Clive J. Ward

Fatal microcystin intoxication in haemodialysis unit in Caruaru, Brazil

BACKGROUND After a drought in February, 1996, all 126 patients in a haemodialysis unit in Caruaru, north-east Brazil, developed signs and symptoms of acute neurotoxicity and subacute hepatotoxicity following the use of water from a lake with massive growth of cyanobacteria (blue-green algae). 60 patients died. METHODS Besides recording clinical details and outcome at follow-up, we arranged laboratory, radiological, and histological investigations on the patients and toxicological studies of serum and haemodialysis water filters. FINDINGS The acute presentation was with malaise, myalgia and weakness, nausea and vomiting, and tender hepatomegaly, with a range of neurological symptoms from tinnitus, vertigo, headaches, and deafness to blindness and convulsions. Liver injury ranged from abnormal liver-function test results to rapidly progressive and fatal hepatic failure. Biochemical investigations revealed gross hyperbilirubinaemia, abnormal liver enzyme activities, and hypertriglyceridaemia, but there was no evidence of haemolysis or microangiopathy. Histology revealed a novel acute toxic hepatitis with diffuse panlobular hepatocyte necrosis, neutrophil infiltration, canalicular cholestasis, and regenerative multinucleate hepatocytes. Samples of serum, dialysis filters, and water-treatment columns contained microcystins, the highly toxic low-molecular-weight hepatotoxins produced by cyanobacteria. INTERPRETATION Cyanobacteria present water-borne hazards to health via drinking water and recreational water. Haemodialysis presents an additional high-risk exposure route: when they enter directly into the circulation, microcystins can lead to fatal clinical syndromes ranging from acute neurotoxic illness to subacute liver failure.

Cyanobacterial toxins, exposure routes and human health

The production of potent toxins by bloom-, scum- and mat-forming cyanobacteria, in fresh-, brackish and marine waters, appears to be a global phenomenon. Cyanobacterial toxins can also be produced by cyanobacteria from terrestrial sources. The range and number of known cyanobacterial toxins are increasing apace as associated poisoning incidents are investigated, and increasingly powerful analytical methods are applied to complement toxicity-based studies on both natural samples and laboratory isolates of cyanobacteria. Water quality management to reduce toxic cyanobacterial mass developments, and schemes to mitigate the potential effects of cyanobacterial toxins, require an understanding of the occurrence and properties of the toxins and of the exposure routes via which the toxins present risks to health. Here, we review advances in the recognition of cyanobacterial toxins and their toxicity, and of the exposure routes with reference to human health, namely via skin contact, inhalation, haemodialysis and ...

Cyanobacterial toxins: occurrence, modes of action, health effects and exposure routes.

Cyanobacterial toxins are produced by terrestrial- fresh-, brackish- and sea-water cyanobacteria of cosmopolitan occurrence. These toxins present acute and chronic hazards to human and animal health and are responsible for isolated, sporadic animal fatalities (mammals, fish, birds) each year. Human health problems are associated with the ingestion of, and contact with cyanobacterial blooms and their toxins. Modes of action of cyanobacterial neurotoxins, hepatotoxins and skin irritants are considered. Recent indications of the accumulation of cyanobacterial toxins in fish, their effect on crop plants and their association with the deaths of human dialysis patients are discussed. These findings and events indicate an incomplete understanding of the exposure routes of these natural toxins and the need for greater awareness of their occurrence and properties among users of waterbodies which are prone to cyanobacterial bloom development.

Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active site residues in quinidine binding

We have previously shown that Phe120, Glu216, and Asp301 in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021–4027 and Flanagan, J. U., Maréchal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353–360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (Kd) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25–0.50 μm. The amide substitution of Glu216 or Asp301 resulted in 30–64-fold increases in the Kd for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a Kd of 65 μm. Alanine substitution of Phe120, Phe481,or Phe483 had only a minor effect on the inhibition of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations.

INFECTIVE ENDOCARDITIS AS PART OF PSITTACOSIS

Abstract Two fatal cases of endocarditis due to the psittacosis agent ( Bedsonia or Chlamydia psittaci ) are described. When a case of infective endocarditis gives repeatedly negative blood cultures, and especially when there are both pneumonitis and vague cerebral signs, a history of contact with birds should be sought and serological tests done for psittacosis. A high initial titre probably justifies the immediate administration of chlortetracycline. In the fatal case, prominent finger-like vegetations on heart valves should suggest to the pathologist the possibility of bedsonial infection; successful microscopic diagnosis is more likely to be made on imprints from the vegetations than on histological sections. Tissues should be sent for culture to the virologist.

