A comparison of metal levels and antioxidant enzymes in freshwater snails, Lymnaea natalensis, exposed to sediment and water collected from Wright Dam and Lower Mguza Dam, Bulawayo, Zimbabwe.

We compared the bioaccumulation of lead (Pb), cadmium (Cd), zinc (Zn), copper (Cu), nickel (Ni) and iron (Fe) with antioxidant enzyme activity in tissues of the snails, Lymnaea natalensis, exposed to elements of two differently polluted dams. 45 snails were exposed to sediment and water collected from Wight Dam (reference) whilst another 45 snails were also exposed to sediment and water collected from Lower Mguza Dam (polluted dam). Except for Fe in sediment and Pb in water, metal concentrations were statistically higher in sediment and water collected from Lower Mguza Dam. Lead, Cd and Zn were two times higher in tissues of snails exposed to Lower Mguza Dam elements. On one hand, superoxide dismutase (SOD), diphosphotriphosphodiaphorase (DTD) and catalase (CAT) activities were significantly lower whilst malondialdehyde (MDA) levels were significantly higher in tissues of snails exposed to Lower Mguza Dam sediment and water. On the other hand, selenium-dependent glutathione peroxidase (Se-GPX) activity was significantly elevated in tissues of snails exposed to Lower Mguza Dam sediment and water. Snails exposed to Lower Mguza Dam elements seem to have responded to pollution by increasing CAT and Se-GPX specific activity in an effort to detoxify peroxides produced as a result of metal induced oxidative stress.

Metal accumulation and antioxidant enzyme activity in C. gariepinus, catfish, and O. mossambicus, tilapia, collected from Lower Mguza and Wright Dams, Zimbabwe.

The aim of this study was to measure antioxidant enzyme activities as biological indicators of pollution in tissues of two species of fish. Five Clarius gariepinus and three Oreochromis mossambicus were collected from Umguza Dam (polluted dam) whilst seven C. gariepinus and eight O. mossambicus were collected from Wright Dam (relatively pristine dam). Diphosphotriphoshodiaphorase and catalase activities were consistently lower (42 +/- 2% and 78 +/- 20%, respectively) in liver whilst malondialdehyde levels were two times higher in muscles of both species of fish collected from Umguza Dam. However, seleniumdependent glutathione peroxidase (Se-GPX) activity was elevated four-fold in liver and gills of O. mossambicus collected from Umguza Dam. Metal levels were two to five times higher in muscles of both species of fish collected from Umguza Dam. Fish from Umguza Dam seem to have responded to pollution by increasing Se-GPX specificactivity in an effort to detoxify peroxides produced as a result of metal induced oxidative stress.

Potential marker enzymes and metal-metal interactions in Helisoma duryi and Lymnaea natalensis exposed to cadmium.

In this study, we investigate the effects of exposure to cadmium and copper on Lymnaea natalensis and Helisoma duryi. The snails were dosed with Cd2+ or Cu2+ for a period of 96h. Snails dosed with Cd accumulated the metal significantly (P<0.05) in tissues but not in shells. Mortality was observed at approximately 1mg Cd/l of culture water. In tissues and shells of snails dosed with Cd or Cu, synergistic and antagonistic metal-metal interactions involving Cd, Cu, Zn, and Pb were observed and these may affect metal toxicity. Glutamate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase were assayed in whole snail tissue sub-cellular fractions of Cd-dosed snails. Generally, enzyme activity significantly increased at lower concentrations of Cd but decreased at high concentrations of the metal. However, mitochondrial alanine aminotransferase activity progressively declined with increasing Cd concentration. The changes in some of the enzymes' activities suggest biomarker potential.

Effects of chloroquine treatment on antioxidant enzymes in rat liver and kidney.

The effect of chloroquine (CHQ) administration on antioxidant enzymes in rat liver and kidney was studied. Male Sprague-Dawley rats were administered 20 mg/kg CHQ once a week for 4 weeks (chronic treatment) or a single dose at 10 or 20 mg/kg (acute treatment). Antioxidant enzyme activities were determined in cytosolic fractions of liver and kidney, whereas reduced glutathione (GSH) and malondialdehyde (MDA) were determined in tissue samples. Results indicate minimal effects of acute CHQ treatment, whereas chronic treatment with CHQ differentially affected antioxidant enzymes in the two organs. Superoxide dismutase activity was increased nearly twofold, while activities of selenium glutathione peroxidase (GPX), catalase, and NAD (P) H: quinone oxidoreductase were decreased in livers of CHQ-treated rats compared to controls. No significant effects of CHQ on glutathione reductase, GSH, and MDA levels were seen in the liver. Fewer effects of CHQ were observed in the kidney where a decrease in GPX activity and an increase in MDA levels was seen. Lowering of antioxidant enzymes activities in the liver by CHQ could render the organ more susceptible to subsequent oxidative stress; while increased MDA production after CHQ treatment in the kidney indicate that the organ is being subjected to oxidative stress. This could have implications for prolonged chloroquine intake.

