbekannter Verbindungen möglich. Dabei genügte ein Probevolumen von nur 10 mL, was zugleich die Voraussetzung zur Analyse biologischer Matrices schaffte. Trotz der zusätzlichen Derivatisierung waren die Reproduzierbarkeiten mit denen etablierter Verfahren zur Bestimmung aromatischer Amine vergleichbar. Mit dem Verfahren konnten zahlreiche aromatische Amine erstmals mit SPME im Abwasser von Rüstungsaltlasten bestimmt werden. Die Iod-Verbindungen ließen sich nach gaschromatographischer Trennung gezielt über einen elementselektiven Atomemissionsdetektor (GC-AED) detektieren, ohne dass Beeinträchtigungen durch Matrixkomponenten auftraten. Auf diese Weise ließen sich aromatische Amine schnell und einfach im Abwasser von Rüstungsaltlasten ausfindig machen. Die Übertragung auf komplexe Matrices erfordert zum Schutz der SPME-Faser die Extraktion aus dem Headspace. Dabei wurde das Gleichgewicht rascher erreicht, so dass bereits nach 25 min die Extraktion vieler Analyten unter Gleichgewichtsbedingungen erfolgte. Dadurch ließen sich für die Mehrzahl der Analyten die Empfindlichkeit und die Reproduzierbarkeit weiter verbessern. Mit dem Ziel einer umfassenden Untersuchung aromatischer Amine wurde das in-situ Derivatisierung-SPME-Verfahren auf Urin angewendet. Nach saurer Hydrolyse wurde eine Vielzahl iodierter Verbindungen im Urin von Rauchern gefunden. Die Derivatisierung erwies sich dabei als hochgradig selektiv, da selbst in einer derart komplexen Matrix ausschließlich die Derivate aromatischer Amine auftraten. Bei der Detektion der Iod-Verbindungen über GC-AED wurden keinerlei Störungen durch biogene Iod-Verbindungen oder andere Amine beobachtet, so dass die Belastung mit aromatischen Aminen leicht beurteilt werden konnte. Beim Vergleich der Urinproben von Rauchern und Passivrauchern wurden nur geringe Unterschiede beobachtet, wohingegen im Urin von Nichtrauchern wesentlich weniger aromatische Amine enthalten waren. Durch die parallele Detektion mit GC-AED/MS konnte anhand der Massenspektren jedem Iod-Signal ein aromatisches Amin zugeordnet werden. Insgesamt konnte die Zahl der in Urin nachgewiesenen aromatischen Amine erheblich erweitert werden. So wurden über das in-situ Derivatisierungsverfahren mit GC-MS im Urin eines Rauchers über 200 aromatische Amine als iodierte Derivate identifiziert. Es handelte sich vorwiegend um alkylierte und chlorierte Aniline. Erstmals wurden neben den Aminobiphenylen und Naphthylaminen zahlreiche alkylierte Homologe identifiziert. Das Verfahren eignete sich zudem auch für die polaren Diamino-Verbindungen und Aminophenole, von denen einige in Urin nachgewiesen wurden. Gleichzeitig wurden mit Aminopyridinen und Aminochinolinen auch einige heteroaromatische Amine gefunden. Aromatic amines are an important class of pollutants due to their wide spread and high toxicity. The enrichment that is required for trace analysis is difficult due to the polarity of the amino compounds. For this reason, a derivatization method was developed consisting of a diazotisation of the aromatic amines directly in an aqueous solution and the conversion to aromatic iodine compounds by addition of iodide. The polarity of the analytes decreases on average by two orders of magnitude per amino group as measured by the octanol-water partition coefficients. Therefore even polar aromatic amines can be enriched by solid-phase microextraction (SPME). The method is applicable to a broad range of aromatic amines that can be derivatized and analysed simultaneously. Apart from alkylated and halogenated amines the derivatization is also suitable for deactivated nitrated and dinitrated anilines and diamino compounds. Liquid and gas chromatography were both used for the separation of the derivatives that are stable in aqueous solution for several weeks. For GC-MS analysis the limits of detection of the investigated aromatic amines were in the lower ng/L range. The detection in the fullscan-mode permitted an unequivocal verification of the target compounds as well as a general non-target analysis for aromatic amines. A sample volume of only 10 mL was sufficient for a sensitive determination so that biological matrices are also accessible. Despite the additional derivatization the reproducibility was comparable with those of established methods for the analysis of aromatic amines. The developed in-situ derivatization SPME GC-MS method was successfully applied to the determination of aromatic amines in contaminated groundwater at the site of a former ammunition plant. The iodine compounds were detected selectively after gas-chromatographic separation by an element-selective atomic-emission detector (GC-AED), providing a powerful screening method even for unknown aromatic amines after derivatization. By this method aromatic amines were easily traced in the runoff-water of a former ammunition plant, without any interference by matrix components or iodinated reaction products. The method transfer to complex matrices required the extraction from the headspace to protect the SPME-fiber. For most derivatives the sensitivity and reproducibility of HS-SPME was even improved compared to direct extraction. The equilibrium is reached quickly, so that after only 25 min the extraction of many analytes is carried out under equilibrium conditions. The in-situ derivatization SPME method was applied to urine to give an comprehensive overview of the occurring aromatic amines. After acidic hydrolysis and derivatization numerous iodine compounds were found in the urine of smokers. The derivatization proved to be highly selective since even in such a complex matrix exclusively the derivatives of aromatic amines were detected. Thus, GC-AED measurements were not inferred by natural iodine compounds or aliphatic amines that also form iodine derivatives so that the exposure to aromatic amines can be easily judged. For urine samples of smokers and passive smokers only slight differences were found, whereas in the urine of nonsmokers without known exposure to cigarette smoke significantly less compounds were found. By parallel atomic-emission and mass-spectrometric detection with GC-AED/MS for each iodine signal a mass spectrum of the related derivative was assigned. Finally the number of known aromatic amines in urine could be remarkably extended. By in-situ derivatization SPME GC-MS in the urine of a smoking person more than 200 aromatic amines were identified as iodinated derivatives. Most of them were alkylated or chlorinated anilines. For the first time several alkylated homologues of aminobiphenyls and naphthylamines have been found. Furthermore, the method was used to identify the more polar diamino compounds and aminophenols in urine. At the same time, some nitrogen-substituted aminopyridines and aminoquinolines occurred.
Microcapsules fabricated by layer-by-layer self-assembly have unique physicochemical properties that make them attractive for drug delivery applications. This study chiefly investigated the biocompatibility of one of the most stable types of microcapsules, those composed of poly-(sodium 4-styrene sulfonate) [PSS] and poly-(allylamine hydrochloride) [PAH], with cells cultured on two-dimensional (2D) substrates and in three-dimensional (3D) matrices. C6 glioma and 3T3 fibroblast cell morphology was observed after 24h of co-culture with PSS/PAH microcapsules on a 2D substrate. Cells were also cultured with four other types of microcapsules, each composed of at least one naturally occurring polyelectrolyte. At microcapsule to cell ratios up to 100:1, it was found that PSS/PAH microcapsules do not affect number of viable cells more substantially than do the other microcapsules investigated. However, differences in number of viable cells were found as a function of microcapsule composition, and our results suggest particular biochemical interactions between cells and internalized microcapsules, rather than mechanical effects, are responsible for these differences. We then investigated the effects of PSS/PAH microcapsules on cells embedded in 3D collagen matrices, which more closely approximate the tumor environments in which microcapsules may be useful drug delivery agents. Matrix structure, cell invasion, and volumetric spheroid growth were investigated, and we show that these microcapsules have a negligible effect on cell invasion and tumor spheroid growth even at high concentration. Taken together, this work suggests that PSS/PAH microcapsules have sufficiently high biocompatibility with at least some cell lines for use as proof of principle drug delivery agents in in vitro studies.
