Characterization of changes in the proteome in different regions of 3D multicell tumor spheroids.

Three dimensional multicell tumor spheroids (MCTS) provide an experimental model where the influence of microenvironmental conditions on protein expression can be determined. Sequential trypsin digestion of HT29 colon carcinoma MCTS enabled segregation into four populations comprising proliferating cells from the surface (SL), an intermediate region (IR), nonproliferating hypoxic cells from the perinecrotic region (PN), and a necrotic core (NC). Total protein was extracted from each population and subjected to iTRAQ-based quantitative proteomics analysis. From a total of 887 proteins identified, 209 were observed to be up-regulated and 114 were down-regulated in the PN and NC regions relative to the SL. Among the up-regulated proteins, components of glycolysis, TCA cycle, lipid metabolism, and steroid biosynthesis increased progressively toward the PN and NC regions. Western blotting, immunohistochemistry, and enzyme assays confirmed that significant changes in the expression of proteins involved in cellular metabolism occur in the nonproliferating fraction of cells within the viable rim. The presence of full length, functional proteins within the NC was unexpected, and further analysis demonstrated that this region contains cells that are undergoing autophagy. This study has identified possible targets that may be suitable for therapeutic intervention, and further studies to validate these are required.

Tumor-selective drug activation: a GDEPT approach utilizing cytochrome P450 1A1 and AQ4N.

Drug metabolizing transgene products, which activate bioreductive cytotoxins, can be used to target treatment-resistant hypoxic tumors. The prodrug AQ4N is bioreduced in hypoxic cells by cytochrome P450s (CYPs) to the cytotoxin AQ4. Previously we have shown that intra-tumoral injection of CYP3A4 and CYP2B6 transgenes with AQ4N and radiation inhibits tumor growth. Here we examine the ability of other CYPs, in particular CYP1A1, to metabolize AQ4N, and to enhance radiosensitization. Metabolism of AQ4N was assessed using microsomes prepared from baculovirus-infected cells transfected with various CYP isoforms. AQ4N metabolism was most efficient with CYP1A1 (66.7 nmol/min/pmol) and 2B6 (34.4 nmol/min/pmol). Transient transfection of human CYP1A1+/-CYP reductase (CYPRED) was investigated in hypoxic RIF-1 mouse cells in vitro using the alkaline comet assay. There was a significant increase in DNA damage following transient transfection of CYP1A1 compared to non-transfected cells; inclusion of CYPRED provided no additional effect. In vivo, a single intra-tumoral injection of a CYP1A1 construct in combination with AQ4N (100 mg/kg i.p.) and 20 Gy X-rays caused a 16-day delay in tumor regrowth compared to tumors receiving AQ4N plus radiation and empty vector (P=0.0344). The results show the efficacy of a CYP1A1-mediated GDEPT strategy for bioreduction of AQ4N.

Advanced microscopy solutions for monitoring the kinetics and dynamics of drug-DNA targeting in living cells.

Many anticancer drugs require interaction with DNA or chromatin components of tumor cells to achieve therapeutic activity. Quantification and exploration of drug targeting dynamics can be highly informative in the rational development of new therapies and in the drug discovery pipeline. The problems faced include the potential infrequency and transient nature of critical events, the influence of micropharmacokinetics on the drug-target equilibria, the dependence on preserving cell function to demonstrate dynamic processes in situ, the need to map events in functional cells and the confounding effects of cell-to-cell heterogeneity. We demonstrate technological solutions in which we have integrated two-photon laser scanning microscopy (TPLSM) to track drug delivery in subcellular compartments, with the mapping of sites of critical molecular interactions. We address key design concepts for the development of modular tools used to uncover the complexity of drug targeting in single cells. First, we describe the combination of two-photon excitation with fluorescence lifetime imaging microscopy (FLIM) to map the nuclear docking of the anticancer drug topotecan (TPT) at a subset of DNA sites in nuclear structures of live breast tumor cells. Secondly, we demonstrate how we incorporate the smart design of a two-photon 'dark' DNA binding probe, such as DRAQ5, as a well-defined quenching probe to uncover sites of drug interaction. Finally, we discuss the future perspectives on introducing these modular kinetic assays in the high-content screening arena and the interlinking of the consequences of drug-target interactions with cellular stress responses.

Tumour cytochrome P450 and drug activation.

