Bat population genetics and Lyssavirus presence in Great Britain.

Most lyssaviruses appear to have bat species as reservoir hosts. In Europe, of around 800 reported cases in bats, most were of European bat lyssavirus type 1 (EBLV-1) in Eptesicus serotinus (where the bat species was identified). About 20 cases of EBLV-2 were recorded, and these were in Myotis daubentonii and M. dasycneme. Through a passive surveillance scheme, Britain reports about one case a year of EBLV-2, but no cases of the more prevalent EBLV-1. An analysis of E. serotinus and M. daubentonii bat genetics in Britain reveals more structure in the former population than in the latter. Here we briefly review these differences, ask if this correlates with dispersal and movement patterns and use the results to suggest an hypothesis that EBLV-2 is more common than EBLV-1 in the UK, as genetic data suggest greater movement and regular immigration from Europe of M. daubentonii. We further suggest that this genetic approach is useful to anticipate the spread of exotic diseases in bats in any region of the world.

A comparison of warfarin resistance and liver microsomal vitamin K epoxide reductase activity in rats.

Vitamin K-1 epoxide reductase activity was investigated in liver microsomal preparations from warfarin-resistant and -susceptible rats. One rat strain (TAS) is susceptible to the anticoagulant and lethal effects of warfarin and the other two strains are homozygous for warfarin resistance genes from either wild Welsh (HW) or Scottish (HS) rats. The enzyme in microsomal preparations from HW rat livers apparently has a reduced affinity for both warfarin and vitamin K-1 2,3-epoxide. The kinetic parameters for the enzyme activity in HS microsomal preparations indicated, however, that vitamin K-1 epoxide reductase in this warfarin-resistant strain was very similar, in respect of substrate and inhibitor affinities, to that prepared from susceptible (TAS) animals. Analysis of vitamin K-1 epoxide reductase activity in the livers of animals that had been orally treated with sodium warfarin (20 mg/kg body wt.) indicated that enzyme activity was inhibited in all three strains, although this dose is lethal only to animals of the TAS strain.

Tumour-initiating activities of dihydrodiols of dibenz[a,c]anthracene.

The tumor-initiating activities of dibenz[a,c]anthracene (DBA) and of the related trans-1,2-, 3,4- and 10,11-dihydrodiols have been examined on mouse skin subsequently promoted with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The 1,2- and 10,11-dihydrodiols were active and were more active than equivalent doses of either the parent hydrocarbon or the 3,4-dihydrodiol. The data are discussed in relation to possible mechanisms that may be involved in the metabolic activation of DBA.

The involvement of a non-'bay-region' diol-epoxide in the metabolic activation of benza[a]anthracene in hamster embryo cells.

The major hydrocarbon-nucleoside adduct present in hydrolysates of DNA from hamster embryo cells that had been treated with 3H-labelled benz[a]anthracene in culture has been examined by chromatography on Sephadex LH-20 columns and by high-pressure liquid chromatography. The results show that this adduct most probably arises from r-8,t-9-hydroxy-t-10,11-oxy-8,9,10,11-tetrahydrobenz[a]anthracene (anti-BA-8,9.-diol 10,11-oxide). On the basis of this and other evidence, this non-bay-region diol-epoxide appears to be a reactive intermediate involved in the metabolic activation of benz[a]anthracene.

The metabolic activation of benz[alpha]anthracene in three biological systems.

The 3,4- and 8,9-dihydrodiols of benz[alpha]anthracene (BA) are formed as metabolites of the parent hydrocarbon by rat-liver microsomes, by mouse skin and by hamster embryo cells. In incubations with rat-liver microsomal fractions, only small amounts of the 3,4-dihydrodiol of BA were detected relative to other dihydrodiol metabolites and only small amounts of BA-deoxyribonucleoside adducts derived from the related diol-epoxide, t-3, r-4-dihydroxy-t-1,2-oxy-1,2,3,4-tetrahydrobenz[alpha]anthracene (anti-BA-3,4-diol 1,2-oxide), were detected relative to adducts derived from r-8,t-9-dihydroxy-t-10,11-oxy-8,9,10,11-tetrahydrobenz[alpha]anthracene (anti-BA-8,9-diol 10,11-oxide). However, in studies with mouse skin and hamster embryo cells, larger amounts of free 3,4-dihydrodiol were detected and a larger proportion of the hydrocarbon-deoxyribonucleoside adducts resulted from the reaction of anti-BA-3,4-diol 1,2-oxide with DNA.

