The characterisation and uptake of paraquat in cultured baboon kidney proximal tubule cells (bPTC).

A primary culture of baboon proximal tubule cells (bPTC) was prepared and characterised using LLC-PK1 cells of proximal tubule origin and MDCK cells of distal tubule origin, as positive and negative references, respectively. The proximal tubular origin of the bPTC was determined by morphological studies, immunoperoxidase staining and the expression of proximal tubule markers alkaline phosphatase and gammaglutamyltransferase. The hypothesis that paraquat (PQ) is transported by the bPTC was investigated. The cytotoxic threshold for PQ in these cells was determined and compared to the LLC-PK1 and MDCK cells. Furthermore, this study investigated the transport of the monovalent cation tetraethyl ammonium (TEA) and the polyvalent cation cimetidine in the bPTC and demonstrated their effect on the cellular uptake of PQ. The cytotoxic threshold of PQ in the bPTC, determined by cellular viability studies using the method of Trypan blue exclusion, is 0.05 mM at 2 h incubation. The LC50 after 24 h is 76, 61 and 455 microM for the bPTC, LLC-PK1 and MDCK cells, respectively. This indicates that proximal tubule cells are more susceptible to PQ toxicity compared to distal tubule cells, which is consistent with clinical PQ toxicity where renal damage is found predominantly in the proximal renal tubules. The cations PQ and cimetidine were actively transported by the bPTC. The uptake of PQ (0.05 mM) commenced after 15 min whereas cimetidine (0.5 mM) uptake was evident after 2 min. Furthermore, cimetidine was shown to compete with PQ for uptake in the bPTC. Coincubating PQ (0.05 mM) and cimetidine (0.5 mM) for 60 min resulted in an approximate 50% decrease in PQ uptake. The cation TEA was not transported by the bPTC suggesting either a genetic mutation or complete absence of the transporter for TEA in the cells. The results suggest that PQ may be transported by the same cation transporter as cimetidine and not TEA, indicating PQ uptake in the bPTC to be via a polyvalent organic cation transporter.

Frequency of cytochrome P450 3A4 variant genotype in transplant population and lack of association with cyclosporin clearance.

OBJECTIVE: Cytochrome P450 3A4 (CYP3A4) plays a vital role in the oxidative metabolism of many xenobiotics. Some recent reports have provided circumstantial evidence in support of an association between a genetic polymorphism (A-->G) in the 5'-flanking region (-290) of CYP3A4 and altered enzyme activity. We sought to determine whether genotyping patients for CYP3A4-G could assist with the dose optimisation of drugs metabolised by this system. METHODS: Normal subjects and renal-transplant patients receiving cyclosporin for immune modulation were genotyped for the CYP3A4-G variant. A surrogate for cyclosporin clearance was estimated from the ratio of the cyclosporin dose, normalised for body weight and the corresponding trough concentration. The association between genotype and clearance was examined in patients who received twice-daily doses of cyclosporin and who were not on concurrent medication known to modify CYP3A4 function. RESULTS: The allelic frequencies of the CYP3A4-G variant were estimated to be 2.6% and 3% in transplant patients and normal subjects, respectively. The median cyclosporin pseudo-clearance of transplant patients with wild-type CYP3A4 was 0.90 l/h/kg (range: 0.35-3.8 l/h/kg; n = 86), whereas the corresponding value for the five patients heterozygotic for the CYP3A4-G variant was 0.71 l/h/kg (range 0.35-0.91 l/h/kg). The distribution of the pseudo-clearance according to genotype was not found to be significant according to a Fisher's exact test (P = 0.15). CONCLUSION: Genotyping for the CYP3A4-G polymorphism is unlikely to assist cyclosporin dose selection in transplant patients.

Low-dose nitro-L-arginine administration in baboon (Papio hamadryas) pregnancy.

1. The purpose of the present study was to examine the effect of nitric oxide (NO) inhibition on mean arterial pressure (MAP), endothelin (ET) and the renin-aldosterone system in pregnancy in the non-human primate (baboon). 2. Twenty pregnant baboons (Papio hamadryas) were examined prospectively after the administration of an oral NO inhibitor in different phases of pregnancy. Haemodynamic responses to NO inhibition, evidence of pre-eclampsia and the renin-aldosterone system were examined under anaesthesia. 3. Oral NL-nitro-L-arginine (NOLA; 5 or 10 mg/kg) was given for 1 week in early (6-8 weeks gestation), middle (14-16 weeks gestation) and late (22-24 weeks gestation) pregnancy and while non-pregnant. Mean arterial pressure, heart rate, haematology, biochemistry, ET, plasma renin activity (PRA) and aldosterone were measured. Foetal effects of NOLA were also examined by ultrasound and neonatal measurements. 4. Nitric oxide inhibition led to an increase in MAP in non-pregnant animals (9 mmHg) and in middle and later pregnancy (6 and 7 mmHg, respectively). Mean arterial pressure in early pregnancy was not affected. A reduction in PRA occurred after NO inhibition in all stages of pregnancy. Significant proteinuria occurred only in late pregnancy. 5. Nitric oxide is involved in the maintenance of lower blood pressure in late pregnancy and inhibition leads to an increase in blood pressure and proteinuria in the baboon. Nitric oxide insufficiency may contribute to the clinical manifestations of human pre-eclampsia. Nitric oxide was not involved in the normal vasodilation of early primate pregnancy.