A solid-phase microextraction GC/MS method for the trace determination of a wide variety of polar aromatic amines in aqueous samples was developed. Prior to extraction the analytes were derivatized directly in the aqueous solution by diazotation and subsequent iodination in a one-pot reaction. The derivatives were extracted by direct-SPME using a PDMS/DVB fiber and analyzed by GC/MS in the full-scan mode. By diazotation/iodination, the polarity of the analytes was significantly decreased and as a consequence extraction yields were dramatically improved. The derivatization proved to be suitable for strongly deactivated aromatic amines and even the very polar diamino compounds can efficiently be enriched after derivatization. We investigated 18 anilines comprising a wide range of functional groups, which could be determined simultaneously. The method was thoroughly validated, and the precision at a concentration of 0.5 microg/L was 3.8-11% relative standard deviation for nonnitrated analytes using aniline-d(5) as internal standard and 3.7-10% for nitroaromatic amines without internal standard. The in situ derivatization/SPME/GC/MS method was calibrated over the whole analytical procedure and was linear over 2 orders of magnitude. Using 10-mL samples, detection limits of 2-13 ng/L were achieved for 15 of the 18 analytes. For two aminodinitrotoluene isomers and a diaminonitrotoluene, detection limits ranged from 27 to 38 ng/L. By allowing quantification at the 0.1 microg/L level, analysis of all target compounds meets EU drinking water regulations. The method provides high sensitivity, robustness, and high sample throughput by automation. Finally, the method was applied to various real water samples and in wastewater from a former ammunition plant the contents of several aromatic amines were quantified.
Alterations in molecular structure are responsible for the differential biological response(s) of a chemical inside a biosystem. Structural and functional parameters that govern a chemical's metabolic course and determine its ultimate outcome in terms of mutagenic/carcinogenic potential are extensively reviewed here. A large number of environmentally-significant organic chemicals are addressed under one or more broadly classified groups each representing one or more characteristic structural feature. Numerous examples are cited to illustrate the influence of key structural and functional parameters on the metabolism and DNA adduction properties of different chemicals. It is hoped that, in the event of limited experimental data on a chemical's bioactivity, such knowledge of the likely roles played by key molecular features should provide preliminary information regarding its bioactivation, detoxification and/or mutagenic potential and aid the process of screening and prioritising chemicals for further testing.
A new parametric quantum mechanical molecular model, AM1 (Austin Model 1), based on the NDDO approximation, is described. In it the major weaknesses of MNDO, in particular failure to reproduce hydrogen bonds, have been overcome without any increase in computing time. Results for 167 molecules are reported. Parameters are currently available for C, H, O, and N.
The relative importance of N-hydroxylation and acetylation of dapsone to the oral clearance of dapsone (100 mg) was investigated in seven healthy volunteers. Plasma dapsone and monoacetyldapsone concentrations rose rapidly with subsequent similar monoexponential elimination. The oral clearance of dapsone was low (33 +/- 14 ml/min), with a threefold variability. Four subjects were identified as fast acetylators; however, differences in acetylation did not explain the variability in oral clearance. The cumulative urinary recoveries of dapsone and its hydroxylamine were approximately 20% of the dose. The formation clearance of hydroxylamine, which exhibited a tenfold intersubject variability, was closely associated with the oral clearance of dapsone (r = 0.96). Thus, the formation of the hydroxylamine is more important than acetylation in determining dapsone's intersubject variability in oral clearance. Variation in N-hydroxylation may have clinical consequences, because the hydroxylamine is considered to be important in dapsone-mediated toxicity.
In vivo and in vitro biotransformation of secondary aromatic amines was investigated using 4-fluoro-N-methylaniline as the model compound. Attention was focused on the role of cytochromes P-450 and the flavin-containing monooxygenase in formation of the various metabolic products. In vitro studies using microsomal preparations, purified reconstituted cytochromes P-450 IA1 and IIB1 and purified flavin-containing monooxygenase, demonstrated that N-demethylation, N-hydroxylation, and 4-hydroxylation accompanied by dehalogenation were all catalyzed by both the cytochrome P-450 system and the flavin-containing monooxygenase. The turnover rates of the two monooxygenase systems for the various metabolite formations and the reaction pathways involved, were shown to vary significantly. This study provides direct experimental support for the conclusion that the aromatic ring hydroxylation of secondary N-methylamines can be a consequence of flavin-containing monooxygenase catalyzed N-hydroxylation rather than of direct aromatic ring hydroxylation. The results obtained in vitro were compared with data from urine analysis of rats exposed to 4-fluoro-N-methylaniline. In this way it could be demonstrated that the major phase I biotransformation reactions for formation of urine excretable metabolites are (i) the cytochrome P-450-catalyzed N-demethylation followed by aromatic ring hydroxylation of the 4-fluoroaniline formed, and (ii) flavin-containing monooxygenase and cytochrome P-450-dependent formation of defluorinated 4-hydroxy-N-methylaniline.
It is generally accepted that aromatic amines belong to a group of compounds which produce ferrihaemoglobin only after biochemical transformation (for reviews see Kiese, 1966, Kiese, 1974). Accordingly, aromatic amines are activated, primarily in the liver, to yield proximate reactive derivatives like aminophenols, N-hydroxyarylamines and N-hydroxy-N-arylacetamides. These compounds are then oxidized by oxyhaemoglobin to give the ultimate ferrihaemoglobin-forming species. Moreover, Mieyal et al. (1976) have shown that oxyhaemoglobin, both in reconstituted systems and in intact erythrocytes can also act as a mono-oxygenase-like catalyst of aniline hydroxylation to give mainly p-aminophenol. Similarly, Golly and Hlavica (1983) reported that 4-chloronitrosobenzene was formed in the reaction of oxyhaemoglobin with 4-chloroaniline in the presence of reduced pyridine nucleotides and cytochrome c reductase, indicating that oxyhaemoglobin can mediate N-oxygenations, in addition to the previous reports on aromatic carbon hydroxylations. These results show that the red cell has a dual function: it is a sensitive target for toxic actions of aromatic amines and plays a metabolic role in the biotransformation of aromatic amine derivatives (Eyer, 1983). Haemoglobin, therefore, has been recognized as a drug-metabolizing ‘enzyme’ (Mieyal et al., 1976; Lenk and Riedl, 1989).
Dapsone toxicity is putatively initiated by formation of a hydroxylamine metabolite by cytochromes P450. In human liver microsomes, the kinetics of P450-catalyzed N-oxidation of dapsone were biphasic, with the Michaelis-Menten constants of 0.14 0.05 and 0.004 0.003 mmol/L and the respective maximum velocities of 1.3 0.1 and 0.13 0.04 nmol/mg protein/min (mean SEM). Troleandomycin (40 mol/L) inhibited hydroxylamine formation at 100 mol/L dapsone by 50%; diethyldithiocarbamate (150 mol/L) and tolbutamide (400 mol/L) inhibited at 5 mol/L dapsone by 50% and 20%, respectively, suggesting that the low-affinity isozyme is CYP3A4 and the high-affinity isozymes are 2E1 and 2C. Disulfiram, 500 mg, 18 hours before a 100 mg oral dose of dapsone in healthy volunteers, diminished area under the hydroxylamine plasma concentration-time curve by 65%, apparent formation clearance of the hydroxylamine by 71%, and clearance of dapsone by 26%. Disulfiram produced a 78% lower concentration of methemoglobin 8 hours after dapsone.