Inhibition by warfarin of liver microsomal vitamin K-reductase in warfarin-resistant and susceptible rats.

The NADH-dependent vitamin K-reductase activity of liver microsomes from three closely related rat strains has been studied. One strain (TAS) is susceptible and two strains (HW and HS) resistant to the anticoagulant and lethal effects of warfarin. The effects of cofactors, temperature, detergent and dithiothreitol on vitamin K1 reduction and solvent extraction of substrate and product have been investigated. Vitamin K-reductase activity was inhibited by approximately 13 and 8% respectively when microsomal preparations from TAS and HW animals were incubated with 50 microM vitamin K1 and 10 microM warfarin. In HS rat liver microsomes the enzyme was highly resistant to inhibition by warfarin. Evidence is presented and discussed that suggests that NADH-dependent vitamin K-reductase may be inhibited in the anticoagulant effect of warfarin and may be altered as a result of expression of the warfarin-resistance gene in HS rats. The enzyme activity studied was probably not a DT-diaphorase although both NADH and NADPH acted as cofactors for the reaction.

The metabolism of a series of polycyclic hydrocarbons by mouse skin maintained in short-term organ culture.

Investigations on the metabolism of 3H-labelled chrysene, benz[a]anthracene, 7-methylbenz[a]anthracene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, benzo[a]pyrene, dibenz[a,c]anthracene and dibenz[a,h]anthracene by mouse skin maintained in short-term organ culture were carried out. Estimations of the distribution of the metabolites of each hydrocarbon present after 24 h showed that there were wide variations both in the rates at which the hydrocarbons were metabolised and in the amounts of metabolites covalently bound to skin macromolecules. All the hydrocarbons were metabolised to dihydrodiols, which were identified by comparison on high pressure liquid chromatography (HPLC) with the authentic compounds, and these were the same diols as those that were formed in previous experiments with rat-liver microsomal fractions. However, free dihydrodiols represented only relatively small proportions of the total amounts of metabolites formed. All the hydrocarbons yielded dihydrodiols of the type that could give rise to bay-region diol-epoxides, when further metabolised, some of which are thought to be involved in hydrocarbon carcinogenesis.

Hepatic microsomal warfarin metabolism in warfarin-resistant and susceptible mouse strains: influence of pretreatment with cytochrome P-450 inducers.

In the present paper, the heterogeneity of hepatic cytochrome P-450 isoenzymes in the mouse has been probed, using warfarin as the substrate. Both sex and strain differences in the in vitro microsomal metabolism of warfarin have been investigated in male and female warfarin-resistant HC and warfarin-susceptible LAC-grey mouse strains. Animals were either untreated or treated with the cytochrome P-450 inducers phenobarbitone, beta-napthoflavone or clofibrate. In both sexes and strains of mice, metabolism of warfarin was stereoselective in favour of the R(+) enantiomer. However, regioselectively was different in both strains and sexes of untreated animals. After pretreatment with phenobarbitone, increases in the rate of formation of 4' and 7-hydroxy R(+) and S(-) warfarin metabolites in HC mice were observed, compared with untreated animals. In LAC-grey mice increases in 4'-, 6-, 7- and 8-hydroxy R(+) and S(-) warfarin metabolites were noted, compared with untreated animals. This data indicated that different amounts or forms of cytochrome P-450s were responsible for warfarin metabolism after phenobarbitone treatment in the two strains. Pretreatment of animals with beta-napthoflavone resulted in significant decreases in the rat of R(+) warfarin metabolism in both strains and sexes of mice indicating that the beta-naphthoflavone-inducible cytochrome P-450 isoenzymes were less active in the metabolism of warfarin, as compared to the uninduced isoenzymes. In addition, the cytochrome P-450 isoenzyme composition in the two mouse strains was different after clofibrate pretreatment, as reflected in reduced levels of some warfarin metabolites and a reduced total metabolism of warfarin, consistent with the narrow substrate specificity of clofibrate-induced cytochrome P450IVA1 for fatty acid hydroxylation. Accordingly, it is clear that both the basal and xenobiotic inducible hepatic cytochrome P-450 isoenzymes in warfarin-resistant and susceptible mice are different and therefore have implications for the in vivo disposition of warfarin.