The expression of drug metabolising cytochrome P450s (CYPs) notably 1A, 1B, 2C, 3A, 2D subfamily members have been identified in a wide range of human cancers. Individual tumour types have distinct P450 profiles as studied by detection of P450 activity, identification of immunoreactive CYP protein and detection of CYP mRNA. Selected P450s, especially CYP1B1, are overexpressed in tumours including cancers of the lung, breast, liver, gastrointestinal tract, prostate, bladder. Several prodrug anti-tumour agents have retrospectively been identified as P450 substrates for which tumour CYP activation may hitherto have been underestimated. Those in clinical use include prodrug alkylating agents (cyclophosphamide, ifosphamide, dacarbazine, procarbazine), Tegafur, a prodrug fluoropyrimidine, methoxymorphylinodoxorubicin, a metabolically activated anthracycline, as well as flutamide and tamoxifen, two non-steroidal hormone receptor antagonists that are significantly more active following CYP-hydroxylation. More exciting is the prospect of developing new agents designed to be selectively dependent on tumour CYP activation. This can be illustrated with P450 activation of the 2-(4-aminophenyl)benzothiazoles exclusively in CYP1A1 inducible tumours. Also of interest is the bioreductive antitumour prodrug AQ4N, a CYP3A substrate that is activated to a cytotoxic metabolite specifically in hypoxic tumour regions.

Identification of cytochrome P450 enzymes in human colorectal metastases and the surrounding liver: a proteomic approach.

We describe the direct identification of multiple cytochrome P450 (CYP) enzymes in healthy and cancerous tissue. CYPs in human liver colorectal metastases were compared with those in the surrounding liver using a mass spectrometry-based proteomic approach. Nano-scale reversed phase liquid chromatography combined with electrospray ionisation tandem mass spectrometry has been used to identify CYPs with no pre-selection of the proteins anticipated. Fourteen distinct CYP enzymes from the subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 4F, 8B and 27A were positively identified; 13 in the liver samples and 12 in the tumour tissue. It was found that three of the colorectal metastases expressed essentially the same drug-metabolising pattern of CYPs as the surrounding liver, whilst three tumour samples from different individuals showed no CYP expression. This was likely in at least one case to be due to low sample mass. The CYP expression profile in an individual tumour is likely to be an important determinant in predicting the outcome of cancer chemotherapy.

A novel cell permeant and far red-fluorescing DNA probe, DRAQ5, for blood cell discrimination by flow cytometry.

The deep red fluorescing agent (DRAQ5) is a synthetic anthraquinone with a high affinity for DNA and a high capacity to rapidly enter living cells or stain fixed cells. DRAQ5 is optimally excited by red-light emitting sources and yields a deep red emission spectrum which extends into the low infra-red. DRAQ5 shows excitation at sub-optimal wavelengths including the 488 nm line and the multi-line UV wavelengths emitted by argon-ion lasers. Single beam (488 nm) flow cytometry has been used to demonstrate the utility of DRAQ5-nuclear DNA fluorescence as a discriminating parameter for human leucocytes and lymphoma cells, in combination with fluorochrome-labelled antibodies for the detection of surface antigens and subpopulation recognition. DRAQ5 fluorescence was found to reflect cellular DNA content as evidenced by cell cycle distribution profiles for asynchronous and cell cycle-perturbed populations. Importantly, DRAQ5 can be used in combination with FITC and RPE-labelled antibodies, without the need for fluorescence compensation.

Involvement of human cytochromes P450 (CYP) in the reductive metabolism of AQ4N, a hypoxia activated anthraquinone di-N-oxide prodrug.