An analysis is presented in which are evaluated correlations among chemical structure, mutagenicity to Salmonella, and carcinogenicity to rats and mice among 301 chemicals tested by the U.S. NTP. Overall, there was a high correlation between structural alerts to DNA reactivity and mutagenicity, but the correlation of either property with carcinogenicity was low. If rodent carcinogenicity is regarded as a singular property of chemicals, then neither structural alerts nor mutagenicity to Salmonella are effective in its prediction. Given this, the database was fragmented and new correlations sought between the derived sub-groups. First, the 301 chemicals were segregated into six broad chemical groupings. Second, the rodent cancer data were partially segregated by target tissue.
Pneumocystis carinii pneumonia (PCP) is a major cause of morbidity and the leading cause of death in patients with the acquired immunodeficiency syndrome. The prevention of the occurrence and recurrence of PCP is a cornerstone in the treatment of patients infected with the human immunodeficiency virus. There are few studies comparing PCP prophylactic regimens.
The efficacy of three regimens for prophylaxis against PCP was assessed in a retrospective chart review of 211 human immunodeficiency virus-infected patients at risk for the disease. Over the course of the 2-year study period, 133 patients were prescribed trimethoprim-sulfamethoxazole (one double-strength tablet twice a day, thrice weekly) for a mean of 7.4 months (range, 1 to 25 months). Seventy-seven patients received dapsone (50 mg daily) for a mean of 5.7 months (range, 1 to 23 months), and 125 patients received aerosolized pentamidine (300 mg via nebulizer once monthly) for a mean of 9.3 months (range, 1 to 21 months). The majority of patients (62%) received primary prophylaxis; 38% had one or more previous episodes of PCP; and 73% were receiving concomitant antiretroviral therapy.
Pneumocystis carinii pneumonia did not develop in any patient receiving trimethoprim-sulfamethoxazole in 981 patient-months. Five patients receiving dapsone for 437 patient-months and 17 patients receiving aerosolized pentamidine for 1166 patient-months developed PCP. Fifty-six percent of the trimethoprim-sulfamethoxazole group and 55% of the dapsone group changed drug due to adverse reactions, while only 2% in the aerosolized pentamidine group required drug change.
Despite its adverse reaction profile, trimethoprim-sulfamethoxazole is the most effective agent to prevent the occurrence and recurrence of PCP.
With microsomes prepared from a single human liver, 4,4'-diaminodiphenyl sulphone (DDS), 4-acetyl-4-aminodiphenyl sulphone (MADDS), 4-acetyl-4-aminodiphenyl thioether (MADDT) and 4,4'-diacetyldiphenyl thioether (DADDT) caused significantly greater methaemoglobin formation compared with control. In-vitro in the rat, the pattern of toxicity was slightly different:DADDT was not haemotoxic, whilst 3,4'-diaminodiphenyl sulphone (3,4'DDS) and 3,3'-diaminodiphenyl sulphone (3,3'DDS) as well as DDS, MADDS and MADDT were significantly greater than control. 4,4' Acetyl diphenyl sulphone (DADDS), 4,4' diaminodiphenyl thioether (DDT), 4,4'-diaminodiphenyl ether (DDE) and 4,4' diaminooctofluorodiphenyl sulphone (F8DDS) did not cause significant methaemoglobinaemia in either human or rat liver microsomes. DDS, MADDS, and MADDT were not significantly different in haemotoxicity generation in-vitro in the presence of human microsomes. In the rat in-vitro, DDS, MADDS, and 3,4'DDS did not differ significantly in red cell toxicity, and were the most potent methaemoglobin formers. The 3,3'DDS and MADDT derivatives were both significantly less toxic compared with DDS. None of the compounds tested caused haemoglobin oxidation in the absence of NADPH in-vitro. In the whole rat, DDS, MADDS and MADDT caused significantly higher levels of methaemoglobin compared with control. None of the remaining compounds caused methaemoglobin formation which was significantly greater than control. DDS and MADDS were the most potent methaemoglobin formers tested, in-vivo and in-vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
Treatment of cytochrome P450LM2 with fluorescein isothiocyanate to introduce up to two equivalents of fluorophore per polypeptide chain resulted in the selective derivatization of lysine residues. CD-spectral measurements revealed the overall conformation as well as the immediate heme environment of the hemoprotein to remain unaffected by attachment of the label. Modification caused decreased affinity of p-phenylenediamine and other 4-substituted anilines for the heme site, whereas there was a rise in the extent of substrate interaction. Experiments with pigment containing acetylated lysines gave analogous results, suggesting that the observed phenomenon was due to charge neutralization. There was linear correlation between the Hammett sigma P values and both the optical dissociation constants for arylamine binding to intact enzyme and the dipole moments of the anilines, indicating that basicity along with electronic factors controlled heme liganding; lipophilicity appeared to be of minor importance. Introduction of fluorescein isothiocyanate into the oxygenase was found to influence the bond-making process through modulating basicity of the nitrogenous compounds, but perturbation of optimal spacial orientation of the amine nitrogen toward the heme iron also might have been operative. The lysines studied seem to represent metabolically inactive elements of the substrate channel located on the cytosolic surface of the aggregates, as evidenced by steady-state fluorescence measurements. A hydrophilic segment in the cytochrome P450LM2 molecule that would accommodate the critical residues is discussed.
Aromatic amines are important intermediates in industrial manufacturing. N-oxidation to the N-hydroxyarylamines is a key step determining the genotoxic properties of aromatic amines. N-hydroxyarylamines can form adducts with DNA, with tissue proteins and with the blood proteins albumin and hemoglobin in a dose-dependent manner. The determination of hemoglobin adducts is a useful tool for biomonitoring exposed populations. We have established the hemoglobin binding index (HBI) [(mmol compound/mol Hb)/(mmol compound/kg body wt)] of several aromatic amines in female Wistar rats. Including the values obtained by other researchers in the same rat strain, the logarithm of hemoglobin binding (log HBI) was plotted against the following parameters: the sum of the Hammett constants (sigma sigma = sigma p + sigma m), pKa, log P (octanol/water), the half wave oxidation potential (E1/2) and the electronic descriptors of the amines and their corresponding nitrenium ions obtained by semiempirical calculations (MNDO, AM1 and PM3), such as atomic charge densities, energies of the HOMO and LUMO and their coefficients, the C-N bond order, the dipole moments and the 'reaction enthalpy' [MNDOHF, AM1HF or PM3HF = Hf(nitrenium) - Hf(amine)]. The correlation coefficients were determined from the plots of all parameters against log HBI for all amines by means of linear regression analysis. The amines were classified into three groups: group 1, all para-substituted amines, group 2, all amines with halogens and group 3, all amines with alkyl groups. For the amines of group 1, log HBI correlates with sigma sigma, MNDOHF, E1/2, the pKa and the log P with r = 0.84, 0.71, 0.73, - 0.69 and 0.50, respectively. For the amines of group 2, log HBI correlates with pKa, sigma sigma, MNDOHF, E1/2 and log P with r = 0.81, -0.76, -0.55, -0.46 and -0.20, respectively. For the amines of group 3, log HBI correlates with the E1/2, PM3HF, sigma sigma, pKa and log P with r = 0.92, 0.89, 0.76, 0.19 and 0.12, respectively. The apparent Michaelis-Menten constants Km and Vmax of the N-acetyltransferase of liver cytosol were determined for several amines. Km and Vmax do not correlate with any of the electronic descriptors. Female Wistar rats were dosed with nitroarenes. Hemoglobin binding of nitroarenes correlates with the energy levels of the LUMO. This investigation determines for a large variety of aromatic amines the bioavailability of the N-hydroxyarylamine--the genotoxic metabolite--and the utility of electronic descriptors for prediction of the N-oxidation.