The formation of dihydrodiols by the chemical or enzymic oxidation of 7-hydroxymethyl-12-methylbenz[alpha]anthracene and the possible role of hydroxymethyl dihydrodiols in the metabolic activation of 7,12-dimethylbenz[alpha]anthracene.

The formation of dihydrodiols from 7-hydroxymethyl-12-methylbenz[alpha]anthracene by rat-liver microsomal fractions, by mouse skin in short-term organ culture and by chemical oxidation in an ascorbic acid/ferrous sulphate/EDTA system has been studied using a combination of thin-layer chromatography and high pressure liquie chromatography. The 3,4-, 8,9- and 10,11-dihydrodiols were formed in all three systems. The 5,6-dihydrodiol was formed in rat-liver microsomal fractions and in chemical oxidation but was not detected as a metabolite of [7-3H]hydroxymethyl-12-methylbenz[alpha]anthracene when this compound was incubated with mouse skin in short-term organ culture. The possible role of hydroxymethyl dihydrodiols in the in vivo metabolic activation of 7,12-dimethylbenz[alpha]anthracene in mouse skin has been studied using Sephadex LH-20 column chromatography. The results show that the hydrocarbon-nucleic acid products formed following the treatment of mouse skin in vivo with [7,12-3H]dimethylbenz[alpha]anthracene are not the same as those that are formed following the treatment of mouse skin under the same conditions with either 7-hydroxymethyl-12-methylbenz[alpha]anthracene or 7-methyl-12-hydroxymethylbenz[alpha]anthracene.

The formation of dihydrodiols in the chemical or enzymic oxidation of dibenz[a,c]anthracene, dibenz[a,h]-anthracene and chrysene.

The formation of trans-dihydrodiols from dibenz[a,c]anthracene, dibenz[a,h]anthracene and chrysene by chemical oxidation in an ascorbic acid-ferrous sulphate-EDTA system and by rat-liver microsomal fractions has been studied using a combination of thin-layer (TLC) and high pressure liquid chromatography (HPLC) to separate the mixtures of isomeric dihydrodiols. The 1,2- and 3,4-dihydrodiols of dibenz[a,c]anthracene, the 1,2-,3,4- and 5,6-dihydrodiols of dibenz[a,h]anthracene and the 1,2-, 3,4- and 5,6-dihydrodiols of chrysene were formed in chemical oxidations. These dihydrodiols were also formed when the three parent hydrocarbons were metabolized by rat-liver microsomal fractions and, in addition, dibenz[a,c]anthracene yielded the 10,11-dihydrodiol. The 1,2- and 3,4-dihydrodiols of dibenz[a,c]anthracene have not been reported previously either as metabolites of the hydrocarbon or as products of chemical syntheses and the 5,6-dihydrodiol of chrysene was not detected in earlier metabolic studies.

The formation of dihydrodiols by the chemical or enzymic oxidation of benz[a] anthracene and 7,12-dimethylbenz[a] anthracene.