PURPOSE: To establish the role of the human cytochromes P450 (CYPs) in the reductive metabolism of the novel anthraquinone di-N-oxide prodrug AQ4N. METHODS AND MATERIALS: Metabolism of AQ4N was conducted in a panel of 17 human phenotyped liver microsomes. AQ4N and metabolites were detected by reverse phase isocratic HPLC. CYP inhibitors and Spearman rank correlation were used to determine the significance of AQ4N metabolism versus specific CYP activity and/or expression. RESULTS: Anaerobic metabolism of AQ4N to the 2-electron reduction product, AQM, and the 4-electron reduced tertiary amine, AQ4, occurred in all 17 human liver microsome preparations. The range (+/- SE) for total AQ4N turnover was 14.26 +/- 1.43 nmol/incubate (highest) to 3.65 +/- 1.05 nmol/incubate (lowest). Metabolism was not detected in the absence of NADPH or microsomes. In aerobic incubates, AQM was less than 4% of anaerobic values whereas AQ4 was undetectable. CYP-mediated metabolism of AQ4N was inhibited completely by ketoconazole (KET) and carbon monoxide (CO), two global inhibitors of CYP-mediated metabolism. AQ4N metabolism correlated significantly with probes for CYP 3A, specifically benzoxylresorufin O-dealkylation [r(s) = 0.70,p <0.01] and tamoxifen N-demethylation (r(s) = 0.85, p < 0.01), but not with probes for CYPs 2C, 2D, and 1A. CYP 3A involvement was confirmed by the use of the CYP 3A specific inhibitor, triacetyloleandomycin (TAO), which repressed the formation of AQM to 13% of the uninhibited value and abolished completely the formation of AQ4. Alpha-naphthoflavone (ANF), an inhibitor of CYP 2C and 1A, had no significant effect on AQ4N metabolism. CONCLUSIONS: These data suggest that the human CYP 3A enzymes can contribute to the reductive metabolism of AQ4N. CYP 3A enzymes are highly expressed in a broad spectrum of human cancers. The results show that AQ4N requires anaerobic conditions for CYP 3A-mediated reduction and hence this subfamily of enzymes is likely to selectively activate AQ4N in hypoxic tumors.

Comparative computer graphics and solution studies of the DNA interaction of substituted anthraquinones based on doxorubicin and mitoxantrone.

1-[[(Diethylamino)ethyl]amino]- and 1,4-, 1,5-, and 1,8-bis[[(diethylamino)ethyl]amino]anthraquinones are shown to intercalate into DNA. Computer graphics modelling of their intercalation into the self-complementary deoxydinucleoside d(CpG) showed differences in binding properties. While the 1-substituted compound can bind from either groove, the 1,8-disubstituted compound binds with both substituents in the major groove. In the low-energy state of the complex with the 1,5-disubstituted compound, this ligand "straddles" the site with a substituent in each groove--to do this, the compound must bind to a non-base-paired region, so inducing base pairing. The 1,4-compound binds from the major groove; "straddling" is also possible if full minimization of deoxydinucleoside geometry is performed. The differences in binding mode and interaction energies are reflected in the affinities of interaction (1,5- greater than 1,4- much greater than 1,8- greater than 1-); also the antiproliferative effects in vitro are in general agreement with this ranking.

Electron paramagnetic resonance spectrometry evidence for bioreduction of tirapazamine to oxidising free radicals under anaerobic conditions.

Tirapazamine (SR 4233) is a bioreductive antitumour drug in Phase III clinical trial which is activated in hypoxic tumour regions to generate a cytotoxic species. Electron paramagnetic resonance (EPR) spectrometry was used to investigate directly the formation of free radicals as the result of tirapazamine reduction by NADPH-supplemented liver microsomes. Under anaerobic conditions, the tirapazamine nitroxide free radical EPR signal was not evident over a range of rat or human liver microsomal protein (1-5 mg) concentrations. However, in combination with 1,1',5, 5'-dimethylpyrolline-1-N-oxide (DMPO), a spin trap for short-lived free radicals, tirapazamine resulted in formation of a 1:1:1:1:1:1 spectrum with hyperfine splitting A(N) = 15.8 G A(H) = 22.3 G consistent with generation of DMPO-R, a carbon-centered radical adduct. Addition of DMSO increased the signal intensity of the carbon-centred radical by at least twofold. The hyperfine splitting constants associated with DMPO-R could be indicative of a tirapazamine carbon-centred radical per se or, more likely, carbon radicals from endogenous materials (or DMSO) in the biological matrix as a result of oxidative attack by the tirapazamine primary radical. Formation of DMPO-OH, the hydroxyl radical spin adduct, by tirapazamine in the absence of air indicates that liberation of a hydroxyl radical may be a consequence of tirapazamine bioreduction under anaerobic conditions. The reactivity of tirapazamine free radicals with endogenous microsomal substances to generate reactive carbon-centred radicals indicates that tirapazamine may disrupt a wide range of cellular activities.

Generation of a free radical from calphostin C by microsomal cytochrome P450 reductase in rat and human liver.

Calphostin C (cal C) is a potent and specific protein kinase C inhibitor that is thought to be photoactivated to generate a reactive perylenesemiquinone free radical intermediate. In this electron spin resonance spectrometry study, we show, for the first time, an enzymatic light-independent mechanism of cal C free radical formation. Such a free radical was generated in liver microsomes from rat and humans, and its formation was dependent on the presence of NADPH, functional microsomes, and protein and cal C concentrations. Inhibitor and purified enzyme studies showed that cytochrome P450 reductase is responsible for the cal C free radical observed, which is probably a perylenesemiquinone.

