Glutathione and trypanothione in several strains of Trypanosoma cruzi: effect of drugs.

Glutathione (GSH), trypanothione (T(SH)2) and glutathionyl spermidine (GSH-SP) concentrations were determined in the Tulahuén and LQ strains and the DM 28c clone of Trypanosoma cruzi. The concentrations of GSH, T(SH)2 and GSH-SP, expressed as nmol of GSH per g of parasite fresh weight, were 60.1, 397.8 and 103.9, respectively, for the Tulahuén strain. For the DM 28c clone, the values were 113.9, 677.9 and 164.1, respectively, and for the LQ strain they were 199.1, 1100.5 and 55.3, respectively. When the parasites were treated with 10 microM nifurtimox or 50 microM benznidazole for 2 h, the concentrations of all three reduced thiols decreased strongly. The total amount of T(SH)2 decreased by more than 50%. Treatment of the parasites with 5 mM buthionine sulfoximine, an inhibitor of GSH synthesis, for 6 h diminished the concentrations of the reduced thiols by between 27% and 53% with respect to the controls. Cyclohexylamine, an inhibitor of spermidine synthesis, decreased the concentrations of T(SH)2 and GSH-SP but not that of GSH. It is possible to conclude from this study that trypanothione is the most important thiol involved in the detoxication of nifurtimox and benznidazole in T. cruzi and that electrophilic reduced metabolites of both drugs are most probably conjugated with GSH, T(SH)2 and GSH-SP, thus decreasing their concentrations. GSH biosynthesis is an important drug target.

Role of microsomal cytochrome P-450 in the formation of ecdysterone in larval house fly.

1. Six cytochrome P-450 species have been purified to varying extents from microsomes obtained from ecdysone-induced house fly larvae by the use of octylamino Sepharose-4B, Synchropak AX-300, Synchropak CM-300 and TSK-DEAE-5 PW column chromatography. 2. One of the fractions apparently corresponded to a mixture of low- and high-spin cytochrome P-450 as judged by spectral characteristics. 3. Molecular weights of the cytochrome P-450 species ranged from 50,000 to 57,000. 4. In a reconstituted system, all the microsomal species hydroxylated ecdysone at rates within the range of microsomal suspensions, as it occurs with mitochondrial fractions 1, 2, 3, 5, and 6 (Srivatsan et al., 1990, Biochem, biophys. Res. Commun. 166, 1372-1377); whereas, mitochondrial fraction 4 hydroxylates ecdysone at significantly higher rates. 5. It is postulated that the 20-monooxygenation of ecdysone is a mitochondrial event which requires the induction of a low-Km cytochrome P-450 species by ecdysone. 6. Microsomal hydroxylation of ecdysone may not be of physiological significance, as Km values for the reaction are above the normal concentrations of the hormone and the activity is not inducible by ecdysone (Agosin et al., 1988, Arch. Insect Biochem. Physiol. 9, 107-117).

Cytochrome P-450-catalyzed formation of 20-hydroxy-ecdysone in larval housefly mitochondria.

Six forms of cytochrome P-450 in the mitochondria of larvae from Musca domestica were isolated by solubilization with CHAPS followed by ammonium sulfate fractionation and HPLC on an anion-exchange column. Forms 1, 2, 3, 5, and 6 catalyzed the formation of 20-hydroxy-ecdysone from ecdysone in the presence of NADPH and pig adrenal adrenodoxin and adrenodoxin reductase at rates not much different that observed in mitochondria; whereas, fraction 4 showed an activity which was about 10-fold higher than mitochondria. Forms 4 and 5 were further purified by HPLC on a cation-exchange column followed by removal of excess detergent by hydroxyl apatite column chromatography. In vitro reconstitution of the monooxygenase activity confirmed that form 4 is primarily involved in the formation of 20-hydroxy-ecdysone from ecdysone. SDS-polyacrylamide gel electrophoresis indicated a high degree of purity of both forms 4 and 5, with molecular weights of 56 and 58 KDa, respectively.

Immunochemical studies on the FAD-dependent NADPH-cytochrome c reductase from Trypanosoma cruzi.