Poisoning by Amanita phalloides ("deathcap") mushrooms in the Australian Capital Territory.

Amanita phalloides ("deathcap") mushrooms are widespread in south-eastern Australia. Seven patients presented to hospital in the Australian Capital Territory with poisoning by this mushroom between 1988 and 1998. Three developed hepatoxicity and one died. Because A. phalloides is becoming more widespread, increased community and medical awareness is needed to reduce the frequency and morbidity of poisoning.

Serotonin syndrome resulting from drug interactions.

We describe six patients diagnosed with serotonin syndrome after exposure to drugs with serotonergic activity. Drug interactions occurred as a result of a combination of tricyclic antidepressants, selective serotonin reuptake inhibitors, selective noradrenaline reuptake inhibitors or monoamine oxidase inhibitors. Management included supportive care and the use of non-specific serotonin antagonists (cyproheptadine, benzodiazepines and chlorpromazine). All patients made uneventful recoveries.

The renal excretory mechanisms and the role of organic cations in modulating the renal handling of paraquat.

Paraquat (PQ), a cationic herbicide, is predominantly excreted by the kidneys, but it is also nephrotoxic. It is thought to cause damage to proximal renal epithelial cells, which results in acute renal failure. The precise mechanism by which PQ is excreted by the kidney has not been fully elucidated, although current evidence indicates that it is actively secreted via a cation transport system. This review examines the renal cytotoxic effect and excretory mechanisms of PQ, and the role of organic cations in modulating the renal handling of PQ.

Lead poisoning from drinking Kombucha tea brewed in a ceramic pot.

Kombucha tea is an alternative therapy that is gaining popularity as a remedy for a diverse range of ailments. We report two cases of symptomatic lead poisoning requiring chelation therapy in a married couple who had been drinking Kombucha tea for six months, brewing the tea in a ceramic pot. We postulate that acids in the tea eluted lead from the glaze pigment used in the ceramic pot, in a manner analogous to elution of lead from crystal decanters by wine and spirits.

The mechanism of excretion of paraquat in rats.

Paraquat (PQ)(1,1'-dimethyl-4,4'-bipyridinium) is a toxic herbicidal cation. The renal excretory mechanisms of PQ and its interactions with organic cations and anions were investigated in anaesthetised rats. The renal clearance of PQ was studied in male Wistar rats using inulin as the marker of glomerular filtration rate. The fractional excretion of paraquat (FEpq) decreased from 2.1+/-0.01 to 1.2+/-0.03 as the plasma concentration rose from 0.4+/-0.02 to 21.2+/-1.6 microM. These results demonstrated that the excretion of PQ was greater than glomerular filtration, concentration dependent and saturable, indicating that it was secreted by an active transport system. The excretion of PQ was dependent predominantly on the glomerular filtration rate with a small secretory component (Km = 8.5+/-3.1 microM, Vmax = 114+/-19 nmol/kg per min). The clearance of PQ was not inhibited by high doses of cimetidine, or p-aminohippurate. However, quinine (P = 0.001) and N-methylnicotinamide (NMN) (P = 0.03) reduced the FEpq, suggesting that they share a similar cation transport system with PQ. In summary, PQ is actively secreted by the rat kidney via a cation transport system.

Survival after a massive hydrofluoric acid ingestion.

BACKGROUND: Hydrofluoric acid ingestion is known to have a very high mortality rate secondary to the rapid development of hypocalcemia and fatal arrhythmias. CASE REPORT: A 33-year-old man ingested an estimated dose of hydrofluoric acid 6 times that considered to be lethal. The patient survived with minimal morbidity despite having multiple ventricular fibrillation arrests. His survival is attributed to early, high dose calcium therapy given via the nasogastric and intravenous routes.

Transport of paraquat by a renal epithelial cell line, MDCK.

Transport of paraquat (PQ), a herbicidal cation, was previously investigated in a proximal (LLC-PK1), renal epithelial cell line using permeable collagen-coated filters. PQ was actively transported from the basolateral side via a cation transport system by the LLC-PK1 cells. In the present study, the transport of PQ was investigated in a distal renal epithelial cell line, MDCK. PQ was predominantly transported from the basolateral to apical (B to A) side. The basolateral transport of PQ in MDCK cells was not saturable with increasing concentrations and not energy dependent. The flux and uptake of PQ was much lower in the MDCK than LLC-PK1 cells. It is concluded that MDCK, a distal renal tubular cell line, does not have an active transport system for PQ.