NAD(P)H (quinone acceptor) oxidoreductase (DT-diaphorase)-mediated two-electron reduction of anthraquinone-based antitumour agents and generation of hydroxyl radicals.

The anthraquinone-based antitumour agents mitoxantrone, daunorubicin and ametantrone were found to be substrates for NAD(P)H (quinone acceptor) oxidoreductase (DT-diaphorase) [QAO] isolated from rat liver. This was indicated by the stimulation of QAO-dependent NADPH oxidation by these agents. This effect followed Michaelis-Menten kinetics and was dependent on the concentration of QAO, inhibited by the specific QAO inhibitor dicumarol (15 microM) and enhanced by the QAO activators bovine serum albumin (0.01%) and Triton X-100 (0.03%). As indicated by the Vmax/Km ratio, mitoxantrone (26.53) was considerably more active than ametantrone (11.25) or daunorubicin (7.35). Metabolism of these anthraquinones was associated with the formation of superoxide anions, hydrogen peroxide and hydroxyl radicals as indicated by electron spin resonance spin trapping studies with 5,5-dimethyl-1-pyrroline-N-oxide. This is likely to be due to the slow auto-oxidation of the respective dihydroquinones in the presence of molecular oxygen. QAO needs to be considered as a possible route of bioreductive activation of these agents.

The effect of the anthrapyrazole antitumour agent CI941 on rat liver microsome and cytochrome P-450 reductase mediated free radical processes. Inhibition of doxorubicin activation in vitro.

The anthrapyrazole CI941 is one of a new series of DNA complexing drugs which displays high level broad spectrum antitumour activity in mice. In view of the proposed role of drug free radical formation, superoxide generation and lipid peroxidation in anthracycline and anthraquinone induced toxicities, the redox biochemistry of CI941 has been investigated. Studies have been performed in vitro using rat liver microsomes and purified cytochrome P-450 reductase. In addition, the ability of CI941 to undergo chemical reduction has been examined. Pulse radiolysis of CI941 demonstrated that the drug can undergo chemical reduction with a one electron reduction potential of E1(7) = -538 +/- 10 mV. However, electron spin resonance (ESR) spectroscopy studies using either NADPH fortified microsomes or cytochrome P-450 reductase, failed to detect a drug free radical signal. Unlike doxorubicin, CI941 (150 microM) inhibited basal rate microsomal NADPH consumption by 45%. Furthermore, CI941 (50-200 microM) antagonised doxorubicin stimulated (1.8-fold) NADPH oxidation by over 50%. CI941 also antagonised the formation of a doxorubicin free radical ESR signal in a concentration dependent manner. CI941 induced minimal superoxide generation in the presence of either microsomes or cytochrome P-450 reductase and inhibited doxorubicin induced (50 microM) superoxide formation by up to 80% (50-200 microM CI941). Importantly, CI941 inhibits both basal rate and doxorubicin (100 microM) stimulated lipid peroxidation (52% inhibition at 5 microM CI941). These data suggest that CI941 is unlikely to induce free radical mediated tissue damage in vivo. On the contrary, CI941 may have a protective role if used in combination with doxorubicin.

Doxorubicin-3'-NH-oestrone-17-oxime-ethyl-carbonyl, a doxorubicin-oestrone conjugate that does not redox cycle in rat liver microsomes.

In summary doxorubicin-3'-NH-oestrone-17-oximethyl-carbonyl (Dox-Oes) is a covalent adduct of the anthracycline antitumor agent doxorubicin and oestrogen. Dox-Oes does not generate free radicals in rat liver microsomes as detected by electron spin resonance spectroscopy or redox cycle as shown by lack of superoxide anion formation and NADPH oxidation. Furthermore Dox-Oes actually inhibits free radical formation by doxorubicin used in equimolar amounts. The lack of free radical formation by doxorubicin when covalently linked to oestrone supports the development of Dox-Oes as a non-cardiotoxic derivative whilst potentially improving its targeting to oestrogen positive breast tumour cells.

Involvement of NADPH: cytochrome P450 reductase in the activation of indoloquinone EO9 to free radical and DNA damaging species.