The cytosolic flavin enzyme from Trypanosoma cruzi was isolated by a modification of the previously reported method (T. Kuwahara, R. A. White, Jr., and M. Agosin (1985) Arch. Biochem. Biophys. 239, 18-28). In the present study, rabbits were inoculated with the purified enzyme and antibodies were purified from the sera. Ouchterlony double-diffusion analysis indicated that the antibodies reacted specifically with the flavoenzyme and not with other T. cruzi proteins. At the equivalence point, 1 ml of antibody neutralized about 4 nmol of enzyme. The IgG fraction had a small inhibitory effect on the catalytic activity of the enzyme as measured by cytochrome c reduction but only at IgG concentrations well above the equivalence point. Immunotitration of the enzyme in T. cruzi cultures showed that the enzyme corresponds to about 1% of the total protein during the logarithmic phase of growth, but this value decreases to about 0.6% during the stationary phase. Among various trypanosomatids tested, T. cruzi had the highest enzyme concentration; whereas, in other species it ranged from 0.25 to 2.4 micrograms/mg protein. These marked differences suggest that the antibody may be suitable for taxonomic purposes. The presence of the enzyme in amastigotes maintained in tissue culture cells was demonstrated by indirect immunofluorescence. The enzyme was found localized in the periphery of the cell, just beneath the subpellicular microtubules. However, distribution of the enzyme in epimastigotes was more diffuse. As immunofluorescence could be detected only in amastigotes and not in the tissue culture cells, it is suggested that the antibody may be suitable for histopathological diagnosis of Chagas' disease.

The effect of alpha-ecdysone and phenobarbital on the alpha-ecdysone 20-monooxygenase of house fly larva.

The NADPH-dependent cytochrome P-450 20-monooxygenation of alpha-ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica, L. larvae, but about 50% of the activity is associated with mitochondria and 37% with microsomes. The mitochondrial activity is increased by pretreatment with alpha-ecdysone with a concomitant decrease in Km values. This effect is not observed in microsomes. Induction with phenobarbital represses the mitochondrial 20-monooxygenase but does not change the microsomal activity, although a large increase in cytochrome P-450 is observed in the latter fraction. It is concluded that only the mitochondrial 20-monooxygenase appears to be regulated by alpha-ecdysone which suggests that mitochondrial cytochrome P-450 forms are involved in the moulting phenomenon; whereas, microsomal cytochrome P-450 activity may be of a nonspecific nature and not relevant to development.

Conversion of N,N-dimethylaniline to N,N-dimethylaniline-N-oxide by a cytosolic flavin-containing enzyme from Trypanosoma cruzi.

A cytosolic NADPH-dependent FAD-containing enzyme purified from Trypanosoma cruzi epimastigotes [Kuwahara, White, and Agosin: Biochem. Biophys. Res. Commun. 124, 121 (1984); Arch. Biochem. Biophys. 239, 18 (1985)] catalyzes the conversion of N,N-dimethylaniline to its corresponding N-oxide. The identity of the product has been established by high performance liquid chromatography and paper chromatography elution patterns and by electron impact spectrometry. The oxidation of N,N-dimethylaniline was determined by following the oxidation of NADPH spectrophotometrically, and a double reciprocal plot of reaction velocity vs. substrate concentration was prepared. At an optimum pH of 8.0, the plot resulted in Km and Vmax values of 56 microM and 114 nmol X min-1 X mg of protein-1, respectively. The oxidative activity of the enzyme suggests that it may be involved in detoxication processes which may contribute to the resistance of T. cruzi to known antiprotozoal drugs.

Comparative aspects of hepatic UDP-glucuronosyltransferases and glutathione S-transferases in bluegill and channel catfish.

1. Microsomal UDP-glucuronosyltransferases (UDPGTs) and cytosolic glutathione S-transferases (GSTs) were examined in bluegill (Lepomis macrochirus R.) and channel catfish (Ictalurus punctatus R.) liver. 2. Hepatic UDPGT activity was of a similar magnitude in both species and was markedly increased by the addition of 0.05-0.2% Triton X-100, however, optimal estimates of activity were obtained when 0.1% detergent was used. 3. Both species exhibited hepatic GST activity toward several structurally dissimilar substrates, suggesting the presence of multiple GSTs. GST activity ranged over three orders of magnitude, depending upon substrate, and was approx. 3-fold higher in the channel catfish than in the bluegill.

A cytosolic flavin-containing enzyme catalyzing reduction of cytochrome c in Trypanosoma cruzi: kinetic studies with cytochrome c as substrate.

