Stephen Murray

Specific C-Terminal Cleavage and Inactivation of Interleukin-8 by Invasive Disease Isolates of Streptococcus pyogenes

Lethal necrotizing fasciitis caused by Streptococcus pyogenes is characterized by a paucity of neutrophils at the site of infection. Interleukin (IL)-8, which is important for neutrophil transmigration and activation, can be degraded by S. pyogenes. Blood isolates of S. pyogenes were better able to degrade human IL-8 than throat isolates. Degradation of IL-8 was the result of a single specific cleavage between 59glutamine and 60arginine within the IL-8 C-terminal alpha helix. Cleaved IL-8 reduced neutrophil activation and migration. IL-8-cleaving activity was found in partially purified supernatant of a necrotizing fasciitis isolate, and this activity was associated with an approximately 150-kDa fraction containing S. pyogenes cell envelope proteinase (SpyCEP). IL-8-cleaving activity corresponded with the presence of SpyCEP in the supernatant. Cleavage of IL-8 by S. pyogenes represents an unprecedented mechanism of immune evasion, effectively preventing IL-8 C-terminus-mediated endothelial translocation and subsequent recruitment of neutrophils.

The specificity of inhibition of debrisoquine 4-hydroxylase activity by quinidine and quinine in the rat is the inverse of that in man

The kinetics of inhibition of debrisoquine 4-hydroxylase activity by quinidine and quinine in rat and human liver microsomes have been compared. Quinidine is a potent inhibitor of debrisoquine 4-hydroxylase activity of human liver (IC50: 3.6 microM). However, its stereoisomer, quinine, is some 60 times less potent (IC50:223 microM). Both compounds are able to inhibit greater than 95% of 4-hydroxylase activity. In rat liver microsomes quinine is approximately 50 times more potent an inhibitor (IC50:2.4 microM) than quinidine (IC50:137 microM). Again, 4-hydroxylase activity is inhibited by greater than 95%. Inhibition of debrisoquine 4-hydroxylase activity by both quinine and quinidine in human and rat liver is competitive. Values of Ki for quinidine in human and rat were 0.6 microM and 50 microM, whereas with quinidine the Ki values were 13 microM and 1.7 microM, respectively. The data in this paper are consistent with 4-hydroxylation of debrisoquine in both rat and human liver catalysed by a specific form of cytochrome P-450. Although both quinidine and quinine are competitive inhibitors of debrisoquine 4-hydroxylase activity in rat and man, their potency is reversed. This suggests that the nature of the active site of cytochrome P-450dbl differs between the two species, and indicates that data on the specificity of this isoenzyme in the rat should be extrapolated to man with extreme caution.

Species variation in the response of the cytochrome P-450-dependent monooxygenase system to inducers and inhibitors

1. In the safety evaluation of drugs and other chemicals it is important to evaluate their possible inducing and inhibitory effects on the enzymes of drug metabolism. 2. While many similarities exist between species in their response to inducers and inhibitors, there are also important differences. Possible mechanisms of such variation are considered, with particular reference to the cytochrome P-450 system. 3. Differences in inhibition may be due to differences in inhibitory site of the enzyme involved, which is not always the active site of the enzyme, in competing pathways or in the pharmacokinetics of the inhibitor. 4. Differences in induction could be due to differences in the nature of the induction mechanism, in the isoenzyme induced, in tissue- or age-dependent regulation, in competing pathways for the substrate or its products, or in the pharmacokinetics of the inducing agent. 5. Examples of each of these possible differences are considered, often from our own work on the P450 IA subfamily, and results in animals are compared with those in humans, where possible. 6. At present, the differences between species in their response to inducers and inhibitors make extrapolation to humans from the results of animal studies difficult, so that ultimately such effects should be studied in the species of interest, humans.

Quantification of the carcinogens 2-amino-3,8-dimethyl- and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in food using a combined assay based on gas chromatography—negative ion mass spectrometry

A gas chromatographic-mass spectrometric assay has been developed for the measurement of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in food. Stable isotope-labelled analogues of MeIQx and PhIP are used as internal standards and the synthesis of deuterated PhIP is described. The mass spectrometer is operated in the electron-capture negative ion chemical ionisation mode and the amines are chromatographed as their di-3,5-bistrifluoromethylbenzyl derivatives. All three compounds can be measured in a single chromatographic run and detection limits of 0.05, 0.1 and 0.2 ng/g for MeIQx, DiMeIQx and PhIP, respectively, in food are obtained. Various home-cooked and commercially prepared foodstuffs were analysed with this assay and several were found to contain measurable amounts of one or more of the three amines. These results are presented and discussed.