The relative energetics of arylamine N-hydroxylation and N-O heterolysis (ArNH2----ArNHOH----ArNH+) for condensed systems of two, three and four rings were calculated using semiempirical AM1 molecular orbital theory. The overall thermodynamics of N-hydroxylation were almost insensitive to the structure of the amine while differences in the energetics of nitrenium ion formation varied from 0 to 35 kcal mol-1. Limited correlations between the latter and the experimental TA98 and TA100 mutagenicities of the amines are presented.
Quantitative structure-activity relationships of the metabolism of drugs by uridine diphosphate (UDP)-glucuronosyltransferase have been investigated. It is observed that most of the variation in the rate of glucuronidation of phenols, thiols, and some other miscellaneous compounds can be accounted for by the lipophilic property of the molecules. The results are consistent with the previous finding with primary and secondary alcohols, and benzoic acids. The optimum lipophilicity for these compounds undergoing metabolism by UDP-glucuronosyltransferase appears to be a log P of 2.
Administration of dapsone (33 mg kg-1) to intact rats resulted in a marked elevation of methaemoglobin levels in male (435.0 +/- 105.2% met Hb h) compared with female rats (59.0 +/- 17.2% met Hb h). However, the clearance of dapsone was significantly faster in males compared with females. Female rats showed very low levels of methaemoglobin which were accompanied by significantly higher blood concentrations of parent drug. Clearance of dapsone in castrated animals was less than one-third of that of the intact sham-operated males (252.2 +/- 67.2 vs 81.4 +/- 33.0 mL h-1). Likewise, clearance of dapsone in ovarectomized rats was approximately half that of intact females. There were no significant differences in the disposition of dapsone between the ovarectomized (AUC 431.0 +/- 31.7 micrograms h mL-1; t1/2, 15.62 +/- 1.8 h) and castrated (AUC, 450.6 +/- 150.9 micrograms h mL-1; t1/2, 17.6 +/- 7.9 h) animals. However, methaemoglobin levels in castrated males, although less than a third of those of intact males, significantly exceeded those of ovarectomized animals. There was no significant difference between the four groups of animals with respect to red cell sensitivity to the methaemoglobin-forming capacity of the toxic metabolite of dapsone, the hydroxylamine. Metabolic conversion of dapsone to the hydroxylamine in the presence of NADPH was 7.6 +/- 1.5% for liver microsomes from intact males and was significantly greater (P less than 0.05) than the corresponding values for liver microsomes from castrated rats (5.3 +/- 0.59%). Conversion of dapsone to dapsone-NOH by liver microsomes from intact females and ovarectomized animals was below 1% in both cases.(ABSTRACT TRUNCATED AT 250 WORDS)
The author tried in a somewhat limited work to quantitatively correlate the electronic and steric intramolecular interactions of substituents on the amino group (influencing the enzymatic reactions of aromatic amines) and the mutagenic event. It was assumed that there is a correlation between these biotransformations and the electronic state of aromatic amines at the ionic dissociation equilibrium. The approach is rather empirical and arbitrary but the overall agreement between experimental mutagenic potencies and the values calculated was encouraging and led the author to further developments. It is hoped that the concepts used in this work may be applied to other aromatic molecules bearing an amino group.
1. We have utilized a two compartment system in which two teflon chambers are separated by a semi-permeable membrane in order to investigate the role of metabolism in dapsone-induced methaemoglobinaemia. Compartment A contained a drug metabolizing system (microsomes prepared from human liver +/- NADPH), whilst compartment B contained target cells (human red cells). 2. Incubation of dapsone (1-100 microM) with human liver microsomes (2 mg protein) and NADPH (1 mM) in compartment A (final volume 500 microliters) led to a concentration-dependent increase in the methaemoglobinaemia (15.4-18.9% at 100 microM) compared with control (2.3 +/- 0.4%) detected in the red cells within compartment B. In the absence of NADPH dapsone had no effect. 3. Of the putative dapsone metabolites investigated, only dapsone-hydroxylamine caused methaemoglobin formation in the absence of NADPH (40.6 +/- 6.3% with 100 microM). However, methaemoglobin was also detected when monoacetyl-dapsone, 4-amino-4'-nitro-diphenylsulphone and 4-aminoacetyl-4'-nitro-diphenylsulphone were incubated with human liver microsomes in the presence of NADPH. 4 Dapsone-dependent methaemoglobin formation was inhibited by addition of ketoconazole (1-1000 microM) to compartment A, with IC50 values of 285 and 806 microM for the two liver microsomal samples studied. In contrast, methaemoglobin formation was not inhibited by cimetidine or a number of drugs pharmacologically-related to dapsone. The presence of glutathione or ascorbate (500 microM) did not alter the level of methaemoglobin observed.
The determination of the covalently bound reaction products of 4,4'-methylenedianiline (MDA) to hemoglobin was investigated as a possible method for biological dosimetry in humans. The extent of binding to rat hemoglobin of MDA was determined by dosing animals with the 14C-ring-labeled compound. Two adducts were released from the hemoglobin on hydrolysis under mildly basic conditions which were identified as MDA and N-acetyl-MDA and accounted for between 36 and 45% of the total radioactivity bound to the protein. A quantitative assay procedure was subsequently developed for measuring both of the base released adducts in rat hemoglobin. The method utilized solvent extraction followed by derivatization with pentafluoropropionic anhydride and subsequent separation and quantitation by capillary gas chromatography with selective ion monitoring mass spectrometry using deuterium-labeled analogues of MDA and N-acetyl-MDA as internal standards. A dose-response relationship was established in orally dosed rats between production of each of the hemoglobin released adducts and dose of MDA (1-12 mg/kg). The possible use of such adduct determinations as dosimeters for industrial workers exposed to MDA is discussed.
A series of new 4,4'-diaminodiphenylsulfones substituted at 2 and 3 position and also at primary amino group of the phenyl rings have been synthesized and evaluated for their antimalarial activity against Plasmodium berghei infection in mice. Some of these compounds were active and showed complete inhibition of parasitaemia which included 7a1-7a4, 7b3, 7b4 and 16a at 1 mg/kg i.p. for 4 days and 16a, at 0.3 mg/kg for 4 days. Some compounds tested for their synthetase inhibitory action in cell-free system isolated from P. berghei (7b1, 7b2 and 8b2) were found to be more active than diaminodiphenylsulphone. The difference in order of activity between these in vivo and in vitro tests may be due to differences in their pharmacokinetic properties.