The kinetic mechanism of cytochrome c reduction by a Trypanosoma cruzi cytosolic flavoenzyme was investigated by initial velocity determinations, by product inhibition patterns, and by the characteristics of inhibition by analogs. The data suggest a two-site ping-pong mechanism in which NADPH reduces the flavin, which is then reoxidized in two one-electron steps by reaction with two molecules of cytochrome c. The two-site nature of the mechanism is probably related to the dimeric nature of the enzyme, and the binding sites of cytochrome c and NADPH are probably on opposite sites of the FAD.

A cytosolic FAD-containing enzyme catalyzing cytochrome c reduction in Trypanosoma cruzi. I. Purification and some properties.

A cytosolic flavoprotein enzyme for the protozoan, Trypanosoma cruzi, has been purified essentially to homogeneity by DEAE-cellulose and 2',5'-ADP-agarose column chromatography. The native enzyme has a molecular weight of 100,000 +/- 6,000, is composed of two identical subunits of molecular weight 52,000 +/- 1,000, and contains FAD in the ratio of 1 mol of FAD per mol of enzyme subunit. The enzyme is NADPH-dependent and is capable of reducing cytochrome c, ferricyanide, 2,6-dichloroindophenol, and menadione, but not adrenalin. It does not hydroxylate either sodium salicylate or sodium p-hydroxybenzoate, but N-methylaniline and N,N-dimethylaminobenzaldehyde-supported oxidation of NADPH has been demonstrated. Plots of initial velocity against NADPH concentration give hyperbolic curves with Km values of 6.289 X 10(-5) M. The enzyme is clearly different from the microsomal NADPH-cytochrome c reductase in its intracellular distribution, molecular weight, dimeric nature, presence of only FAD, and activity against secondary and tertiary aromatic amines.

NADPH-cytochrome c reductases of Trypanosoma cruzi.

NADPH-dependent reduction of cytochrome c is catalyzed both by microsomes and the cytosolic fraction isolated from Trypanosoma cruzi homogenates. About one-third of the activity is microsomal and two-thirds is cytosolic. The microsomal activity is increased by Lubrol and sodium cholate, but pretreatment with phenobarbital has negligible effect. On the other hand, detergents do not affect the cytosolic activity but it is increased by phenobarbital. From these observations, it is concluded that the NADPH-dependent reduction of cytochrome c by microsomes and the cytosol corresponds to two distinct enzymes. The cytosolic enzyme has been purified to a single SDS-PAGE band of about 53,000 da and partially characterized.

Cytochrome P-450 in culture forms of Trypanosoma cruzi.

Trypanosoma cruzi epimastigote and trypomastigote forms contain microsomal peptides in the 40-60,000 mol. wt region, some of which are heme-staining-positive and are induced by phenobarbital, as indicated by SDS-gel electrophoresis and by double-labeling experiments. Epimastigotes show induced peptides of mol. wt 56,000, 52,000, 49,000, 44,000, 42,000 and 40,500 whereas only one peptide (52,500 mol. wt) is increased in trypomastigotes. Fractionation of microsomes derived from epimastigotes by octylamine Sepharose-4B column chromatography reveals the presence of two heme peptides with mol. wt of 55,800 and 56,600. The pooled fraction has a typical cytochrome P-450 CO-difference spectrum and appears to correspond to a high spin form. The demonstration of the existence of this family of hemoproteins in T. cruzi further supports the idea that resistance to chemotherapeutic agents is due to active metabolism. The active metabolism, however, may not be similar in the various developmental forms of this organism since differences exist in the patterns of induction of heme-positive microsomal peptides.

Ultrastructural modifications during the metabolism of metronidazole by Trypanosoma cruzi.

Trypanosoma cruzi epimastigotes incubated in the presence of [14C] metronidazole are capable of a rapid uptake of the drug as shown by timecourse experiments and by autoradiography of the cells. The drug is metabolized to a more polar compound which has the chromatographic behavior of 2-methyl-5-nitroimidazole-1-yl-acetic acid. Mass spectral analysis of the metabolite shows diagnostic mass values (185, 184, 126) which are compatible with the 2-methyl-5-nitroimidazole-1-yl-acetic acid derivative. Flavone dramatically increases the production of the metabolite both in control and cells pretreated with phenobarbital. The cells show the presence of vesicles whose number is not significantly increased by phenobarbital. Metronidazole, on the other hand, significantly increases the number of vesicles in both control and cells grown in phenobarbital. The vesicles do not contain acid phosphatase and/or polyphosphates. It is concluded that the vesicles may correspond to a marked proliferation of the endoplasmic reticulum. A secondary effect of flavone is the proliferation of the mitochondrial membranes.

