Assessment of protein phosphatase in a re-usable rapid assay format in detecting microcystins and okadaic acid as a precursor to biosensor development.

The feasibility of developing an immobilised protein phosphatase (PP) biosensor was tested by immobilising PP onto CNBr-activated Sepharose beads placed in Millipore microfilter plate wells. Under optimised immobilised enzyme assay conditions, okadaic acid (OA) and microcystin LR (MC-LR) inhibited Upstate Biotechnology PP (PP-2A), with IC50 values of 12.5 and 11nM respectively. Similarly, immobilised recombinant PP type 1 (rec PP-1) was inhibited by MC-LR and OA, with IC50 values of 150 and >1000nM respectively. The IC50 values for free PP-2A against OA and MC-LR were 2.5 and 3.5nM, and 0.7nM and 200nM for rec PP-1 against the same substrates respectively. For free and immobilised Neptunea arthritic PP (PP-2Ana) against OA the IC50 values were 0.45 and >1000nM respectively. Of the three immobilised enzyme systems, PP-2A showed greatest sensitivity to OA and MC-LR followed by rec PP-1 and PP-2Ana. In assessments for re-usability (determined by removal of > or =70% OA or MC-LR inhibition of PP-2A by washing), <50% of the original activity remained after 20 washings. Including 1M NaCl in the wash buffer did not increase enzyme activity with wash frequency, but rather "salted in" the inhibitor. The LoD of immobilised PP-2A to MC-LR meets the WHO guideline of 1microgl(-1) for drinking water, and the sensitivity to OA (3.5microgl(-1)) would allow detection of DSP during the peak of some phytoplankton blooms.

Sublethal responses of wolf spiders (Lycosidae) to organophosphorous insecticides.

The activities of cholinesterase (ChE) and glutathione S-transferase (GST) enzymes were assessed in the wolf spider (Lycosa hilaris) as biomarkers of organophosphate contamination in agricultural ecosystems. Spiders were exposed to simulated field rates of two commercially available organophosphorous insecticides [Basudin (diazinon) and Lorsban (chlorpyrifos)] under laboratory conditions. In terms of survival, chlorpyrifos and diazinon were more toxic to male than to female wolf spiders, but gender-specific differences in ChE activities were not evident. Cholinesterase activity in male spiders was inhibited to 14% and 61% of control activity by Basudin and Lorsban, respectively. Gluthathione S-transferase activity was not affected by either pesticide. Mortality and biomarker responses in the wolf spider were further investigated following the application of Basudin to pasture. Wolf spiders were deployed into field mesocosms; after 24 h mortality was 40%, and surviving spiders displayed significant inhibition of ChE activity (87%) compared with controls. Cholinesterase activity in spiders exposed for subsequent 24- or 48-h time periods was monitored until it returned to control levels 8 days post-application. Inhibition of ChE activity after a single application of Basudin indicate the potential use of this enzyme in wolf spiders as a biomarker for evaluating organophosphate contamination.

In vivo evidence for accelerated generation of hydroxyl radicals in liver of Long-Evans Cinnamon (LEC) rats with acute hepatitis.

The Long-Evans Cinnamon (LEC) rats accumulate excess copper (Cu) in the liver in a manner similar to patients with Wilson's disease (WD) and spontaneously develop acute hepatitis with severe jaundice. Although hydroxyl radicals (*OH) have been proposed to be a cause of hepatitis by the accumulation of Cu, it is not clear whether or not *OH can be produced in the liver of hepatitic LEC rats in vivo and also can be involved in the onset of hepatitis. In the present study, *OH production in plasma and liver of hepatitic LEC rats was quantified by trapping *OH with salicylic acid (SA) as 2, 3-dihydroxybenzoic acid (2, 3-DHBA). The ratios of 2, 3-DHBA/SA were significantly higher in plasma and liver of hepatitic LEC rats than those of Wistar rats and LEC rats showing no signs of hepatitis. Furthermore, the ratios of 2, 3-DHBA/SA in plasma and liver of hepatitic LEC rats were almost the same as those of Wistar rats treated orally with CuSO(4) (0.5 mmol/kg) 2 h before acetylsalicylic acid (ASA) injection. We also evaluated the protective effects of D-mannitol (a *OH scavenger) treatment against acute hepatitis in LEC rats. D-mannitol (500 mg/kg) was administered intraperitoneally to 10-week-old LEC rats for 3 weeks. D-mannitol treatment suppressed the increases in serum aspartate aminotransferase activity and total bilirubin concentration. In addition, D-mannitol treatment significantly reduced hepatic mitochondrial lipid peroxidation, which is thought to be important in the pathogenesis of Cu-induced hepatotoxicity. These observations suggest that accelerated generation of *OH catalyzed by free Cu in the liver may, at least in part, play a role in the pathogenesis of acute hepatitis in LEC rats.