Characterisation and uptake of paraquat by rat renal proximal tubular cells in primary culture.

1 Uptake of the herbicide paraquat (PQ), by rat proximal tubular cells (PTC) in primary culture grown on a collagen coated support was investigated. 2 The uptake of PQ by PTC was predominantly from the basolateral side. The basolateral uptake of PQ was saturable with time and increasing concentrations, energy dependent and could be inhibited by certain organic cations. Using Michaelis Menten kinetics, the apparent K(m) was 778 +/- 241 microM and Vmax was 0.97 +/- 0.24 pmol/microgram protein/15 min for the basolateral uptake of PQ. Cimetidine (5.7 +/- 0.4 pg/microgram protein/ 30 min, P < 0.001) was the most potent inhibitor of PQ uptake, followed by quinine (6.5 +/- 0.4 pg/microgram protein/30 min, P < 0.01) and then tetraethylammonium (8.2 +/- 0.5 pg/microgram protein/30 min, P < 0.05) when compared with control (11 +/- 1 pg/microgram protein/30 min). N-methylnicotinamide, p-aminohippurate and putrescine did not inhibit the basolateral uptake of PQ. The sodium hydrogen exchange inhibitors, amiloride and its analogue, 5-(N,N hexamethylene) amiloride (HMA) inhibited both the apical and basolateral uptake of PQ. 3 The apical uptake of PQ was not saturable with increasing concentrations and was not inhibited by 2,4-dinitrophenol, but it was reduced by cimetidine (P < 0.01), quinine (P < 0.05) and a sodium potassium ATPase inhibitor, ouabain (P < 0.01). 4 It is concluded that PQ was taken up from the basolateral side of primary cultured rat PTC by an energy dependent transport system.

Transport of paraquat in a renal epithelial cell line LLC-PK1.

Transport of paraquat (PQ), a cationic herbicide, was investigated in a proximal renal epithelial cell line, LLC-PK1. Collagencoated permeable filters were used to study the direction of PQ transport. PQ was transported predominantly from the basolateral to apical (B-->A) membrane of these cells. The B-->A flux and uptake of PQ were saturable with time and increasing concentrations, energy dependent and inhibited by several cations. Quinine was the most potent inhibitor of basolateral PQ uptake, followed by cimetidine and then tetraethylammonium acetate (P < .0001). The noninhibitable basolateral uptake of PQ has an apparent K(m) of 357 microM and a Vmax of 1.47 pmol/micrograms protein/2 min. For flux studies, only quinine inhibited the B-->A flux of PQ (P = .02). Putrescine, p-aminohippurate, probenecid, N-methylnicotinamide and choline did not inhibit the flux or uptake of PQ. 5-N,N-Hexamethylene amiloride, a cationic amiloride analog and a potent inhibitor of the Na/H exchanger, significantly inhibited the uptake of PQ from either side (P < .0001). Acidic pH in the apical medium inhibited the uptake of PQ from either side. The studies demonstrated that PQ was actively transported by the LLC-PK1 cells. PQ shared a similar transport system with several cations, which appeared to have a more significant inhibition on the transcellular uptake than the flux of PQ.

Cyclical changes in the renin-angiotensin-aldosterone system during the menstrual cycle of the baboon (Papio hamadryas).

This study characterizes the renin-angiotensin-aldosterone system during the normal menstrual cycle in the baboon. Ten animals received a daily dose of an ACE inhibitor or placebo in a randomized blind cross-over design. Data were obtained during the mid-follicular and early luteal phases of normal non-pregnant menstrual cycles. All examinations and blood collections were performed with ketamine sedation: 7-kg by im injection. Blood pressure was recorded by sphygmomanometer. Serum ACE activity was measured by spectrophotometry. Aldosterone (ALDO), angiotensin I (AI), and angiotensin II (AII) were measured by radioimmunoassay. Plasma renin activity (PRA) was measured by AI generation. The renin-angiotensin-aldosterone system was found to be activated in the follicular phase and suppressed during the luteal phase of the normal non-pregnant menstrual cycle in the baboon.

Combination analgesic-induced kidney disease: the Australian experience.