Primaquine alters antioxidant enzyme profiles in rat liver and kidney.

The effects of primaquine treatment on antioxidant enzyme activities were investigated in rat liver and kidney. Male Sprague-Dawley rats were treated with 0.21 mg/kg daily for two weeks (chronic treatment) or a single dose at 0.21 or 0.63 mg/kg. Antioxidant enzyme activities were determined in liver and kidney cytosolic fractions whereas glutathione (GSH) and malondialdehyde (MDA) levels were determined in tissue samples. Results for the liver showed increases in cytosolic superoxide dismutase (SOD) and glutathione peroxidase (GPX) enzymatic activities after chronic primaquine treatment. Levels of MDA, a marker for lipid peroxidation, were also increased by more than 50% indicating enhanced oxidative damage in the liver. In the single dose study, 0.63 mg/kg primaquine caused a more than 100% increase in liver SOD and a 36% increase in NAD (P) H: quinone oxidoreductase (NQOR) activities. Results for the kidney, however, showed fewer primaquine-induced changes in antioxidant enzyme activities when compared to the liver in both the chronic and single dose studies. Overall, our results indicate that primaquine treatment causes an oxidative stress in the two rat organs. These results are consistent with the known pro-oxidant effects of primaquine in vivo, and supplement current knowledge on the effects of antimalarial drugs on various enzyme systems.

Proposed reductive metabolism of artemisinin by glutathione transferases in vitro.

Artemisinin is a sesquiterpene lactone containing an endoperoxide bridge. It is a promising new antimalarial and is particularly useful against the drug resistant strains of Plasmodium falciparum. It has unique antimalarial properties since it acts through the generation of free radicals that alkylate parasite proteins. Since the antimalarial action of the drug is antagonised by glutathione and ascorbate and has unusual pharmacokinetic properties in humans, we have investigated if the drug is broken down by a typical reductive reaction in the presence of glutathione transferases. Cytosolic glutathione transferases (GSTs) detoxify electrophilic xenobiotics by catalysing the formation of glutathione (GSH) conjugates and exhibit glutathione peroxidase activity towards hydroperoxides. Artemisinin was incubated with glutathione, NADPH and glutathione reductase and GSTs in a coupled assay system analogous to the standard assay scheme with cumene hydroperoxide as a substrate of GSTs. Artemisinin was shown to stimulate NADPH oxidation in cytosols from rat liver, kidney, intestines and in affinity purified preparations of GSTs from rat liver. Using human recombinant GSTs hetelorogously expressed in Escherichia coli, artemisinin was similarly shown to stimulate NADPH oxidation with the highest activity observed with GST M1-1. Using recombinant GSTs the activity of GSTs with artemisinin was at least two fold higher than the reaction with CDNB. Considering these results, it is possible that GSTs may contribute to the metabolism of artemisinin in the presence of NADPH and GSSG-reductase. We propose a model, based on the known reactions of GSTs and sesquiterpenes, in which (1) artemisinin reacts with GSH resulting in oxidised glutathione; (2) the oxidised glutathione is then converted to reduced glutathione via glutathione reductase; and (3) the latter reaction may then result in the depletion of NADPH via GSSG-reductase. The ability of artemisinin to react with GSH in the presence of GST may be responsible for the NADPH utilisation observed in vitro and suggests that cytosolic GSTs are likely to be contributing to metabolism of artemisinin and related drugs in vivo.

Phenotyping of the glutathione S-transferase M1 polymorphism in Zimbabweans and the effects of chloroquine on blood glutathione S-transferases M1 and A.

The frequency of the null allele phenotype of glutathione S-transferase (GST) M1 was investigated in 114 Zimbabweans and results for a subset of 63 subjects were compared with genotyping by PCR. In addition, the effect of the antimalarial chloroquine on blood levels of GSTM1 and GSTA in 19 subjects was studied. Quantification of GSTs was by enzyme linked immunosorbent assays (ELISA). Thirty percent of the subjects were of the GSTM1 null phenotype. Comparison of results of phenotyping by ELISA and genotyping by PCR showed that 16% of samples were in discordance; unknown mutations in the GSTM1 gene in the Zimbabwean population may explain this observation. Chloroquine decreased levels of blood GSTM1 and GSTA by 50% or more. In populations treated with chloroquine, these decreases in GST activities might lead to compromised ability to detoxify xenobiotics, could confound GSTM1 phenotyping and might invalidate use of GSTA as an indicator of liver damage.

