Gilberte Larrieu

Enhancement of moxidectin bioavailability in lamb by a natural flavonoid: quercetin

Moxidectin is an antiparasitic drug widely used in cattle, sheep and companion animals. Due to the involvement of P-glycoprotein (P-gp) and cytochrome P450 3A in the metabolism of moxidectin, we studied the influence of various P-gp interfering agents (ivermectin, quercetin and ketoconazole) on the metabolism of 14C moxidectin in cultured rat hepatocytes over 72 h. This in vitro study allowed selection of compounds which are able to increase the moxidectin bioavailability in lambs. From this, the modulation of moxidectin pharmacokinetics in plasma of lambs was studied after co-administration of 0.2 mg kg(-1) moxidectin (subcutaneously (SC)) and 0.2 mg kg(-1) ivermectin (SC), or 10 mg kg(-1) quercetin (SC), or 10 mg kg(-1) ketoconazole (orally). Ivermectin and quercetin increased significantly the quantity of 14C moxidectin in the rat hepatocytes. Ketoconazole co-administration led to a higher concentration of moxidectin in the rat hepatocytes. In vivo, only quercetin was able to modify the pharmacokinetics of moxidectin in plasma of lambs by increasing significantly the area under the plasma concentration-time curve. This study allowed the use of a natural agent, quercetin, to improve the bioavailability of moxidectin.

Comparative effects of T-2 toxin and diacetoxyscirpenol on drug metabolizing enzymes in rat tissues

The effects of T-2 toxin and diacetoxyscirpenol on tissue drug-metabolizing enzymes in young male rats were compared. Mycotoxicoses were produced by daily oral administration of toxins at 1.0 mg/kg body weight for 1, 4 or 8 days. Many hepatic, renal and pulmonary oxidative and conjugative enzymes were measured in animals killed 24 hr following the last administration. The effects of the two trichothecene mycotoxins were generally similar. In liver the decrease in microsomal and cytosolic proteins paralleled the decline in total plasma proteins or the increase in plasma GOT activity. Hepatic microsomal cytochrome P-450 decreased in rats receiving trichothecenes for 8 days. This effect was more marked when aminopyrine, benzphetamine, ethylmorphine and ethoxycoumarin dealkylations or aniline and benzopyrene hydroxylations were measured. p-nitrophenol glucuronyltransferase activity was enhanced in animals receiving at least one administration of trichothecenes, whereas there was no change in conjugation to glutathione or acetate. In other tissues, there was no change in any renal enzymes whereas a significant rise in pulmonary monooxygenase was observed in T-2 toxin administered to rats for 4 or 8 days.

RXR activators molecular signalling: involvement of a PPARα-dependent pathway in the liver and kidney, evidence for an alternative pathway in the heart

In this study we compared the molecular signalling elicited by rexinoids, selective retinoid X receptor (RXR)‐activators, in several organs (i.e. liver, kidney, heart) and in hepatocytes of various species. RXR plays the pivotal role of a hetero‐dimerization partner for the members of the class II subset of nuclear receptors which regulate the transcription of numerous target genes, following chemical activation. Several of these selective activators are currently used to treat hyperlipidaemia (fibrates), type II diabetes (glitazones), or skin disorders (retinoic acid). Although these therapeutic pathways are not fully elucidated, receptor activation is considered a pre‐requisite for efficacy. Therefore RXR, which accepts numerous dimeric partners, is considered a worthwhile pharmacological target. We analysed a number of biochemical and molecular responses to rexinoids which were given orally to mice. Our results showed a prominent involvement of the peroxisome proliferator‐activated receptor (PPARα) as a majority of the observed hepatic and renal regulations were abolished in PPARα‐knockout animals. Therefore we documented the species‐specificity of these rexinoid actions which were reproduced in rat primary hepatocyte cultures but not in cultures of rabbit or human origin. Conversely, we established that the regulation of the pyruvate dehydrogenase kinase (PDK4) gene in the heart, by rexinoids, is independent of PPARα expression. Our results support the obligatory expression of the active, although quiescent, PPARα to sustain a subset of relevant regulations attributable to rexinoids in the liver and kidney. Their cardiac molecular signalling unveiled an alternate transduction pathway and therefore opens new prospects in the therapeutic potential of rexinoids.