When benz[a] anthracene was oxidised in a reaction mixture containing ascorbic acid, ferrous sulphate and EDTA, the non-K-region dihydrodiols, trans-1,2-dihydro-1,2-dihydroxybenz[a] anthracene and trans-3,4-dihydro-3,4-dihydroxybenz[a] anthracene together with small amounts of the 8,9- and 10,11-dihydrodiols were formed. When oxidised in a similar system, 7,12-dimethylbenz[a] anthracene yielded the K-region dihydrodiol, trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a] anthracene and the non-K-region dihydrodiols, trans-3,4-dihydro-3,4-dihydroxy-7,12-dimethylbenz[a] anthracene, trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz[a] anthracene, trans-10,11-dihydro-10,11-dihydroxy-7,12-dimethylbenz[a] anthracene and a trace of the 1,2-dihydrodiol. The structures and sterochemistry of the dihydrodiols were established by comparisons of their UV spectra and chromatographic characteristics using HPLC with those of authentic compounds or, when no authentic compounds were available, by UV, NMR and mass spectral analysis. An examination by HPLC of the dihydrodiols formed in the metabolism, by rat-liver microsomal fractions, of benz[a] anthracene and 7,12-dimethylbenz[a] anthracene was carried out. The metabolic dihydriols were identified by comparisons of their chromatographic and UV or fluorescence spectral characteristics with compounds of known structures. The principle metabolic dihydriols formed from both benz[a] anthracene and 7,12-dimethylbenz[a] anthracene were the trans-5,6- and trans-8,9-dihydrodiols. The 1,2- and 10,11-dihydrodiols were identified as minor products of the metabolism of benz [a] anthracene and the tentative identification of the trans-3,4-dihydriol as a metabolite was made from fluorescence and chromatographic data. The minor metabolic dihydriols formed from 7,12-dimethylbenz[a] anthracene were the trans-3,4-dihydrodiol and the trans-10,11-dihydriol but the trans-1,2-dihydrodiol was not detected in the present study.

Comparison of the half-lives and regeneration rates of blood clotting factors II, VII, and X in anticoagulant-resistant and susceptible Norway rats (Rattus norvegicus Berk.).

The half-lives and regeneration rates of clotting factors II, VII, and X in the plasma of anticoagulant-resistant and susceptible rats were determined. There is little or no difference in the half-lives of factors II and X in anticoagulant-resistant rats compared to susceptible rats, but the half-life of factor VII is longer in anticoagulant-resistant rats. In anticoagulant-resistant rats critical clotting factors appear to be carboxylated in preference to factor II, whereas the opposite occurs in susceptible rats; this may contribute to an animal's resistance status.

Difenacoum poisoning as a cause of haematuria.

A man presented with frank haematuria and a grossly prolonged prothrombin time. He was later found to have taken an overdose of difenacoum--a 'superwarfarin' rodenticide. The diagnosis was confirmed by a serum concentration of difenacoum of 0.6 micrograms ml-1. Overdosage with superwarfarins is discussed and the need for prolonged treatment with vitamin K1 highlighted.

The stability of ergocalciferol in rodenticidal baits.

Concentrations of the rodenticide ergocalciferol (vitamin D2) in samples of rodent baits laid in foodstores and in the laboratory were monitored over several months. Bait samples were solvent extracted and ergocalciferol concentration determined by high pressure liquid chromatography (HPLC). Ergocalciferol levels were constant for more than 21 days in dry samples and did not fall by more than 30% in 100 days. When water (10% w/w) was added to the baits in the laboratory the ergocalciferol concentration fell by approximately 30% in 30 days. In these wet laboratory samples there was a rapid visible growth of fungus and in normal rodent control use baits should have been replaced when such deterioration became evident.

Simple and rapid method for the determination of the diastereomers of difenacoum in blood and liver using high-performance liquid chromatography with fluorescence detection.

A rapid and sensitive high-performance liquid chromatographic method for the analysis of cis and trans diastereomers of the anticoagulant rodenticide difenacoum has been described. The methodology demonstrates potential for the analysis of diastereomers of related 4-hydroxycoumarin anticoagulants. Separations were achieved by reversed-phase chromatography on a Zorbax ODS column with gradient elution using acetonitrile-water, modified with 0.1% acetic acid, as the mobile phase. Detection of the analytes was effected by fluorescence at excitation and emission wavelengths of 310 and 390 nm, respectively. Sample preparation from both plasma and liver has been simplified to reduce preparation time and manipulation. The minimum detectable concentration of each diastereomer was 5 ng/ml. Recoveries of 100% were obtained from plasma and 93% from liver tissue. This method has been used for the investigation of the pharmacokinetics of difenacoum diastereomers in rats, and for investigation of unexplained hypoprothrombinaemic events encountered clinically.