Evidence suggests that DT-diaphorase is involved in the activation and mechanism of cytotoxicity of the investigational indoloquinone anticancer drug EO9 under aerobic conditions. Data also implicate a role for other enzymes including NADPH: cytochrome P450 reductase, especially in low DT-diaphorase tumour cells and under hypoxic conditions. Here, we used purified rat NADPH: cytochrome P450 reductase to provide additional evidence in support of a role for this enzyme in activation of EO9 to generate free radical and DNA-damaging species. Electron spin resonance spectrometry studies showed that NADPH: cytochrome P450 reductase reduced EO9 to a free radical species, including a drug radical (most likely the semiquinone) and reactive oxygen species. Plasmid DNA experiments showed that reduction of EO9 catalysed by NADPH: cytochrome P450 reductase results in single-strand breaks in DNA. The information obtained may contribute to the understanding of the molecular mechanism of DNA damage and cytotoxicity exerted by EO9 and may be useful in the design of future bioreductive drugs.

Reduction of the indoloquinone anticancer drug EO9 by purified DT-diaphorase: a detailed kinetic study and analysis of metabolites.

DT-diaphorase has been implicated in the activation and mechanism of cytotoxicity of the investigational indoloquinone anticancer drug EO9. Here, we have used a highly purified DT-diaphorase isolated from rat Walker tumour cells to provide unambiguous evidence for the ability of this enzyme to catalyze reduction of EO9 and to provide a more detailed characterization of the reaction. Under the conditions used hypoxia had no effect on the initial rate of this reduction but did effect the nature and stability of metabolites formed. Electron spin resonance (ESR) spectrometry studies showed that DT-diaphorase reduced EO9 to a highly oxygen-sensitive metabolite that is probably the hydroquinone. In the presence of air, this metabolite is auto-oxidized to generate both drug- and oxygen-based radicals. Comproportionation:disproportionation reactions may also be involved in the generation of these radical species. The identification of these metabolites may contribute to the understanding of the molecular mechanism of DNA damage and cytotoxicity exerted by EO9.

Comparative metal content profiling of parasitic helminths by electron paramagnetic resonance spectrometry: significance for metalloprotein content.

Variation in co-ordination geometries of metal ions bound to proteins imposes electronic states different from free (hydrated) ions in solution. Electron paramagnetic resonance spectroscopy has been used to analyse a selection of parasitic helminths for metal content as an initial step to determination of metallo-enzymes in their ES products under immune stress conditions. Characteristic paramagnetic resonance spectroscopy spectra show clear evidence for the presence of iron, copper, and manganese centres and in the selected parasites. The metals ions are identified as protein-bound as distinct from free metal ions present in aqueous solution, and distinguishable from parasite dietary components derived from host sources. Indication is given that superoxide dismutases may, in part, account for the metal ions observed. The use of electron paramagnetic resonance spectroscopy to identify specific protein-bound metals without prior isolation of the suspected protein is here applied.

Lack of involvement of reactive oxygen in the cytotoxicity of mitoxantrone, CI941 and ametantrone in MCF-7 cells: comparison with doxorubicin.

The MCF-7 cell S9 fraction and whole MCF-7 cells can mediate one-electron-redox cycling of doxorubicin, giving rise to concomitant oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH), formation of a drug semiquinone free radical, consumption of molecular oxygen and formation of superoxide anions and hydroxyl radicals. Doxorubicin redox cycling was consistent with DNA strand breakage and cell kill in MCF-7 cells. In contrast, no evidence for redox cycling was found for mitoxantrone (MIT), CI941 or ametantrone (AMET) in MCF-7 cells. Despite the absence of redox cycling, the CI941, MIT, and AMET concentrations resulting in 50% mortality (LC50; 1.5 x 10(-10), 5.2 x 10(-9) and 1.2 x 10(-6) M, respectively) of MCF-7 cells were lower than that of DOX (3.0 x 10(-6) M). Furthermore, the higher cytotoxicity of MIT and CI941 as compared with AMET or DOX was associated with greater efficiency in inducing DNA strand breakage in MCF-7 cells as determined by alkaline elution. Since MIT and CI941 proved to be the most potent DNA-damaging and cytotoxic agents in this study, the ability of DOX to undergo redox cycling does not appear to confer increased cytotoxic potential on this agent. The present study revealed several important aspects with regards to the structural modification of anthraquinone antitumour agents. Firstly, the C1 and C4 positioning of the hydroxyethylamino side chains on MIT, CI941 and AMET is associated with a lack of flavin reductase-mediated activation of these agents. Secondly, the possession of a C5 or C8 aromatic hydroxyl group appears to be intimately involved in the enhanced DNA strand breakage and cytotoxic potency of MIT and CI941, since AMET does not possess these groups. These findings indicate that future development of quinone antitumour agents should concentrate on compounds that do not undergo redox cycling but do possess aromatic hydroxyl groups, since the latter appear to be responsible for the enhanced cytotoxicity of MIT and CI941.