Identification of human and rabbit cytochromes P450 1A2 as major isoforms involved in thiabendazole 5-hydroxylation

Summary— This report characterized one of the major cytochrome P450 isozyme involved in thiabendazole metabolism. This study was undertaken by using both cultured rabbit hepatocytes treated or not with drugs known to specifically induce various cytochromes P450 isoenzymes (ie, P450 1A1/2 by β‐naphthoflavone, P450 2B4 by phenobarbital, P450 3A6 by rifampicine and P450 4A by clofibrate) and human liver (THLE‐5) and bronchial (BEAS‐2B) epithelial cells expressing or not the major constitutive human cytochromes P450 (ie, CYP1A2, 2A6, 2B6, 2C9, 2D6, 2E1 or 3A4). Only hepatocytes exposed to β‐naphthoflavone and clofibrate significantly metabolized thiabendazole to 5‐hydroxythiabendazole. Extensive biotransformation of this anthelmintic only occurred in human cells expressing CYP1A2. Moreover, experiments performed on rabbit preparations showed good correlations between thiabendazole 5‐hydroxylase activity and both ethoxyresorufin and methoxyresorufin O‐dealkylase activities. Thus, CYP1A2 is a major isoenzyme involved in thiabendazole 5‐hydroxylation.

Comparative effects of cytokines on constitutive and inducible expression of the gene encoding for the cytochrome P450 3A6 isoenzyme in cultured rabbit hepatocytes: consequences on progesterone 6β-hydroxylation

Cultured rabbit hepatocytes were used to compare the relative activities of cytokines to inhibit the constitutive or rifampicin (RIF)-induced expression of the cytochrome P450 3A6 gene (CYP3A6). Human recombinant cytokines tested were interleukin-1beta (IL-1beta) (2 U/mL), interleukin-2 (IL-2) (5,000 U/mL) and interferon-gamma (IFN-gamma) (50 U/mL). Hepatocytes were cultured in the presence or absence of 25 microM RIF for 24 hr, with or without cytokines alone or in combination. All these cytokines inhibited RIF-induced P4503A6 expression without apparent cellular toxicity. By contrast, only IFN-gamma treatment provided a significant decrease (41%) in the constitutive P4503A6 protein level. Moreover, cytokines differed in their ability to repress RIF-dependent transcriptional induction of CYP3A6: IL-1beta and IL-2 were approximately equipotent, causing an almost 40-50% suppression of CYP3A6 mRNA and protein levels, whereas IFN-gamma exerted repressive effects only on P4503A6-related erythromycin N-demethylase activity and inducible protein expression. In fact, although strongly reducing P4503A6 protein content (an approximate 70% decrease), IFN-gamma did not exhibit any influence on CYP3A6 mRNAs with the exception of its association with interleukins. All these results suggest that IL-1beta and IL-2 mainly promote a transcriptional repression mechanism, given the absence of effect of these cytokines on the basal P4503A6 level, whereas IFN-gamma exerts a post-transcriptional suppressive action on both induced and constitutive P4503A6 expression. Consequently, P4503A6-dependent progesterone 6beta-hydroxylase activity also presented a cytokine-specific pattern of inhibition, with a much greater sensitivity than P4503A6 immunoreactive protein to IL-1beta and IL-2 + IFN-gamma treatments. Thus, this study underlines the significant impact of inflammation on steroid metabolism.

Effect of exposure of rabbit hepatocytes to sulfur-containing anthelmintics (oxfendazole and fenbendazole) on cytochrome P4501A1 expression.

The expression of cytochrome P4501A1 and 1A2 was investigated in rabbit hepatocytes maintained in primary cultures for 96 hr in the absence or presence of 100 mum of the benzimidazole anthelmintics oxfendazole or fenbendazole. Total cytochrome P-450, ethoxyresorufin O-deethylase and acetanilide hydroxylase activities were significantly increased in cell cultures receiving benzimidazoles. These increases were more marked after exposure of cultured hepatocytes to oxfendazole (OFZ) than to fenbendazole (FBZ). Western and Northern blot analysis of microsomes and RNA prepared from these cultures revealed increased levels of both protein and specific mRNA for P4501A1. The inhibition of these inductions in the presence of actinomycin D suggests a transcriptional way of activation of this gene. The ability of OFZ to bind to the Ah receptor has been examined. Data obtained from competition experiments with dioxin demonstrated that OFZ and other compounds in the benzimidazole series are not ligand of the Ah receptor. From saturation experiments and Scatchard plot analysis, rabbit hepatocyte Ah receptor (K(d) = 10.6 nm) seems to belong, as does the human Ah receptor, to a low-affinity category. Different induction rates obtained with several benzimidazole drugs suggest that the sulfur atom within the molecule is critical for CYP1A1 induction. The widely used benzimidazole anthelmintics OFZ and FBZ may exert an inducing effect through an original pathway that does not require a specific binding step to the Ah receptor.

Fasciola hepatica: Liver enzymes in rats and interaction with chemical inducers

Adult male rats were sorted into control and infected groups, the latter receiving an oral dose of 20 metacercariae of Fasciola hepatica. In Weeks 3 and 6 after infection, some rats received phenobarbital or 3-methylcholanthrene which induced drug metabolizing enzymes. The parasitic pathology was ascertained by clinical observation of the rats and at autopsy. Hepatic microsomal cytochrome P-450 content was significantly decreased in infected rats compared to untreated phenobarbital treated groups. In all infected rats, the simultaneous increase in cytosolic calcium and decrease in cytosolic glutathione corresponded to oxidative cell injury occurring in the course of fascioliasis. Both arylamine acetyltransferase (EC 2.3.1.5.) and glutathione transferase (EC 2.5.1.18) activities were decreased in all newly infected and 6 week infected groups. Fascioliasis did not alter the substrate related uridine diphosphate glucuronosyltransferase activities (EC 2.4.1.17) of any rat group.