Dapsone is an effective anti-inflammatory agent in conditions in which inflammation is mediated by neutrophils. Dapsone also has been associated with agranulocytosis. We found that neutrophils, which had been activated by a phorbol ester or opsonized zymosan, oxidized dapsone to its nitroderivative. It appears as if this is due to oxidation of dapsone by myeloperoxidase to the hydroxylamine, followed by nonenzymatic oxidation of the hydroxylamine to the nitroderivative. The hydroxylamine can be isolated if ascorbic acid is added to the incubations. Monocytes also contain myeloperoxidase and activated mononuclear leukocytes also metabolize dapsone to the hydroxylamine. Dapsone also causes a mononucleosis-like syndrome. The reactive hydroxylamine could be responsible for both the pharmacologic and toxic properties of dapsone.
An integrated molecular graphics and computational chemistry framework is described which has been designed primarily to handle small molecules of up to 300 atoms. The system provides a means of integrating software from any source into a single framework. It is split into two functional subsystems. The first subsystem, called COSMIC, runs on low-cost, serial-linked colour graphics terminals and allows the user to prepare and examine structural data and to submit them for extensive computational chemistry. Links also allow access to databases, other modelling systems and user-written modules. Much of the output from COSMIC cannot be examined with low level graphics. A second subsystem, called ASTRAL, has been developed for the high-resolution Evans & Sutherland PS300 colour graphics terminal and is designed to manipulate complex display structures. The COSMIC minimisers, geometry investigators, molecular orbital displays, electrostatic isopotential generators and various interfaces and utilities are described.
Bio-monitoring the covalent binding of nitrosoarenes to the SH groups of human hemoglobin has been proposed as a reliable approach to get an integral parameter for exposure control and possibly risk assessment of persons exposed to aromatic amines and nitro compounds. Availability of nitrosoarenes to bind to the cysteine residues is greatly influenced by the competition of hemoglobin iron with nitrosoarenes. In contrast to earlier reports, we found that nitrosobenzene has a 14 fold higher affinity for "stripped" human hemoglobin than oxygen. The binding mode is similar to gaseous ligands and exhibits the same free energy of cooperation and sensitivity to heterotropic effectors like inositol hexaphosphate. To elucidate the electronic influence of para substituents, 4-chloronitrosobenzene, 4-nitrosotoluene and 4-nitrosophenetole were tested. A linear free energy relationship was found for all equilibrium parameters with a reaction constant rho = 3, when using Hammett sigma p constants. Similarly, the apparent second order rate constants for binding of para-substituted nitrosobenzenes to the cysteine residues (Cys beta 93) in hemoglobin followed the Hammett relationship with lg k-lg k0 = 1.7 X sigma p (r2 = 0.99). In case of 4-chloronitrosobenzene covalent binding proceeded biphasically and a "semimercaptal"-like intermediate was observed. The affinities for hemoglobin iron and for the SH groups were highest with 4-chloronitrosobenzene and lowest with 4-nitrosophenetole. All nitrosobenzenes were capable to produce ferrihemoglobin. In the absence of oxygen, 4-chloronitrosobenzene hemoglobin decayed with formation of ferrihemoglobin. Presumably the nitroxide radical anion is formed as an intermediate which comproportionates into the azoxy derivative. It is assumed that the efficiency of the microscopic compartmentation of nitrosoarenes by binding to hemoglobin iron has important impacts on the toxicokinetics of these compounds.
Hemolytic anemia after aniline and aniline-related drugs such as dapsone and primaquine is thought to be mediated by active/reactive metabolite(s) formed during the hepatic clearance of the parent compounds. To determine whether any of the known metabolites of aniline contribute to the hemolytic response seen in rats given aniline, rats were infused with isologous 51Cr-labeled erythrocytes 24 hr before administration of aniline or aniline metabolites. The time course of blood radioactivity was followed in individual rats by serial sampling from the orbital sinus and the time required for blood radioactivity to fall by 50% (T50Cr) was used as a measure of in vivo erythrocyte survival. Aniline HCl produced a dose-dependent reduction in the T50Cr. Acetanilide also reduced the T50Cr, but was less potent than aniline. Aminophenols (2-, 3- and 4-) in similar doses did not significantly alter the T50Cr. In contrast, phenylhydroxylamine produced a dose-dependent decrease in the T50Cr with approximately 10 times the potency of aniline. The T50Cr was also decreased in a concentration-dependent manner for labeled erythrocytes incubated in vitro with phenylhydroxylamine, then readministered to rats, indicating a direct toxic effect of phenylhydroxylamine on erythrocytes. In addition, the area under the blood time course curve for phenylhydroxylamine plus nitrosobenzene was equivalent in rats administered equitoxic doses of aniline or phenylhydroxylamine, indicating that sufficient phenylhydroxylamine is formed in vivo during aniline clearance to account for aniline's toxicity. These results suggest that phenylhydroxylamine is the active metabolite that mediates aniline-induced hemolytic anemia.
1.
During the incubation of Dapsone1 with rabbit liver microsomes, NADPH and bovine erythrocytes, rapid haemoglobin oxidation was observed. The velocity increased 2–3 times with liver microsomes of rabbits pretreated with phenobarbital.
2.
Liver microsomes of rabbits catalyzed the N-hydroxylation of DDS in the presence of O2 and NADPH. The oxidation was dependent upon microsomal protein, DDS concentration, NADPH concentration and pH. The velocity of N-hydroxylation in incubates with microsomes from rabbits pretreated with phenobarbital was 2–3 times greater than the velocity with microsomes from control animals. Carbon monoxide and metyrapone inhibited the microsomal N-hydroxylation of DDS. The reaction must be included in the cytochrome P-450 dependent N-hydroxylations of primary arylamines.
3.
In dogs, very low amounts of free DDS-NOH were found in the urine. 7–10% of an oral dose of 50 mg/kg DDS was excreted in the urine in the form of conjugated DDS-NOH liberated by acid hydrolysis (1 N HCl at 20°C).
4.
Human volunteers receiving 200 mg DDS in capsules excreted 0.9–3.4% of the dose as free DDS-NOH and 6–20% as conjugated, acid labile DDS-NOH within 24h. After 72 h 5–7% of the dose was excreted as free DDS-NOH and 25–33% as conjugated, acid labile DDS-NOH. 80–90% of the DDS-NOH conjugates were liberated by treatment with 1 N HCl at 20°C. The total amount of conjugates in the urine was split by glusulase treatment under anaerobic condition. N-Hydroxy metabolites of DDS in the urine can reach 50% of the dose. Dapsone metabolism in humans is the first example in which N-hydroxylation is the principal metabolic pathway.
This investigation was made to clarify the mechanism of the increased methaemoglobin formation in erythrocytes that follows the administration of 4,4′diaminodiphenylsulphone.The drug itself does not induce the formation of methaemoglobin, but the results of incubation of erythrocytes with plasma obtained during administration of the drug indicate that a metabolite is responsible for this complication. The metabolite activates the pentose-phosphate shunt of the erythrocytes.A hypothesis is put forward according to which the aromatic amino compound is converted to a nitroso- or hydroxylamino-compound. A coupled oxidation involving the hydroxylamino compound and haemoglobin results in the formation of a nitroso-compound and methaemoglobin, respectively. The hydroxylamino-compound is regenerated by means of a pyridine-nucleotide-dependent enzyme. Rapid methaemoglobin formation after addition of the drug to a system of erythrocytes, microsomes, and NADPH2 supports the theory.