Effects of phenobarbital and 3-methylcholanthrene pretreatment on the pharmacokinetics of praziquantel in rats.

The effects of phenobarbital (PB) and 3-methylcholanthrene (MC) pretreatment on the pharmacokinetics of praziquantel (PZQ), a schistosomicide were studied in Sprague-Dawley rats. Blood samples at different time intervals were obtained by severing the tail vein and were analyzed for unchanged PZQ by HPLC. The PB-pretreated rats showed a 6-fold decrease in AUC, a 5-fold decrease in Cmax and an 8-fold increase in CLtot compared to the saline treated controls. The MC-pretreated rats and their olive-oil treated controls did not show any statistically significant differences in the above parameters. These results suggest that PZQ is extensively metabolised by PB-inducible cytochrome P-450 isoforms and not by MC-inducible isoforms. These findings also suggest that the bioavailability of praziquantel could be altered to a significant extent in humans taking drugs that are phenobarbital type inducers.

Intensity of Schistosoma mansoni infection determines alterations in hepatic drug metabolism.

Zoxazolamine paralysis times have been used as a probe to measure the activities of cytochrome P450 1A1 in mice in vivo. The results indicate that male and female mice of the BALB/c and CBA/J strain do not show altered paralysis times if infected with less than 4 worm pairs. Alterations were observed only in animals harbouring more than 5 worm pairs irrespective of the sex or strain of mouse being used. The study has extended the findings of other workers showing that mice infected with fewer than 4-5 worm pairs of S. mansoni do not show any alteration in the metabolism of pentobarbital in vivo.

The effect of chloroquine on the pharmacokinetics and metabolism of praziquantel in rats and in humans.

It is likely that a proportion of people treated with the anti-schistosomicidal drug praziquantel (PZQ) is also taking other drugs such as chloroquine (CHQ), a widely used anti-malarial. The effect of CHQ on the pharmacokinetics and metabolism of PZQ in rats and in humans was therefore studied. CHQ decreased the bioavailability of PZQ and reduced its maximum serum concentrations to a significant extent in rats and in humans. The clearance was increased to a statistically significant extent in rats but not in humans because of the wide interindividual variation. The effect of CHQ on PZQ pharmacokinetics was unexpected since drugs that inhibit hepatic drug metabolism usually increase the bioavailability of PZQ. We found that CHQ inhibits non-competitively the metabolism of PZQ to its major metabolite, 4-hydroxy-praziquantel, with a Ki of 1.65 mM in rat hepatic microsomes. Maximum concentrations attained by CHQ in serum, however, are low compared to the Ki value and significant inhibition is therefore unlikely in vivo. The explanation for CHQ's effect on the pharmacokinetics of PZQ may be due to other effects of CHQ rather than to a direct effect on drug-metabolizing enzymes.

Activities of glutamate dehydrogenase and aspartate and alanine aminotransferases in freshwater snails Helisoma duryi and Lymnaea natalensis exposed to copper.

In this paper we investigate the potential of glutamate dehydrogenase (GDH) and aspartate and alanine aminotransferases (AST and ALT) as biomarkers of water pollution due to copper in the freshwater snails Helisoma duryi and Lymnaea natalensis. Snails were dosed with copper(II) ion concentrations of 0.01, 0.1 and 1 mg kg(-1) breeding water for a period of 96 h, after which those surviving were shelled. The copper content in the breeding water, in whole snail tissue and in the snail shells was determined at the end of the period of exposure. For enzyme determinations, whole snail tissue was first homogenized and fractionated by centrifugation at 500 g to remove the nuclei. The resulting supernatant was then centrifuged at 10,000 g to give a pellet fraction representing the mitochondrial fraction and a supernatant representing the cytosolic fraction. Copper was very toxic to both snail species at concentrations above 0.2 mg l(-1), with only 3% of the Helisoma and 12% of the Lymnaea surviving at concentrations of approximately 1 mg l(-1). The copper content in the shells and tissues of snails rose with increasing copper concentration in the breeding water, and was 2.1- to 4.9-fold in snails exposed to copper ion at a dose of 1 mg kg(-1) water compared with undosed snails. Similarly, the activities of GDH and AST rose by up to 4.7-fold in the homogenate and the mitochondrial and cytosolic fractions with increasing concentrations of copper. These activities, however, fell at copper concentrations of approximately 1 mg l(-1), which coincided with massive death of snails. Mitochondrial ALT disappeared at copper ion concentrations of approximately 0.2 mg l(-1) for Lymnaea and 1 mg l(-1) for Helisoma, possibly indicating mitochondrial degeneration. These results show that GDH, AST and ALT have the potential to be biomarkers of sublethal copper pollution in these two snail species, since their activities were significantly altered by low copper concentrations.