Evidence for cytochrome P4501A2-mediated protein covalent binding of thiabendazole and for its passive intestinal transport: use of human and rabbit derived cells.

Thiabendazole (TBZ), an anthelmintic and fungicide benzimidazole, was recently demonstrated to be extensively metabolized by cytochrome P450 (CYP) 1A2 in man and rabbit, yielding 5-hydroxythiabendazole (5OH-TBZ), the major metabolite furtherly conjugated, and two minor unidentified metabolites (M1 and M2). In this study, exposure of rabbit and human cells to 14C-TBZ was also shown to be associated with the appearance of radioactivity irreversibly bound to proteins. The nature of CYP isoforms involved in this covalent binding was investigated by using cultured rabbit hepatocytes treated or not with various CYP inducers (CYP1A1/2 by beta-naphthoflavone, CYP2B4 by phenobarbital, CYP3A6 by rifampicine, CYP4A by clofibrate) and human liver and bronchial CYP-expressing cells. The covalent binding to proteins was particularly increased in beta-naphthoflavone-treated rabbit cells (2- to 4-fold over control) and human cells expressing CYP1A2 (22- to 42-fold over control). Thus, CYP1A2 is a major isoenzyme involved in the formation of TBZ-derived residues bound to protein. Furthermore, according to the good correlation between covalent binding and M1 or 5OH-TBZ production, TBZ would be firstly metabolized to 5OH-TBZ and subsequently converted to a chemically reactive metabolic intermediate binding to proteins. This metabolic activation could take place preferentially in liver and lung, the main biotransformation organs, rather than in intestines where TBZ was shown to be not metabolized. Moreover, TBZ was rapidly transported by passive diffusion through the human intestinal cells by comparison with the protein-bound residues which were not able to cross the intestinal barrier. Consequently, the absence of toxicity measured in intestines could be related to the low degree of TBZ metabolism and the lack of absorption of protein adducts. Nevertheless, caution is necessary in the use of TBZ concurrently with other drugs able to regulate CYP1A2, particularly in respect to liver and lung tissues, recognised as sites of covalent-binding.

Cytochrome P450 decreases are correlated to increased microsomal oxidative damage in rabbit liver and primary cultures of rabbit hepatocytes exposed to AFB1.

Although numerous studies report hepatic drug metabolizing enzyme alterations during aflatoxicosis, the mechanisms involved in P450 decreases remain to be established. The purpose of this work is to investigate whether increased oxidative damage revealed by the detection of malondialdehyde (MDA), lipofuscin substances, and conjugated dienes in microsomes, could explain the decreased P450 content. Studies were conducted with two different doses of aflatoxin B1 (AFB1), both in vivo in rabbits and ex vivo in primary cultures of rabbit hepatocytes, in the presence or absence of beta-naphthoflavone or rifampicin used as respective P450 inducers. Strong negative correlations were observed between MDA and P450 contents, both in vivo and ex vivo, whereas rifampicin appears to protect the hepatocytes from oxidative damage but not AFB1 toxicity. Positive correlation were also obtained between MDA formation and lactate dehydrogenase (LDH), aspartate aminotransferase (ASAT) or alanine amino-transferase (ALAT) releases, used as non-specific markers of AFB1 toxicity. Taken together these results suggest that the dramatic decreases of cytochrome P450 observed in vivo during aflatoxicosis could be linked, at least in part, to microsomal oxidative damage.

PHARMACOKINETICS OF AMPICILLIN AND PENTOBARBITAL IN THE COURSE OF SUBCLINICAL FASCIOLIASIS IN SHEEP

Pharmacokinetics of two common veterinary drugs, ampicillin and pentobarbital, were determined in sheep before and four, eight, 12, 17 and 21 weeks after infestation of animals by an oral administration of 150 metacercariae of Fasciola hepatica. The parasite infestation was ascertained by clinical observation of the animals. The pharmacokinetics of ampicillin were not significantly affected by the liver parasitism but the disposition of pentobarbital changed. A significant increase in elimination half-life (around 180 per cent), volume of distribution (130 per cent) and mean residence time (154 to 170 per cent) was observed in sheep infected by the parasite for four to 12 weeks. In these animals, duration of narcosis caused by pentobarbital was prolonged 1.8-fold. The results suggested that both reduced elimination of pentobarbital and impaired distribution of the drug would be responsible for the prolonged duration of narcosis in infected animals.