Pharmacokinetics of antipyrine, warfarin and paracetamol in the brushtail possum.

The plasma pharmacokinetics of antipyrine, warfarin and paracetamol have been studied in the Australian brushtail possum (Trichosurus vulpecula). The plasma elimination half-lives (t1/2) were 1.2 h for antipyrine, 11.9 h for warfarin and 5.2-12.9 h for paracetamol. Our data indicate that the clearance of these three xenobiotics in the possum is similar to that reported in eutherian mammals. There was no dose-dependent increase in paracetamol plasma t1/2 over the dose range 100-1000 mg kg(-1), indicating a lack of capacity saturation. This observation may in part explain the unusual resistance of the possum to the hepatotoxic effect of high doses of paracetamol.

Inhibition of cytochrome P450 enzymes in the Australian brushtail possum, Trichosurus vulpecula: comparison with that of the rat, rabbit, sheep and chicken.

A comparative study was conducted of the inhibition of liver microsomal cytochrome P450 phase I biotransformation enzyme activity of the Australian brushtail possum, rat, rabbit, sheep and chicken. The possum has caused considerable agricultural and ecological problems since its introduction to New Zealand. This work investigated species differences in cytochrome P450 inhibition by selected imidazole derivatives that may be exploited for designing a more species-specific method of toxicological control of the New Zealand possum population. The imidazole derivatives used were ketoconazole, clotrimazole, miconazole and cimetidine. The potency of these inhibitors varied, with clotrimazole and miconazole being most potent, followed by ketoconazole. Cimetidine was the least effective inhibitor. The inhibitory effect of imidazole derivatives on cytochrome p450 phase I biotransformation enzymes appeared more effective in the possum than in other species. All inhibitors used produced type II spectra upon interaction with cytochrome P450 preparations. Possum and chicken microsomal preparations showed absorbancy maxima at 428 nm, rabbit and rat and 429 nm, and sheep at 431 nm.

Cytochrome P450 enzyme activities in the Australian brushtail possum trichosurus vulpesula: a comparison with the rat, rabbit, sheep and chicken.

A comparative study of cytochrome P450 phase 1 biotransformation enzyme activities of the Australian brushtail possum was conducted. Since its introduction to New Zealand this animal pest has caused considerable agricultural and ecological problems. The objective of this work was to probe for potential biochemical weaknesses that may be exploited for designing a more species-specific method of toxicological control of the possum population in New Zealand. Liver microsomal enzyme content and the kinetics of in vitro biotransformation reactions in the possum were quantified and compared with those of the rabbit, rat, chicken and sheep. Significant (p > 0.05) species variations in the liver cytochrome P450 enzyme content were observed. All mammals, including the possum, had considerably higher cytochrome P450, cytochrome b5 and cytochrome reductase levels than the chicken. The chicken had the highest specific phase 1 biotransformation activity (Vmax) for all except 1 of the 8 xenobiotics tested. Among the mammals studied, P450 (Vmax) values in the possum were similar or higher than in the rabbit, rat or sheep. The Km values varied significantly (p > 0.05) between species. The Km for 7-hydroxylation of coumarin was highest in the rat. In comparison with other species, the possum had a lower Km for aromatic hydroxylation of aniline, N-demethylation of N,N-dimethyl aniline, and N-dimethyl aniline, but a higher Km for 0-demethylation of 7-methoxycoumarin, 7-hydroxylation of coumarin, and 0-deethylation of acetophenetidin. It was concluded that phase 1 biotransformation of xenobiotics in the possum is similar to or more efficient than in the other animals tested.

Outcome of stroke in patients undergoing hemodialysis.

BACKGROUND: While elevated levels of serum creatinine have been shown to be a risk factor for diminished survival after stroke, it is unknown how renal replacement therapy may affect the outcome. METHODS: Strokes occurring in 26 consecutive patients undergoing hemodialysis at our institution were reviewed and clinical and laboratory variables and outcome were compared with those of patients who had a stroke but had normal renal function. RESULTS: Twenty-four strokes in the patients undergoing hemodialysis were ischemic while only 2 were hemorrhagic. Virtually all the patients had hypertension, half had diabetes mellitus, and most had some prior evidence of cardiovascular disease at the time of their stroke. Fifty percent of the patients undergoing hemodialysis had a good outcome (defined as being discharged home) while the remainder had a poor outcome (defined as dying or being discharged to a nursing facility). The combined presence of hypertension and coronary artery disease had a sensitivity of 91.2% for identifying patients with a poor outcome, while male sex, the presence of coronary artery disease, and the combined presence of hypertension, coronary artery disease, and/or congestive heart failure had sensitivities greater than 80% but low specificity. The outcome of patients undergoing hemodialysis was comparable with that of a control group of patients who had a stroke but had normal renal function, although the length of hospital stay was greater (mean [+/-SEM] 29.8+/-6.4 days vs 12.7+/-1.1 days, respectively; P<.01). CONCLUSIONS: Hospitalized patients undergoing hemodialysis in whom stroke occurs appear to have as good an outcome as that of patients with normal renal function, although they are hospitalized longer. In addition, certain clinical variables seem to be associated with a worse outcome. Aggressive measures to prevent and treat stroke seem as warranted for patients undergoing hemodialysis as for patients with normal renal function, although interventions to reduce the length of hospital stay are needed.