Aspects of anticoagulant action: a review of the pharmacology, metabolism and toxicology of warfarin and congeners.

Warfarin is the most widely used anticoagulant in the treatment of thromboembolism in man. It has also been used extensively as a rodenticidal agent. Insofar as its clinical use is concerned, it is now clear that many of the drug interactions observed in patients are mediated via metabolic or pharmacokinetic factors. An understanding of the disposition of warfarin is therefore essential if one is to predict the likely response in patients undergoing anticoagulant therapy with this compound. Warfarin-resistance has been reported in both man and rodents. Understanding resistance in both man and rodents is important for effective anticoagulant therapy, and in control of resistant strains of rodents. Warfarin resistance in rat strains does not appear to have a metabolic or pharmacokinetic basis; in this species, resistance is thought to be due to differences in permeability to, or affinity for a receptor. Apart from its clinical and rodenticidal uses, warfarin is an excellent substrate for probing the heterogeneity of cytochrome P.450, since its metabolic oxidation is mediated by this mixed function oxidase. This review draws together much of the current published literature on the pharmacology, metabolism and toxicology of warfarin and related congeners.

Polycyclic hydrocarbon activation and metabolism in epithelial cell aggregates prepared from human mammary tissue.

The metabolism of benz(a)anthracene (BA), 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (BP) by human mammary epithelial cell aggregates in culture has been investigated using non-neoplastic tissues obtained from eight patients undergoing reduction mammoplasty. All three hydrocarbons were metabolized to water-soluble and organic solvent-soluble products and the latter included both K-region and non-K-region dihydrodiols. The major dihydrodiols detected as metabolites of the parent hydrocarbons were the 8,9-dihydrodiols of BA and DMBA and the 9,10-dihydrodiol of BP. The 1,2-dihydrodiols of BA and DMBA and the 11,12-dihydrodiol of BP were not detected. The hydrocarbons also became bound to the proteins and DNA of the epithelial cells but there were wide differences in the extents of binding occurring with the different hydrocarbons and in the extents of metabolism and binding occurring with tissue preparations from different patients. Some of the hydrocarbon-deoxyribonucleoside adducts formed from DMBA and BP appeared to have arisen through reactions of "bay-region" diol-epoxides with DNA, but only very low levels of reaction with DNA were detected in tissue preparations treated with BA.

The development of a blood clotting response test for discriminating between difenacoum-resistant and susceptible Norway rats (Rattus norvegicus, Berk.).

1. A new test for identifying levels of difenacoum resistance in the Norway rat is described, based upon the differential physiological response to difenacoum administration. 2. This test is based on changes in blood clotting activity over 4 days, following administration of the rodenticide difenacoum in conjunction with menadione (vitamin K3). 3. The anticoagulant effect is reduced only in rats that are resistant or tolerant to difenacoum. 4. This test procedure is quicker than traditional feeding tests, and identifies the degree of resistance in both laboratory and wild rats that have difenacoum resistance genes.

Liver microsome-mediated mutagenicity of dihydrodiols derived from dibenz(a,c)anthracene in S. typhimurium TA100.

The mutagenic activities of the trans-1,2- and 3,4-diols of dibenz(a,c)anthracene, both of which could yield 'bay-region' diol-epoxides on further metabolism, were lower than those of the parent hydrocarbon and of the related 10,11-diol.

Utilization by the isolated perfused rat liver of N-acetyl-D-[1-14C]galactosamine and N-[3H]acetyl-D-galactosamine for the biosynthesis of glycoproteins.

The isolated perfused rat liver system has been used to monitor the utilization of N-[3H]acetyl-D-galactosamine and N-acetyl-D-[1-14C]galactosamine for the biosynthesis of radiolabelled glycoproteins, which are subsequently secreted into the plasma. Both radiolabels appear in a number of different glycoproteins, predominantly as sialic acid and N-acetylglucosamine. The ratio of labelled sialic acid to labelled N-acetylglucosamine varies for different glycoproteins, but the bulk of N-acetyl-D-galactosamine is incorporated without deacetylation.