Fate of ptaquiloside-A bracken fern toxin-In cattle.

Ptaquiloside is a natural toxin present in bracken ferns (Pteridium sp.). Cattle ingesting bracken may develop bladder tumours and excrete genotoxins in meat and milk. However, the fate of ptaquiloside in cattle and the link between ptaquiloside and cattle carcinogenesis is unresolved. Here, we present the toxicokinetic profile of ptaquiloside in plasma and urine after intravenous administration of ptaquiloside and after oral administration of bracken. Administered intravenously ptaquiloside, revealed a volume of distribution of 1.3 L kg-1 with a mean residence-time of 4 hours. A large fraction of ptaquiloside was converted to non-toxic pterosin B in the blood stream. Both ptaquiloside and pterosin B were excreted in urine (up to 41% of the dose). Oral administration of ptaquiloside via bracken extract or dried ferns did not result in observations of ptaquiloside in body fluids, indicating deglycosolidation in the rumen. Pterosin B was detected in both plasma and urine after oral administration. Hence, transport of carcinogenic ptaquiloside metabolites over the rumen membrane is indicated. Pterosin B recovered from urine counted for 7% of the dose given intravenously. Heifers exposed to bracken for 7 days (2 mg ptaquiloside kg-1) developed preneoplastic lesions in the urinary bladder most likely caused by genotoxic ptaquiloside metabolites.

Intrabronchial Microdialysis: Effects of Probe Localization on Tissue Trauma and Drug Penetration into the Pulmonary Epithelial Lining Fluid.

Recent intrabronchial microdialysis data indicate that the respiratory epithelium is highly permeable to drugs. Of concern is whether intrabronchial microdialysis disrupts the integrity of the respiratory epithelium and thereby alters drug penetration into the pulmonary epithelial lining fluid (PELF). The objective of this study was to investigate the effect of intrabronchial microdialysis on the integrity of the bronchial epithelium. Microdialysis sampling in PELF in proximal (n = 4) and distal bronchi (n = 4) was performed after intravenous inulin and florfenicol administration in anaesthetized pigs. Inulin was used as a marker molecule of permeability of the epithelium, and florfenicol was used as test drug. Bronchial tissue was examined by histopathology (distal and proximal bronchi) and gene expression analysis (RT-qPCR, proximal bronchi) at the termination of the experiment (6.5 hr). The microdialysis probe caused overt tissue trauma in distal bronchi, whereas no histopathological lesions were observed in proximal bronchi. A moderate up-regulation of the pro-inflammatory cytokines IL1B, IL6 and acute-phase reactant serum amyloid A was seen in proximal bronchi surrounding the microdialysis probes suggesting initiation of an inflammatory response. The observed up-regulation is considered to have limited impact on drug penetration during short-term studies. Inulin penetrated the respiratory epithelium in both proximal and distal bronchi without any correlation to histopathological lesions. Likewise, florfenicol penetration into PELF was unaffected by bronchial histopathology. However, this independency of pathology on drug penetration may not be valid for other antibiotics. We conclude that short-term microdialysis drug quantification can be performed in proximal bronchi without disruption of tissue integrity.

Is bupropion a more specific substrate for porcine CYP2E than chlorzoxazone and p-nitrophenol?

Porcine microsomes are able to hydroxylate chlorzoxazone and p-nitrophenol, the most commonly used human test substrates for CYP2E1. However, in pigs, CYP2E appears not to be the only enzyme involved in the hydroxylation of chlorzoxazone and p-nitrophenol, as the enzyme capacity and immunochemical level of the apoprotein do not correlate. The present study shows that the hydroxylation of chlorzoxazone and p-nitrophenol is inhibited 50-65% by anti-human CYP2A6, suggesting that these substrates are metabolized almost equally well by CYP2A and CYP2E in pigs. To find an alternative probe to porcine CYP2E, bupropion, another human substrate, was examined. Incubation with bupropion concentrations ranging from 0.05 to 20 mM and with various inhibitors revealed that this substrate is metabolized by both CYP2A and CYP2E. At the high substrate concentration (5 mM), however, the CYP2A6 inhibition decreased compared to inhibition percentages found using the low substrate concentration (0.5 mM). The opposite was found for CYP2E, as inhibition studies with antibodies and diethyldithiocarbamate indicate that it catalysed a negligible part of the reaction at the low substrate concentration and up to 84% at the high concentration. Thus, hydroxylation of bupropion follows the same pattern in pigs as in human beings and the activity measured in pigs is comparable with the human counterpart. Furthermore, bupropion is a more specific substrate for CYP2E than chlorzoxazone and p-nitrophenol although not perfect.

Porcine CYP2A polymorphisms and activity.

CYP2A6 in man catalyzes the oxidation of nicotine-forming cotinine and 7-hydroxylation of coumarin, which is used as test substrate for CYP2A6 in man. Large interindividual differences are found in man and some are due to genetic polymorphism. The 7-hydroxylation of coumarin is present in pigs, and an inter-individual variation has been found that might be due to polymorphisms. To enable the finding of polymorphism in pigs, the minipig cDNA was sequenced. Two cDNAs were found and translated to a 494 and a 487 amino acid long protein, both cDNAs were found in all but one pig. The 494 a.a. protein showed high homology to the human and 100% homology to the conventional pig CYP2A19 protein. In the wild type protein, all 6 substrate recognition sites were found, whereas the short protein only contained the first 5 substrate recognition sites. SSCP analysis revealed 3 polymorphisms. In order to study the effect of these polymorphisms on enzyme activity, microsomes were incubated with nicotine and coumarin. The polymorphisms appeared to have no effect on either enzyme activity as the specific enzyme activity towards nicotine and coumarin were approximately the same for all pigs. The specificity of pig CYP2A was investigated and it was found that the formation of cotinine correlated with the immunochemical level of CYP2A as did the coumarin hydroxylation. Anti-human CYP2A inhibitory antibody inhibited coumarin 7-hydroxylation by about 90% and formation of cotinine by 44--60% and 85--100% at substrate concentrations of 500 microM and 50 microM respectively, showing that coumarin and nicotine (50 microM) are very specific substrates for CYP2A in pigs, whereas the CYP2A only is responsible for about 50% of the cotinine formation at a 500 microM nicotine incubation concentration. These results show that the large interindividual differences in porcine CYP2A activity are not caused by polymorphisms but transcriptional regulation and the coumarin 7-hydroxylation is as specific a reaction for porcine CYP2A as for human CYP2A6.