The aerobic metabolism of metronidazole by Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi epimastigotes actively metabolize metronidazole under aerobic conditions to a polar compound tentatively identified as 2-methyl-5-nitroimidazole-1-yl-acetic acid. The rate of metabolite formation is increased by more than 50% by pretreatment with phenobarbital and inhibited by SKF-525A and metyrapone. The reaction is dramatically stimulated by the addition of flavone which suggests that the metabolite is produced via the cytochrome P-450 system. Apparently the nitro group in the metabolite is maintained intact. Detoxication reactions catalyzed by cytochrome P-450 appear to be more important than previously suspected as a basis to explain at least partially the resistance of these organisms to known antimicrobial agents. However, other factors such as the fate of nitro substituent in metronidazole require further evaluation.

Methoprene and hydroprene are metabolized by ester cleavage in isolated hepatocytes.

Methoprene (isopropyl [2E,4E]-11-methoxy-3,7,11-trimethyldodeca-2,4-dienoate) and hydroprene (ethyl[2E,4E]-3,7,11-trimethyldodeca-2,4-dienoate) are actively metabolized, mainly to their corresponding acids by isolated rat hepatocytes and rat liver subcellular fractions. Small amounts of conjugates corresponding to glucuronides are also found. The esterase activity is of microsomal origin and is almost completely inhibited by tri-o-tolyl phosphate in isolated hepatocytes and microsomal fractions supplemented with NADPH. No evidence for the participation of the NADPH-dependent cytochrome P-450 system in the metabolism of either compound was observed.

Epoxide hydrase in Trypanosoma cruzi epimastigotes.

1. Microsomal fractions from Trypanosoma cruzi epimastigotes catalyze the hydration of styrene oxide to styrene glycol. The activity is linear up to 45 min of incubation, is proportional to microsomal protein concentration within certain range, and has an optimum pH of 8.5. 2. Double-reciprocal plots indicate a Km value of 5.3 . 10(-4) M for styrene oxide and a V of 29.6 pmol of styrene glycol formed/min per mg protein at 37 degrees C. 4-Chlorophenyl-2,3-epoxypropyl either (Ki = 2.08 . 10(-4) M) and juvenile hormone I (Ki = 2.7 . 10(-4) M) are competitive inhibitors; whereas, 1-chloro-2,3-epoxypropane is a non-competitive inhibitor. The enzyme is induced about three-fold by 5 mM phenobarbital in the growth medium. 3. The epoxide hydrase is not activated by detergents but rather inhibited by concentrations of Tween-80 and Lubrol as low as 0.025%. 4. Experiments with intact cells indicate that about 3% of [8-14C]styrene oxide penetrates after 90 min of incubation; whereas, over 30% of juvenile hormone I is found intracellularly after the same incubation period. Intracellular styrene oxide is hydrated to styrene glycol to a significant extent and the in vivo hydration is increased by pretreatment with phenobarbital and inhibited upon the addition of 4-chlorophenyl-2,3-epoxypropyl ether. Only a small amount of the intracellular juvenile hormone I is recovered as the corresponding diol ester.

Fate of juvenile hormone in mammalian cell culture.

The metabolism of juvenile hormone (JH) I has been examined in fetal mouse liver cells maintained in culture. Diffusion of the hormone into the cells appears to be passive. The hormone is metabolized essentially to organic-soluble metabolites (diol ester, diol acid and acid) by the action of epoxide hydrase and carboxylesterases. Conjugative reactions play a minor role, less than 3% of the hormone being excreted as conjugates (glucuronides, sulfates and mercapturic acid). About 0.8% of the cellular radioactivity is bound to macromolecules, mainly those of nuclear and mitochondrial origin. Metyrapone and SKF 525-A inhibit covalent binding of the hormone to cytoplasmic macromolecules, which suggests participation of the cytochrome P-450 system in covalent binding of the hormone.