An ovine model of acute myocardial infarction and chronic left ventricular dysfunction.

In order to develop and validate an ovine model of myocardial infarction with subsequent impairement of left ventricular function, 15 instrumented sheep underwent selective microembolization of the left coronary arteries with 0.5 mL 90 microns polystyrene beads. Hemodynamics and plasma hormones were measured preembolization (baseline) and then at hours 2, 4, 6, and 12 and days 1, 2, 3, 5 and 7 postembolization. Of the 15 sheep studied, 2 (13%) died on the day of embolization from arrhythmias. In the remaining sheep, left ventricular systolic pressure and stroke work (both P < 0.001) were reduced promptly and remained below basal levels. Mean arterial pressure (P < 0.001) increased initially, then decreased to below basal levels by hour 6. Heart rate (P < 0.001) and left atrial pressure (P < 0.05) were increased while cardiac output was decreased (P < 0.05). Left ventricular ejection fraction at day 7 was reduced (38.8 +/- 3.5 vs 46.0 +/- 3.9% preembolization; P < 0.05). The cardiac enzymes creatine kinase (P < 0.001) and troponin-T (P < 0.001) were increased following microembolization and returned to basal levels by days 2 and 5 respectively. Plasma atrial and brain natriuretic peptides (both P < 0.001) and plasma renin activity (P < 0.005) were all increased following embolization. This ovine model mimics the hemodynamic and neurohumoral features of acute myocardial infarction, resulting in left ventricular dysfunction, and should prove suitable for the study of interventions in a number of these conditions.

Risk of fatal cerebrovascular accident in patients on peritoneal dialysis versus hemodialysis.

Several reports have suggested that the incidence of hemorrhagic stroke may be greater on hemodialysis as compared with that among the general population and that patients with intracranial hemorrhage should be treated with peritoneal dialysis rather than hemodialysis. However, whether the risk of fatal stroke is greater on hemodialysis versus peritoneal dialysis has not been systematically examined. In this study, the case of a diabetic patient with extensive peripheral vascular disease who, after 7 years on hemodialysis, was changed to peritoneal dialysis and subsequently suffered two strokes over a 5-month period, is reported. Recent data from the United States Renal Data System, which allow a comparison of death rates from stroke in large numbers of hemodialysis versus peritoneal dialysis patients, are reviewed. These data suggest that the risk of death from stroke may actually be greater for patients on peritoneal dialysis versus hemodialysis in spite of their having a lesser prevalence of preexisting cerebrovascular disease. This risk was greatest for elderly diabetic black patients and women, who experienced a nearly twofold-greater odds favoring death from stroke on peritoneal dialysis versus hemodialysis. Selection of a dialysis modality for a patient beginning renal replacement therapy may require the consideration of such data, particularly in those patients with extensive preexisting vascular disease.

AIDS-associated membranous nephropathy with advanced renal failure: response to prednisone.

We report the case of a patient with acquired immunodeficiency syndrome (AIDS) who developed nephrotic syndrome and progressive renal failure mimicking human immunodeficiency virus (HIV)-associated focal segmental glomerulosclerosis (FSGS) who required initiation of hemodialysis and was found on renal biopsy to have membranous nephropathy. Hepatitis B and C serologies were negative. Although she required hemodialysis, she was treated with prednisone and experienced a progressive decline in her serum creatinine from 10.1 mg/dL to 1.9 mg/dL, which permitted the discontinuation of hemodialysis. After she abruptly discontinued prednisone, her creatinine level increased to 4.8 mg/dL, and she experienced marked worsening of her nephrotic syndrome. Resumption of prednisone resulted in normalization of serum creatinine and reduction in urine protein excretion. No adverse effects of prednisone occurred during this time. She remains off of hemodialysis for 1 year with a serum creatinine level of 1.0 mg/dL and urine protein excretion of 0.4 g/d. Although most patients with HIV infection, nephrotic-range proteinuria, and renal failure have FSGS, a minority may have membranous nephropathy. Although typically not a steroid-responsive lesion in the setting of advanced renal failure, membranous nephropathy may be a highly steroid-responsive lesion in the HIV-infected patient, and treatment may help avert the need for dialysis in a patient population that generally has a poor outcome on dialysis.