Analgesic nephropathy is a unique drug-induced kidney disease characterized pathologically by renal papillary necrosis and chronic interstitial nephritis, and is the result of excessive consumption of combination antipyretic analgesics. The clinical features of the disorder relate mainly to the papillary necrosis, renal colic, and obstructive uropathy and the development of chronic renal failure in a small percentage of patients. There are significant geographic variations in the clinical features that may be related to the differing combinations of analgesics. The pathogenesis of the disease is in part related to the kidneys' ability to concentrate drugs in the papillae. The following sequence of events presents a plausible explanation for the evolution of the disease. If a combination of phenacetin and aspirin is ingested, the following steps occur. Phenacetin is converted in the gut and liver to acetaminophen by first-pass metabolism. Acetaminophen is then taken up by the kidney and excreted. During its excretion, acetaminophen becomes concentrated in the papillae of the kidney during physiologic degrees of antidiuresis, the concentration being up to five times the intracellular concentration of other tissues. Acetaminophen undergoes oxidative metabolism by prostaglandin H synthase to a reactive quinoneimine that is conjugated to glutathione. If acetaminophen is present alone, there is sufficient glutathione generated in the papillae to detoxify the reactive intermediate. If the acetaminophen is ingested with aspirin, the aspirin is converted to salicylate and salicylate becomes highly concentrated in both the cortex and papillae of the kidney. Salicylate is a potent depletor of glutathione. The mechanism is not completely understood; however, the inhibition of the production of NADPH via the pentose shunt is a possible explanation. With the cellular glutathione depleted, the reactive metabolite of acetaminophen then produces lipid peroxides and arylation of tissue proteins, ultimately resulting in necrosis of the papillae.

Stability of cyclosporine in an extemporaneously compounded paste.

The stability of cyclosporine in an extemporaneously compounded paste was studied. Cyclosporine oral solution 100 mg/mL was mixed with an adhesive gel to prepare six aluminum-lined ointment tubes containing paste with cyclosporine 9.6 mg/g. Two of the tubes were stored at 37 degrees C, two at 21 degrees C, and two at 2 degrees C. Cyclosporine was extracted from samples taken on days 2, 4, 5, 7, 9, 14, 18, 22, 28, and 31, and the concentration was measured by high-performance liquid chromatography (except for samples obtained on day 2) and fluorescence polarization immunoassay. Throughout the study period, the concentration of cyclosporine remaining in the paste was > 90% of the initial concentration according to both assay methods. Cyclosporine 9.6 mg/g in a paste compounded extemporaneously from cyclosporine oral solution and an adhesive gel was stable for at least 31 days when stored in aluminum-lined ointment tubes at 37, 21, and 2 degrees C.

Fetotoxicity of angiotensin-converting enzyme inhibition in primate pregnancy: a prospective, placebo-controlled study in baboons (Papio hamadryas).

OBJECTIVES: Serious concerns have been raised about angiotensin-converting enzyme inhibition in pregnancy. The central question remains: does toxicity of angiotensin-converting enzyme inhibition pertain to pregnant humans? STUDY DESIGN: A prospective, placebo-controlled study was performed to investigate the effect of angiotensin-converting enzyme inhibition on pregnancy outcome in the baboon. Subjects (N = 12) received active and placebo treatments sequentially in a crossover protocol. Data were analyzed with two-sample t tests, analysis of variance, Fisher's exact test, or Kaplan-Meier survival analysis, where appropriate. RESULTS: Chronic administration of enalapril (7.5 mg per day) from before conception achieved moderate but sustained angiotensin-converting enzyme inhibition as determined by repeated measures of renin-angiotensin system parameters (serum angiotensin-converting enzyme activity, plasma renin activity and plasma angiotensin I, angiotensin II, and aldosterone concentrations). Serum angiotensin-converting enzyme activity was significantly reduced throughout (< 10 nmol.ml-1.min-1, p < 0.01), with significant increases in plasma renin activity and angiotensin I (p < 0.01). Angiotensin II and aldosterone were maintained unchanged compared with placebo. There was a significant incidence of fetal death or intrauterine growth retardation in fetuses exposed to enalapril (eight of 13, zero on placebo, p < 0.01). When the definition of adverse pregnancy outcome was restricted to fetal death alone (four of 13) the difference remained significant (p < 0.05). Maternal arterial pressure was unchanged before conception, but a small and significant fall (10 to 15 mm Hg, p < 0.01) was detected throughout pregnancy. There was no fetal malformations. CONCLUSION: The study provides definitive evidence for serious consequences of angiotensin-converting enzyme inhibition in pregnancy of high-order primates.

Inhibition of fibroblast proliferation in L-valine reduced selective media.

A selective cell culture medium, D-valine minimal essential medium (92 mg/l), has been developed to inhibit the proliferation of fibroblasts in cell culture (Gilbert & Migeon 1975). Substitution of D-valine for L-valine prevents fibroblast growth due to the absence of D-amino acid oxidase in these cells. Most cell cultures require foetal bovine serum as an essential component of the culture media, however foetal bovine serum contains L-valine, negating the value of D-valine selective media. To overcome this difficulty, we have produced a modified selective media for cell culture, by the dialysis of foetal bovine serum and confirmed its ability to inhibit fibroblast growth whilst still allowing the proliferation of epithelial cells in culture.