Genetic polymorphism in drug oxidation: In vitro studies of human debrisoquine 4-hydroxylase and bufuralol 1′-hydroxylase activities

A sensitive, specific assay utilizing fluorescence-HPLC has been developed for determining the 1-hydroxylation of bufuralol by human liver. The 1-hydroxylation of the isomers of bufuralol varied threefold, both the Vmax and the Km for the (+) isomer being greater than the corresponding values for the (-) isomer. Debrisoquine was a competitive inhibitor of the 1-hydroxylation of both isomers and of the racemate of bufuralol. Both isomers and the racemate of bufuralol were competitive inhibitors of debrisoquine 4-hydroxylase activity. The competitive inhibition of debrisoquine and bufuralol of each others metabolism, together with the similarity in the values for Km and Ki, support the conclusion that the same form of cytochrome P-450 catalyses these two reactions.

Assessing human risk to heterocyclic amines

Heterocyclic amines such as MeIQx and PhIP are potent genotoxic chemicals which are formed at part per billion levels when meat is cooked. Using assays based on gas chromatography/mass spectrometry with stable isotope labelled analogues as internal standards we have demonstrated MeIQx and PhIP, are efficiently absorbed into the systemic circulation after ingestion of fried beef. Using a potent and selective inhibitor of human CYP1A2, furafylline, we have shown that N-hydroxylation catalysed by this enzyme is the major pathway of metabolism of MeIQx and PhIP and is solely responsible for their oxidation to mutagenic species. This is in contrast to the situation in laboratory animals in which both activation by N-hydroxylation and deactivation by C-oxidation occurs. When furafylline was administered to human volunteers before ingestion of fried beef, we showed that > 90% of MeIQx and approximately 70% of PhIP elimination could be inhibited, demonstrating the extent to which activation occurred in man. MeIQx is a very powerful mutagen in bacterial assays whereas PhIP is a more potent mammalian cell mutagen. Using a mammalian cell target gene, hprt, we have shown that PhIP induces a characteristic mutational fingerprint which is identical to that detected in the Apc gene of 5/8 colonic tumours induced by PhIP in rats. These studies support a biological association between HA exposure and diet-related tumours but emphasise that information obtained from animal studies does not always reflect the situation in humans.

Identification of the epitope of an anti-peptide antibody which binds to CYP1A2 in many species including man

An anti-peptide antibody was raised against the sequence Thr-Gly-Ala-Leu-Phe-Lys-His-Ser-Glu-Asn-Tyr-Lys which occurs at positions 283-294 in the rat cytochrome P450 enzyme CYP1A2. Compared with its binding to the peptide used for immunization, the antibody bound with only slightly reduced affinity to the truncated peptides Thr-Gly-Ala-Leu-Phe-Lys-His-Ser and Leu-Phe-Lys-His-Ser. However, binding to the peptide Ser-Glu-Asn-Tyr-Lys-Asp-Asn, which overlaps with the C-terminal region of the immunizing peptide, was very low. Thus, a major epitope for the anti-peptide antibody is Leu-Phe-Lys-His-Ser, which corresponds to a region of CYP1A2 that is conserved in many species. The antibody was tested by immunoblotting for its ability to bind to hepatic microsomal fractions from a number of species. Where possible animals were treated with compounds which induce CYP1A2 and the results compared with those with untreated animals. It was found that the antibody bound to rat, mouse, rabbit, hamster, guinea pig, pig, marmoset monkey and human CYP1A2. No evidence was found for binding to dog CYP1A2. The region corresponding to the major epitope at residues 286-290 of rat CYP1A2 was identical in mouse, hamster, rabbit and human CYP1A2. The sequence of marmoset and guinea pig CYP1A2 are not known but are predicted to be very similar to the sequence in the rat. The lack of binding of the antibody to dog CYP1A2 may be explained by two differences in this region compared with rat CYP1A2. Maximum inhibition of CYP1A2 activity by this antibody, as measured by high-affinity phenacetin O-deethylase activity, was 20%. This is in contrast to a previously described anti-peptide antibody directed to an adjacent region which caused 65% inhibition of this activity. Thus, the edge of an inhibitory region on the surface of cytochrome P450 has been identified. The ability of the antibody to bind to CYP1A2 from a number of animals should make this antibody of use for studying the levels of CYP1A2 apoprotein in many species.