Beta-naphthoflavone induced CYP1A1 and 1A3 proteins in the liver of rainbow trout (Oncorhynchus mykiss).

1. Two CYP1A proteins, designated HAP 1 and HAP 2, were isolated from the liver of the beta-naphthoflavone (BNF)-treated rainbow trout. The proteins were initially resolved by chromatography on a DEAE sepharose column and were further purified by hydroxyl-apatite chromatography. 2. Both HAP 1 and HAP 2 proteins exhibited high 7-ethoxyresorufin, methoxy-resorufin and phenacetin O-dealkylase activities and were good catalysts for the oxidation of 7,12-dimethylbenz[a]anthracene (DMBA). No qualitative difference was observed between the two proteins in their ability to catalyse the formation of the individual metabolites of DMBA. 3. The two purified proteins showed identical amino acid sequence for the first 13 amino acids. However, the 14th amino acid was valine for HAP 1 protein and alanine for HAP 2 protein. 4. Alignment of the amino acid sequences showed that HAP 1 protein was identical to the deduced protein of the previously reported trout CYP1A2 (renamed CYP1A1) gene for the first 24 amino acids at the N-terminal region. HAP 2 protein corresponded to the deduced protein sequence of CYP1A3 gene for the first 14 amino acids. However, unlike the deduced sequences of CYP1A1 and 1A3 the N-terminal methionine was absent in the purified proteins. 5. We conclude that HAP 1 and HAP 2 are the products of the CYP1A1 and CYP1A3 genes respectively, and are found in the liver of the BNF-treated rainbow trout.

Persistence of sodium monofluoroacetate in livestock animals and risk to humans.

1. Sodium monofluoroacetate (1080), a vertebrate pesticide widely used in New Zealand, was administered orally to sheep and goats at a dose level of 0.1 mg kg-1 body weight to assess risk to humans of secondary poisoning from meat. Blood, muscle, liver, and kidney were analysed for 1080 residues. 2. The plasma elimination half-life was 10.8 h in sheep and 5.4 h in goats. Concentrations of 1080 in muscle (0.042 microgram g-1), kidney (0.057 microgram g-1), and liver (0.021 microgram g-1) were substantially lower than those in plasma (0.098 microgram ml-1) at 2.5 h after dosing. 3. Only traces of 1080 (< 0.002 to 0.008 microgram g-1) were found in sheep tissues after 96 hours. 4. Livestock are normally excluded from areas where 1080 is being used for pest control, reducing the risk of secondary poisoning. Even with accidental exposure to a sublethal dose 1080 would not persist in tissues for more than a few days because it is cleared rapidly from the body. Therefore the occurrence of 1080 in meat intended for human consumption is highly unlikely.

Effect of 67Cu and 99Mo labeled tetrathiomolybdate on the distribution of 67Cu, Cu, and 99Mo in bile fractions in sheep.

The effect of intravenous administration of 67Cu and 99Mo labeled tetrathiomolybdate (TTM) on the appearance of 67Cu, stable Cu, and 99Mo in gel chromatographic fractions of bile was examined in sheep fed either 5 or 35 mg Cu kg-1 DM. Peak excretory periods of biliary 67Cu, stable Cu, and 99Mo were observed at 30 min-1.25 hr, 2-3 hr, and 11-13 hr after 67Cu and after 99Mo labeled TTM. Sephadex G-75 gel filtration of bile samples collected at 1, 3, and 12 hr after 67Cu administration revealed two major protein peaks of molecular weights of greater than 80,000 (peak I) and 7,000 (peak II) containing both 67Cu and Cu. But the ratio of 67Cu in the two peaks varied with time of bile collection. The ratio of areas of peak I:II 1 hr after 67Cu administration was approximately 0.48; at 3 hr, 0.62, and at 12 hr 1.35. Tetrathiomolybdate administration increased both 67Cu and stable Cu in bile by severalfold and induced a major shift of Cu into the higher molecular weight protein fraction. The experiments confirm the effectiveness of TTM as a "decoppering" agent. Furthermore, TTM not only promoted bile Cu excretion, but it also increased the incorporation of Cu into the macromolecular fraction. This may limit enterohepatic circulation of biliary Cu and thereby cause an overall Cu depletion and a negative Cu balance.