Methaemoglobin formation induced in cats by many substituted anilines has been investigated in an attempt to correlate chemical structure with activity. Although no precise relation emerged, some generalizations could be made. 4-Substituents (except methyl and methoxy) increased the activity of aniline. The activity of aniline was either unaltered or reduced by 2- and 3-substituents. Polyhalo-, polymethyl-, methoxy-, ethoxy- and carboxyester substituents reduced activity, and carboxy groups abolished it. Steric effects around the amino-group were not important. The activities of the compounds are discussed in relation to their absorption, distribution, metabolic transformation and the activities of their metabolites.
Methemoglobinemia is an important side effect of dapsone (4,4′-diaminodiphenyl sulfone; DDS) therapy. Previous workers had shown a correlation between methemoglobin formation and time of incubation of DDS in a system involving rat liver microsomes and human red cells (RBC). N-Oxidation of ring 14C labelled DDS by rat liver microsomes was studied both in the presence and absence of RBC. Evidence is presented that in vitro DDS is metabolized to the monohydroxylamine of DDS (DDS-NOH), and maybe to the nitroso-(DDS-NO) and azoxy- (azoxy-DDS) analogues of DDS. The latter compounds could also arise from non-microsomal oxidation of DDS-NOH. Specific isotope dilution procedures were employed to measure the N-oxidation of DDS. These involved conversion to azoxy-DDS or the formation of the pentocyanamine ferroate complex of DDS-NOH (and/or DDS-NO). The extent of total N-oxidation of DDS was always less when experiments were carried out in the presence of RBC than in their absence. This suggests that DDS-NOH is enzymatically reduced by RBC. Like other microsomal oxidations, N-oxidation of DDS was inhibited by SKF-525A. Our studies indicate that DDS-NOH (and/or DDS-NO) is the cause of the methemoglobinemia observed in dapsone therapy.
As an extension of in vitro studies (in which it was shown that dapsone (DDS) is N oxidized by rat liver microsomes to its monohydroxylamine and that this metabolite is responsible for methemoglobinemia) the authors investigated the metabolism of DDS and diformyl dapsone in man and DDS in rats and guinea pigs. In man given DDS 14C or diformyl dapsone 14C, urine represented the major route of elimination of radioactivity. Significant amounts of N oxidation metabolites were found in urine. One of the primary urinary N oxidation metabolites, the monohydroxylamine of dapsone, was isolated and characterized as azoxy DDS. A new metabolite (the monohydroxylamine of 4 acetyl amino 4' aminodiphenylsulfone) was also identified. N oxidation metabolites accounted for a small fraction of the dose of DDS 14C in animal urine and liver; the amount in bile was higher. A number of metabolites and derivatives of DDS were synthesized and tested for their ability to produce methemoglobin.
Nitrobrombenzol (I) und 4-Fluor-thiophenol (II) werden zum Sulfid (III) kondensiert, und dieses wird in das Sulfon (IV) übergeführt.
4,4′-Diaminodiphenylsulfone (DDS) can produce methemoglobinemia in vivo, apparently by the action of an unidentified metabolic product. To discern the chemical nature of such a compound, 13 p-substituted diphenylsulfones have been examined for their ability to oxidize hemoglobin (Hb) to methemoglobin (MHb) in human erythrocytes in vitro. 4-Amino-4′-hydroxyaminodiphenylsulfone (DDS-NOH) is most effective, 1·9 × 10−5 M of drug oxidizing as much as 60 per cent of the Hb, or 100 heme equivalents/mole of DDS-NOH in 2 hr. Oxygen is required for this process, and glucose, while not required, enhances MHb formation. With dialyzed hemolysates and solutions of purified Hb, the heme equivalents oxidized/mole of drug becomes less than 10, but a ratio of 80 : 100 can be restored by adding reduced glutathione (GSH) or NADPH. Apparently DDS-NOH produces MHb by coupled oxidation with Hb and O2 and DDS-NOH can be regenerated by reducing compounds to yield the high heme oxidation ratio. In intact cells, DDS-NOH causes a 7-fold increase in the activity of the hexose monophosphate shunt as measured by 14CO2 production from glucose 1-14C. Incubation with galactose, which is minimally oxidized by the shunt pathway, results in decreased formation of MHb, and the GSH concentration drops by 75 per cent. Depletion of intracellular GSH by a sulfhydryl oxidizing compound also reduces MHb formation by DDS-NOH. These observations implicate GSH or NADPH or both in the recycling of DDS-NOH in erythrocytes. Consistent with these interpretations, DDS-NOH produced only one-half as much MHb in erythrocytes from three individuals with glucose 6-phosphate dehydrogenase deficiency as it did in normal erythrocytes.
The hydroxylation of a variety of halo and alkyl substituted anilines and acetanilides with hepatic microsomal preparations was studied. 4 Chloro- and 4-fluoro-acetanilides and anilines were hydroxylated to the corresponding 4-hydroxy derivatives with loss of halogen. The 4-chloro derivatives were extremely poor substrates for the aryl hydroxylases. 3-Methyl-, 3-chloro-, and 3-fluoro-acetanilides were metabolized to the corresponding 4-hydroxy-3-substituted acetanilide. 4-Methylaniline, 4-methyl-acetanilide, and 4-ethylacetanilide were not substrates for aryl hydroxylation, but instead underwent side-chain oxidation to form, respectively, 4-hydroxymethylaniline, 4-hydroxymethylacetanilide, and 4-(1′-hydroxyethyl)acetanilide. The hydroxymethyl compounds were further oxidized to aldehydes.
Fresh-frozen human liver tissue was assayed for its ability to reduce the nitrogroup of R--NO2 to the amine. All 10 livers examined exhibited demonstrable reductase activity. The reduction was potentiated by NADPH and abolished by boiling the liver homogenates. The nitroreductase activity varied among the different livers by as much as severalfold. These findings show that ability of the human liver to reduce R--NO2 and support the hypothesis that certain toxic intermediates of the nitroreduction of R--NO2 may be responsible for the aplastic anemia associated with this drug.
4-Amino-4'-substituted biphenyls and 4-aminostilbenes substituted in the 3' or 4' position were studied for their in vitro and in vivo genotoxicity. The in vitro mutagenicity of the biphenyls with and without S9 activation was established with Salmonella strains TA98 and TA100 and that of the stilbenes with the same strains plus TA98/1,8-DNP6. The in vivo genotoxicity assay with both series of compounds was for chromosomal aberrations in the bone-marrow cells of mice following intraperitoneal administration of the chemicals. Hammett values of substituents, partition coefficients and frontier orbital energies (ELUMO and EHOMO) of the compounds were used for correlations with mutagenicity. The Salmonella mutagenicity in TA98 and TA98/1,8-DNP6 with S9 was correlated to Hammett sigma + values for the 4-aminostilbene substituents, showing a strong trend of increasing mutagenicity with an increase in the electron-withdrawing capability of the substituent. Hydrophobicity of the stilbenes, however, had little effect on their relative mutagenicity. The 4-aminobiphenyls showed a correlation between their mutagenicity and Hammett sigma + values of their 4'-substituents in stain TA98 with S9, although the trend was not as strong as for the stilbenes. But unlike the stilbenes, TA98 mutagenicity of the biphenyls could also be correlated to hydrophobicity, and structure-activity correlations for the biphenyls was substantially improved when both sigma + and hydrophobicity data were included. For strain TA100 with S9, little correlation was found between mutagenicity of the stilbenes and any of the parameters. However, a limited correlation did exist between the mutagenicity of the biphenyls and their hydrophobicity. There was also limited correlations of the mutagenicity for the stilbenes in TA98 and TA98/1,8-DNP6 with S9 to ELUMO or EHOMO. The in vivo genotoxicity results for the biphenyls and stilbenes could not be correlated to electronic effects as for the in vitro results, nor could they be explained by hydrophobicity. However, it is interesting to note that 3'-substituted 4-aminostilbenes were all substantially more genotoxic in vivo than their corresponding 4'-substituted counterparts. The most genotoxic compound in vivo in either series was 4-aminostilbene which would not have been predicted from the in vitro results.