Differential effects of 3-methylcholanthrene and phenobarbitone treatment on the oxidative metabolism of antipyrine in vitro by microsomal fractions of rat liver

1. The effects of treating rats with the inducers phenobarbitone (4 X 80 mg/kg per day) and 3-methylcholanthrene (80 mg/kg) on the kinetics of the formation of the three major oxidative metabolites of antipyrine in vitro by hepatic microsomal fractions have been investigated. 2. Phenobarbitone treatment significantly increased the Vmax of 4-hydroxyantipyrine formation (by 2.3 fold) and of norphenazone formation (by 2.3-fold). 3-Methylcholanthrene treated caused a slight, but significant (P less than 0.05), reduction in Vmax for 3-hydroxymethylantipyrine formation. 3. Phenobarbitone markedly reduced the Km for 3-hydroxymethylantipyrine formation (from 2.2 +/- 0.5 mM to 0.65 +/- 0.09 mM), whereas 3-methylcholanthrene treatment resulted in an increase (P less than 0.05) in the Km (to 4.9 +/- 1.1 mM). The only other significant change in Km was a slight decrease in that of 4-hydroxyantipyrine formation following phenobarbitone treatment (from 4.6 +/- 1.1 mM to 2.2 +/- 0.3 mM, P less than 0.05). 4. Calculation of the ratio Vmax/Km permitted an estimate of the clearance in vivo to the metabolites. There was good agreement between predicted values and those for total body clearance of antipyrine, and four changes in clearance to individual metabolites in vivo following treatment with enzyme inducers. 5. Kinetics analysis of formation of antipyrine metabolites in vitro has enabled the enzymic basis for changes observed in their excretion to be established. Further evidence was also obtained for the involvement of multiple forms of cytochrome P-450 in antipyrine oxidation.

A non-enzymic procedure for the quantitative analysis of (3-methoxy-4-sulphoxyphenyl)ethylene glycol (MHPG sulphate) in human urine using stable isotope dilution and gas chromatography—mass spectrometry

Abstract A method is described for the quantitative analysis of (3-methoxy-4-sulphoxyphenyl)-ethylene glycol (MHPG sulphate) in human urine, based on selected ion monitoring gas chromatography—mass spectrometry and using a specifically deuterium-labelled analogue of MHPG sulphate as internal standard. The procedure involves extraction of the urine sample on Amberlite XAD-2, followed by isolation of MHPG sulphate by column chromatography on Sephadex LH-20. Cleavage of the sulphate conjugate and formation of the MHPG tris(trifluoroscetate) derivative are carried out in a one-step reaction, without recourse to enzymic hydrolysis.

Reduction by inhibitors of mono(ADP-ribosyl)transferase of chemotaxis in human neutrophil leucocytes by inhibition of the assembly of filamentous actin.

1 Chemotaxis of human neutrophils is mediated by numerous agents [e.g. N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) and platelet activating factor (PAF)] whose receptors are coupled to phospholipase C. However, the subsequent transduction pathway mediating cell movement remains obscure. We now propose involvement of mono(ADP‐ribosyl)transferase activity in receptor‐dependent chemotaxis. 2 Human neutrophils were isolated from whole blood and measurements were made of FMLP or PAF‐dependent actin polymerization and chemotaxis. The activity of cell surface Arg‐specific mono(ADP‐ribosyl)transferase was also measured. Each of these activities was inhibited by vitamin K3 and similar IC50 values obtained (4.67 ± 1.46 μm, 2.0 ± 0.1 μm and 4.7 ± 0.1 μm respectively). 3 There were similar close correlations between inhibition of (a) enzyme activity and (b) actin polymerization or chemotaxis by other known inhibitors of mono(ADP‐ribosyl)transferase, namely vitamin K1 novobiocin, nicotinamide and the efficient pseudosubstrate, diethylamino(benzylidineamino)guanidine (DEA‐BAG). 4 Intracellular Ca2+ was measured by laser scanning confocal microscopy with two fluorescent dyes (Fluo‐3 and Fura‐Red). Exposure of human neutrophils to FMLP or PAF was followed by transient increases in intracellular Ca2+ concentration, but the inhibitors of mono(ADP‐ribosyl)transferase listed above had no effect on the magnitude of the response. 5 A panel of selective inhibitors of protein kinase C, tyrosine kinase, protein kinases A and G or phosphatases 1 and 2A showed no consistent inhibition of FMLP‐dependent polymerization of actin. 6 We conclude that eukaryotic Arg‐specific mono(ADP‐ribosyl)transferase activity may be implicated in the transduction pathway mediating chemotaxis of human neutrophils, with involvement in the assembly of actin‐containing cytoskeletal microfilaments.