DNA topoisomerase II-dependent cytotoxicity of alkylaminoanthraquinones and their N-oxides.

We studied the role of DNA topoisomerase II in the biological actions of a series of novel alkylaminoanthraquinones, including N-oxide derivatives designed as prodrugs liable to bioreductive activation in hypoxic tumour cells. Drug structures were based upon the DNA-binding anticancer topoisomerase II poison mitoxantrone with modifications to the alkylamino side chains. The agents included AQ4, 1,4-bis{[2-(dimethylamino)ethyl] amino}5,8-dihydroxy-anthracene-9,10-dione, and AQ6, 1{[2-dimethylamino)-ethyl]amino}4-{[2[(hydroxyethyl)amino]ethyl]- amino}5,8-dihydroxy-anthracene-9,10-dione, together with the corresponding mono-N-oxide (AQ6NO) and di-N-oxide (AQ4NO). The R3N(+)-O- modification renders the terminal nitrogen group electrically neutral and was found to reduce AQ6NO or effectively abolish AQ4NO-DNA binding. Comparative studies were carried out using two SV40-transformed fibroblast cell lines, MRC5-V1 and AT5BIVA, the latter being a relative overproducer of DNA topoisomerase II alpha. The inhibition of DNA topoisomerase II decatenation activity ranked according to DNA-binding capacity. A similar ranking was found for drug-induced DNA-protein cross-linking in intact cells, depending upon topoisomerase II availability. Inhibition of DNA synthesis in S-phase synchronized cultures ranked in the order of AQ6 > mitoxantrone > > AQ6NO and was independent of topoisomerase II availability. Cytotoxicity of acute 1-h exposures for all agents except the inactive AQ4NO was enhanced in the topoisomerase II-overproducing cell line. The results indicate an important role for enzyme targeting in anthraquinone action. However, DNA synthesis inhibition and cytotoxicity were greater than expected for AQ6, given its topoisomerase- and DNA-interaction properties, and parallel studies have provided evidence of an additional role for enhanced subcellular accumulation and nuclear targeting. The inactivity of AQ4NO and the retention of only partial activity of AQ6NO, allied with the effective topoisomerase II-targeting and high cytotoxic potential of their presumed metabolites, favour their use as prodrugs in tumour cells with enhanced bioreductive potential.

Free radicals in irradiated drugs: an EPR study.

Exposure of dry powder forms of the drugs nitrendipine, nifedipine, felodipine, and nimodipine to gamma-radiation results in the formation of free radicals detected by electron paramagnetic resonance (EPR) spectroscopy. The four structurally related drugs show qualitatively identical EPR spectral features in terms of g-values, the qualitative descriptive parameter. These radicals are very stable, surviving long periods of time in excess of 9 months and possibly beyond conventional shelf-life of the drugs. The residual radical population is high enough to be detectable after long storage. Administration of such radiation-treated drugs may present patients with quantities of free radicals and possibilities of secondary cell damage.

Glutathione S-transferase (GST) expression in the human hookworm Necator americanus: potential roles for excretory-secretory forms of GST.

The difficulty in demonstrating protective immunity to human gastro-intestinal nematodes is thought to be a consequence of the expression of defences by the parasites directed against the toxic metabolites of leukocytes produced during inflammation (Brophy and Pritchard, 1992a). Parasite glutathione S-transferases (GSTs) may provide part of this defence by detoxifying the secondary products of lipid peroxidation produced via immune initiated free-radical attack on host or parasite membranes (Brophy and Pritchard, 1994; Taylor et al., 1988). Neutralisation of parasite immune defence components could tip the molecular balance in favour of the immune response during chronic infections. For example, GSTs have been extensively investigated from the digenean parasites Schistosoma and Fasciola hepatica and provide protection in animal-model systems (Mitchell, 1988; Wijffels et al., 1991). In contrast, although GSTs have been initially characterised in filarial nematodes (Salinas et al., 1994; Leibau et al., 1994; Jaffe and Lambert, 1986), there is limited information on GSTs from human gastro-intestinal nematode parasites. We were particularly interested in analysing the products of hookworms for evidence of the presence of excretory-secretory forms of this putative immune defence protein.