Amodiaquine, a 4-aminoquinoline antimalarial, has been associated with hepatitis and agranulocytosis in humans. Drug hypersensitivity reactions, especially agranulocytosis, have been attributed to reactive intermediates generated by the oxidants discharged from stimulated polymorphonuclear leucocytes (PMN). The metabolism of amodiaquine to both stable and chemically reactive metabolites by human PMN has been investigated in vitro. Incubation of [14C]-amodiaquine with PMN resulted in irreversible binding of radiolabel to protein and depletion of intracellular reduced glutathione, which were enhanced by phorbol myristate acetate (PMA), a PMN activator. Two metabolites were identified: the C-5' glutathione adduct of amodiaquine, derived from both endogenous and exogenous glutathione, and 4-amino-7-chloroquinoline, which was presumed to be formed by hydrolysis of amodiaquine quinoneimine. Desethylamodiaquine, the major plasma metabolite of amodiaquine in humans, also underwent bioactivation to a chemically reactive species in the presence of PMA-stimulated PMN. Substitution of the 4'-hydroxyl group in amodiaquine with fluorine significantly reduced irreversible binding to protein and abolished depletion of intracellular glutathione in the presence of PMA. These findings indicate that the bioactivation of amodiaquine by PMN is associated with the formation of a quinoneimine intermediate. Such a reactive metabolite, if produced in PMN or bone marrow in vivo, may be responsible for the drug's myelotoxicity.
The cytochrome P450 catalysed biotransformation of 4-halogenated anilines was studied in vitro with special emphasis on the dehalogenation to 4-aminophenol metabolites. The results demonstrated that a fluorine substituent at the C4 position was more easily eliminated from the aromatic ring than a chloro-, bromo- or iodo-substituent. HPLC analysis of in vitro biotransformation patterns revealed that the dehalogenation of the C4-position was accompanied by formation of non-halogenated 4-aminophenol, without formation of NIH-shifted metabolites. Changes in the apparent Vmax for the microsomal oxidative dehalogenation appeared to correlate with the electronegativity of the halogen substituent at C4, the fluorine substituent being the one most easily eliminated. A similar decrease in the rate of dehalogenation from a fluoro- to a chloro- to a bromo- to an iodo-substituent was observed in a system with purified reconstituted cytochrome P450 IIB1, in a tertiair butyl hydroperoxide supported microsomal cytochrome P450 system as well as in a system with microperoxidase 8. This microperoxidase 8 is a haem-based mini-enzyme without a substrate binding site, capable of catalysing cytochrome P450-like reaction chemistry. Together, these results excluded the possibility that the difference in the rate of dehalogenation with a varying C4-halogen substituent arose from a change in the contribution of cytochrome P450 enzymes involved in oxidative dehalogenation with a change in the halogen substituent. Rather, they strongly suggested that the difference was indeed due to an intrinsic electronic parameter of the various C4 halogenated anilines dependent on the type of halogen substituent. Additional in vitro experiments with polyfluorinated anilines demonstrated that elimination of the C4-fluorine substituent became more difficult upon the introduction of additional electron withdrawing fluorine substituents in the aniline-ring. 19F-NMR analysis of the metabolite patterns showed that the observed decrease in 4-aminophenol formation was accompanied by a metabolic switch to 2-aminophenols and N-hydroxyanilines, while products resulting from NIH-type mechanisms were not observed. For a C4-chloro-, bromo-, or iodo-substituted 2-fluoroaniline the Vmax for the oxidative dehalogenation was reduced by the additional electron withdrawing fluorine substituent at the C2 position in a similar way.(ABSTRACT TRUNCATED AT 400 WORDS)
The formation of dapsone hydroxylamine (DDS-NOH) and monoacetyldapsone hydroxylamine (MADDS-NOH) was found to be greater in male vs. female rat liver microsomes, suggesting a role for either CYP2C11 or CYP3A2. Preincubation with cimetidine (selective for inhibition of CYP2C11), but not troleandomycin (selective for inhibition of CYP3A1/2), inhibited metabolite formation. Furthermore, incubation with monoclonal antibodies (Mabs) to CYP2C6/2C11 reduced metabolite formation to below the level of detection. Together, these data indicate that N-hydroxylation of DDS and MADDS in rat liver microsomes from untreated male rats is catalyzed by CYP2C6/2C11. Interestingly, dexamethasone pretreatment increased the hydroxylation of both metabolites. Preincubation with cimetidine or Mabs to CYP2C6/2C11 (at an antibody:protein ratio of 26:1) in microsomes from dexamethasone pretreated animals did not reduce the N-hydroxylation of DDS, whereas preincubation with troleandomycin reduced metabolite formation by > or = 50%. Collectively, these data indicate that the constitutive enzymes CYP2C6 and/or CYP2C11, as well as CYP3A1 (nonconstitutive), are capable of catalyzing the hydroxylation of DDS and MADDS.
Two cloned human hepatic UDP-glucuronosyltransferase (UGT) cDNAs were stably expressed in chinese hamster V79 cells. More than 100 drugs and xenobiotics were used as substrates for glucuronidation catalyzed by the cloned human transferases to determine the chemical structures accepted as substrates. UGT HP1 exhibited a limited substrate specificity for planar phenolic compounds, whereas UGT HP4 was more promiscuous in acceptance of non-planar phenols, anthraquinones, flavones, aliphatic alcohols, aromatic carboxylic acids, steroids, and many drugs of varied structure. Levels of HP4 UGT activity toward some substrates were sufficient to allow determination of kinetic parameters for the enzyme reaction. Metabolism of drugs could be studied by addition to the recombinant cell lines in culture, and extraction of the media allowed analysis of glucuronide formation. Data presented herein demonstrate the potential of using these recombinant cell lines for investigation of phase II metabolism by human UGTs.
1. The general mechanism of metabolic oxidation of substrates by cytochromes P450 (P450s) appears to consist of sequential one-electron oxidation steps rather than of a single concerted transfer of activated oxygen species from P450 to substrates. 2. In case of the acetanilides paracetamol (PAR), phenacetin (PHEN), and 4-chloro-acetanilide (4-CLAA), the first one-electron oxidation step consists of a hydrogen abstraction from the acetylamino nitrogen and/or from the other side-chain substituent on the aromatic ring. The substrate radicals thus formed delocalize their spin and the respective reactive centres of the substrate radical recombine with a P450 iron-bound hydroxyl radical to either yield oxygenated metabolites, or undergo a second hydrogen abstraction forming dehydrogenated products. By this mechanism, the formation of all known oxidative metabolites of PAR, PHEN, and 4-ClAA can be explained. Furthermore, this mechanism is consistent with all available experimental data on [18O]PAR/PHEN, [2H]PAR, and [14C]PHEN. 3. The oxidative metabolic reactions proposed for the acetanilides PAR, PHEN, and 4-ClAA are used to generalize P450-mediated oxidations of these and other acetanilides, such as analogues of PAR and 2-N-acetyl-aminofluorene. 4. A further generalization of the hydrogen abstraction, spin delocalization, radical recombination concept is derived for other aromatic substrates with abstractable hydrogen atoms, notably those with activated N-H, O-H, C-H, or S-H bonds directly attached to the aromatic nucleus.