IN SILICO PREDICTION OF DRUG BINDING TO CYP2D6: IDENTIFICATION OF A NEW METABOLITE OF METOCLOPRAMIDE

Patients with cancer often take many different classes of drugs to treat the effects of their malignancy and the side effects of treatment, as well as their comorbidities. The potential for drug-drug interactions that may affect the efficacy of anticancer treatment is high, and a major source of such interactions is competition for the drug-metabolizing enzymes, cytochromes P450 (P450s). We have examined a series of 20 drugs commonly prescribed to cancer patients to look for potential interactions via CYP2D6. We used a homology model of CYP2D6, together with molecular docking techniques, to perform an in silico screen for binding to CYP2D6. Experimental IC50 values were determined for these compounds and compared with the model predictions to reveal a correlation with a regression coefficient of r2 = 0.61. Importantly, the docked conformation of the commonly prescribed antiemetic metoclopramide predicted a new site of metabolism that was further investigated through in vitro analysis with recombinant CYP2D6. An aromatic N-hydroxy metabolite of metoclopramide, consistent with predictions from our modeling studies, was identified by high-performance liquid chromatography/mass spectrometry. This metabolite was found to represent a major product of metabolism in human liver microsomes, and CYP2D6 was identified as the main P450 isoform responsible for catalyzing its formation. In view of the prevalence of interindividual variation in the CYP2D6 genotype and phenotype, we suggest that those experiencing adverse reactions with metoclopramide, e.g., extrapyramidal syndrome, are likely to have a particular CYP2D6 genotype/phenotype. This warrants further investigation.

WIDENING PERCEPTIONS OF THE OCCURRENCE AND SIGNIFICANCE OF CYANOBACTERIAL TOXINS

Cyanobacterial toxins (CTX) are associated annually with episodes of animal-, bird-or fish-poisonings and, almost annually, with examples of human illness, somewhere or other, in the world. However, recognition of the occurrence and impact of CTX has been, and remains, limited among various relevant groups. These include biologists and limnologists, environmental- and health-care professionals and in the water industry. Constraints have included: inadequate investigation and reporting protocols for suspected cyanobacterial intoxications; a lack of adequate and widely available CTX analysis methods; and a lack of awareness among non-professional water-users of the hazards of cyanobacterial blooms and of signs of cyanobacterial poisoning1,2. These shortcomings have been partly addressed by education and information programmes, the introduction of sensitive and quantitative toxin analysis methods and by the performance of state and national CTX surveys. Understanding of the health hazards presented by CTX is gradually being advanced from several sources. These include: fundamental studies of the toxicology of purified CTX, ranging from molecular in vitro studies to toxicity trials with animals3,4 and investigations, albeit limited, of animal poisonings and human health problems associated with—or attributed to—CTX5-8. Data on the toxicity of cyanobacterial microcystin hepatotoxins have been used this year in risk assessments for the derivation of a guideline value for microcystin-LR levels in drinking water. Examples of the effects of CTX on human health continue to emerge, a recent example being the deaths of haemodialysis patients exposed to microcystin hepatotoxins in Caruaru, Brazil in 19969-11.

In silico prediction of drug binding to CYP2D6: identification of a new metabolite of metoclopramide

Patients with cancer often take many different classes of drugs to treat the effects of their malignancy and the side effects of treatment, as well as their comorbidities. The potential for drug-drug interactions that may affect the efficacy of anticancer treatment is high, and a major source of such interactions is competition for the drug-metabolizing enzymes, cytochromes P450 (P450s). We have examined a series of 20 drugs commonly prescribed to cancer patients to look for potential interactions via CYP2D6. We used a homology model of CYP2D6, together with molecular docking techniques, to perform an in silico screen for binding to CYP2D6. Experimental IC50 values were determined for these compounds and compared with the model predictions to reveal a correlation with a regression coefficient of r2 = 0.61. Importantly, the docked conformation of the commonly prescribed antiemetic metoclopramide predicted a new site of metabolism that was further investigated through in vitro analysis with recombinant CYP2D6. An aromatic N-hydroxy metabolite of metoclopramide, consistent with predictions from our modeling studies, was identified by high-performance liquid chromatography/mass spectrometry. This metabolite was found to represent a major product of metabolism in human liver microsomes, and CYP2D6 was identified as the main P450 isoform responsible for catalyzing its formation. In view of the prevalence of interindividual variation in the CYP2D6 genotype and phenotype, we suggest that those experiencing adverse reactions with metoclopramide, e.g., extrapyramidal syndrome, are likely to have a particular CYP2D6 genotype/phenotype. This warrants further investigation.

In Silico Prediction of Drug Binding to Cytochrome P450 2D6: Identification of a new Metabolite of Metoclopramide

Patients with cancer often take many different classes of drugs to treat the effects of their malignancy and the side effects of treatment, as well as their comorbidities. The potential for drug-drug interactions that may affect the efficacy of anticancer treatment is high, and a major source of such interactions is competition for the drug-metabolizing enzymes, cytochromes P450 (P450s). We have examined a series of 20 drugs commonly prescribed to cancer patients to look for potential interactions via CYP2D6. We used a homology model of CYP2D6, together with molecular docking techniques, to perform an in silico screen for binding to CYP2D6. Experimental IC50 values were determined for these compounds and compared with the model predictions to reveal a correlation with a regression coefficient of r2 = 0.61. Importantly, the docked conformation of the commonly prescribed antiemetic metoclopramide predicted a new site of metabolism that was further investigated through in vitro analysis with recombinant CYP2D6. An aromatic N-hydroxy metabolite of metoclopramide, consistent with predictions from our modeling studies, was identified by high-performance liquid chromatography/mass spectrometry. This metabolite was found to represent a major product of metabolism in human liver microsomes, and CYP2D6 was identified as the main P450 isoform responsible for catalyzing its formation. In view of the prevalence of interindividual variation in the CYP2D6 genotype and phenotype, we suggest that those experiencing adverse reactions with metoclopramide, e.g., extrapyramidal syndrome, are likely to have a particular CYP2D6 genotype/phenotype. This warrants further investigation.