Dapsone is useful in the treatment of a number of inflammatory conditions which are characterized by neutrophil infiltration. It is the drug of choice for suppression of the symptoms of dermatitis herpetiformis, as it inhibits the process by which neutrophils leave the circulation and migrate to lesional sites. It also prevents the tissue destruction normally caused by the neutrophils' respiratory burst. Although dapsone can cause a number of serious idiosyncratic reactions, such as agranulocytosis, tolerance of the drug at higher doses is more usually determined by its haematological side-effects of methaemoglobinaemia and haemolysis. These effects are due entirely to the hepatic N-hydroxylation of dapsone to a hydroxylamine metabolite, some of which escapes from the liver and rapidly enters red cells. Attempts have been made to counteract the haemotoxic effects of the metabolite by the use of antioxidants such as vitamins E and C. Recently, the co-administration of a metabolic inhibitor such as cimetidine has been shown to reduce significantly dapsone-dependent methaemoglobinaemia, without any change in drug efficacy. It remains to be seen if this approach will be adopted clinically, to improve patient tolerance of high dapsone dosage.
1. N-hydroxylation is thought to be an essential step in the haemotoxicity of dapsone (DDS). To investigate both metabolism-dependent and cell-selective drug toxicity in vitro we have developed a three-compartment system in which an hepatic drug metabolizing system is contained within a central compartment separated by semipermeable membranes from compartments containing mononuclear leucocytes (MNL) and red blood cells (RBC). 2. Metabolism of dapsone (100 microM) by rat liver microsomes resulted in toxicity to RBC cells (47.3 +/- 2.1% methaemoglobin), but there was no significant toxicity toward MNL (3.7 +/- 1.3% cell death) compared with control values (1.6 +/- 0.9%). However, when RBC were replaced with buffer in the third compartment there was significantly greater (P < 0.001) white cell toxicity (17.6 +/- 0.6% cell death), demonstrating the protection of MNL by RBC. Metabolism of dapsone by human liver microsomes again resulted in RBC toxicity (12.5 +/- 3.3% methaemoglobin) but no significant MNL toxicity (2.9 +/- 0.8% cell death). Replacement of RBC resulted in a significant (P < 0.001) increase in MNL toxicity (6.5 +/- 0.7% cell death). Addition of synthetic dapsone hydroxylamine (30 microM) in the absence of a metabolizing system and with no RBC in the third compartment resulted in significant (P < 0.001) toxicity toward MNL (43.36 +/- 5.82% cell death) compared with control (1.8 +/- 1.1%). The presence of RBC in the third compartment resulted in a significant (P < 0.001) decrease in MNL toxicity (17.6 +/- 2.2% cell death), with 40.1 +/- 3.7% methaemoglobin in the RBC.(ABSTRACT TRUNCATED AT 250 WORDS)
The fate of the toxic metabolite of dapsone, dapsone hydroxylamine, has been studied in the human red cell. Twice-washed red cells were incubated at 37 degrees with dapsone hydroxylamine: at 3 and 5 min, 27.0 +/- 2.2 and 33.2 +/- 2.7% of the haemoglobin had been converted to methaemoglobin, leading to a maximum at 45 min (45 +/- 1.8%). HPLC analysis revealed that parent amine was produced from dapsone hydroxylamine during methaemoglobin formation in the red cells. At 3 min, conversion of dapsone hydroxylamine to dapsone reached 7.0 +/- 3.9% leading to a maximum at 30 min (18.1 +/- 3.7%). There was a linear relationship between hydroxylamine-dependent methaemoglobin formation and conversion of hydroxylamine to dapsone (r = 0.97). At 4 degrees, methaemoglobin and dapsone formation was greatly retarded, and did not exceed 10%. Co-incubation of diethyl dithiocarbamate (DDC) with dapsone hydroxylamine and red cells led to a marked increase in methaemoglobin formation (61.4 +/- 3.4%) compared with hydroxylamine and red cells alone (45.0 +/- 1.8%, P < 0.001) at 45 min, and conversion of dapsone hydroxylamine to dapsone was almost doubled at 45 min (35.7 +/- 5.3%) compared with hydroxylamine and red cells (18.1 +/- 2.5%). A linear relationship between methaemoglobin formation and dapsone formation (r = 0.96) was also shown to occur in the presence of DDC. Incubation of red cells with DDC and dapsone hydroxylamine caused a significantly greater reduction in glutathione levels (98.3 +/- 1.6%) compared with red cells and dapsone hydroxylamine alone (84.8 +/- 2.7%) at 5 min (P < 0.001), although there was no significant difference between the groups at 15 min (96.9 +/- 2.6 vs 98.1 +/- 2.2%). Intra-erythrocytic glutathione was then depleted by 75 +/- 3.4%, by pretreatment with diethyl maleate (6 mM), and these cells in the presence of the hydroxylamine showed a significant fall in both methaemoglobin generation (29.7 +/- 1.2 vs 35.0 +/- 1.7%) and parent amine formation (11.1 +/- 0.2 vs 16.5 +/- 1.1%) compared with untreated red cells at 45 min. It is possible that a cycle exists between hepatic oxidation of dapsone to its hydroxylamine and reduction to the amine within the red cell, which may lead to re-oxidation by hepatic cytochrome P450. This process may contribute to the persistence of the drug in vivo.
1. The adverse reactions associated with the administration of dapsone are believed to be caused by metabolism to its hydroxylamine. Previous reports suggest that CYP3A4 is responsible for this biotransformation [1]. 2. Data presented in this paper illustrate the involvement of more than one cytochrome P450 enzyme in dapsone hydroxylamine formation using human liver microsomes. Eadie-Hofstee plots demonstrated bi-phasic kinetics in several livers. No correlation could be established between hydroxylamine formation and CYP3A concentrations in six human livers (r = -0.47; P = 0.34). 3. Studies with low molecular weight inhibitors illustrate the importance of CYP2C9 and CYP3A in dapsone N-hydroxylation. 4. Differential sensitivity of dapsone N-hydroxylation to selective CYP inhibitors indicated that the contribution of individual CYP enzymes varies between livers. Selective inhibition ranged from 6.8 to 44.1% by 5 microM ketoconazole, and from 24.0 to 68.4% by 100 microM sulphaphenazole. The extent of inhibition, by either ketoconazole or sulphaphenazole was dependent on the CYP3A content of the liver. 5. The levels of expression of these cytochrome P450 enzymes may be an important determinant of individual susceptibility to the toxic effects of dapsone, and may influence the ability of an enzyme inhibitor to block dapsone toxicity in vivo. Because of the inability to produce complete inhibition, selective CYP inhibitors are unlikely to offer any clinical advantage over cimetidine in decreasing dapsone hydroxylamine formation in vivo.
Studies of the metabolism of dapsone in man and experimental animals: formulation of N-hydroxy metabolites
- Israili