Renger F. Witkamp

Congener specific PCB and polychlorinated camphene (toxaphene) levels in Svalbard ringed seals (Phoca hispida) in relation to sex, age, condition and cytochrome P450 enzyme activity

Congener specific PCB and toxaphene (polychlorinated camphene, PCC) analyses were performed in seal blubber, collected in Svalbard, Norway. The concentration, body burden and metabolic index (PCB congener concentration in seal relative to their prey) were calculated. Multiple regression analyses were carried out to evaluate the influence of age, sex, blubber (as a percentage of total body weight) and cytochrome P450 activities on PCB and PCC levels. Levels of total PCBs found were five times higher than in ringed seals from the Canadian Arctic, corresponding with the relatively high contaminant levels in the European Arctic. The dominant PCB congeners (> 70% of the total PCBs measured) were 153, 138, 99, 180 and 101. The observed PCB and PCC accumulation patterns were very similar to patterns in seals from other studies, suggesting a large resemblance in contaminant metabolism. A decrease in the relative abundance of the lower chlorinated PCBs, was associated with higher concentrations of PCB 153. Since there was no indication for selective PCB excretion by lactating females, this suggests metabolism of these PCBs in ringed seals due to xenobiotic metabolising enzymes. The metabolic index confirmed the model of persistency of the different PCBs except for congener 128 and 138. These congeners, considered persistent in seals, could to some extent be metabolised in ringed seals. However, co-elution of PCB 138 with PCB 163 and of PCB 128 with TOX 50 possibly has resulted in an underestimation of the metabolic index for these congeners. Multiple regression analyses revealed a significant positive effect of age and a negative effect of the blubber content on the PCB concentrations. Since large fluctuations of body lipids occur between seasons in pinnipeds, PCB measurements should account for the total blubber content to avoid biased results. PCBs with vicinal H-atoms in the o, m or the m, p positions showed in addition a relation with cytochrome P450 enzyme activities. Surprisingly, no effect of sex on the PCB concentrations was observed, probably because female ringed seals, unlike other pinnipeds, continue feeding during lactation. This results in only small amounts of lipid and lipid-associated contaminants being mobilised from the blubber. Consequently, contaminant excretion with the milk will be low. Toxaphene concentrations found were low compared to levels found in the Canadian Arctic. Two congeners, TOX 26 and TOX 50 were predominant (15 and 18%, respectively of total toxaphene). There was no effect of sex, age, total blubber, or cytochrome P450 activities on the toxaphene levels. There was also no correlation between toxaphene and PCB levels, which may indicate differences in exposure and metabolism between these contaminants. Toxaphenes did not bioaccumulate to any substantial extent in ringed seals.

Participation of β-adrenergic receptors on macrophages in modulation of LPS-induced cytokine release

For several years it is known that beta-adrenergic receptor agonists have anti-inflammatory effects. However, little is known about the role of beta-adrenergic receptors on macrophages in the modulation of cytokine production by beta-agonists during inflammation. In this study, the presence of beta-receptors on PMA-differentiated U937 human macrophages, and the participation of these receptors in the modulation of LPS-mediated cytokine production by beta-agonists was investigated. Total beta-receptor expression on undifferentiated (monocyte) and PMA-differentiated U937 cells was established using receptor binding studies on membrane fractions with a radio ligand. The expression of beta-receptors proved to be significantly lower on monocytes than on macrophages, additionally a predominant expression of beta 2-receptors was found. Production of the cytokines TNF-alpha, IL-6, and IL-10 by LPS-stimulated differentiated U937 cells was measured in time. Peak concentrations for TNF-alpha, IL-6 and IL-10 occurred at 3, 12 and 9 hrs, respectively. When differentiated U937 cells were incubated with both LPS and the beta-agonist clenbuterol the production of TNF-alpha and IL-6 was significantly reduced. However the production of IL-10 was increased. To study the mechanism of modulation of cytokine production in more detail, U937 macrophages were incubated with LPS/clenbuterol in combination with selective beta 1- and beta 2-antagonists. These results indicated that the beta 2- and not the beta 1-receptor is involved in the anti-inflammatory activity of clenbuterol.

Development and validation of a quantitative method for the determination of 12 endocannabinoids and related compounds in human plasma using liquid chromatography-tandem mass spectrometry.

A sensitive and specific LC-MS/MS method for the quantification of the endocannabinoids and related structures anandamide, 2-arachidonoyl glycerol, 2-arachidonyl glycerol ether, O-arachidonoyl ethanolamide, dihomo-gamma-linolenoyl ethanolamide, docosatetraenoyl ethanolamide, N-arachidonoyl dopamine, N-arachidonyl glycine, N-oleoyl dopamine, oleoyl ethanolamide, palmitoyl ethanolamide, and stearoyl ethanolamide in human plasma was developed and validated. Compounds were extracted using acetonitrile followed by solid-phase extraction. Separation was performed on a Xterra C8 column using gradient elution coupled to a triple-quadrupole MS. LLOQ levels ranged from 0.02 to 1.75 microg/mL, LODs ranged from 0.0002 to 0.1266 ng/mL, and accuracies were >80% (except stearoyl ethanolamide at lowest spike level) at all spike levels.

Characterization of cytochrome P450 isoenzymes in primary cultures of pig hepatocytes

Despite the fact that pigs are increasingly used in pharmacological and toxicological studies, knowledge on the enzymes which metabolize xenobiotics, in particular cytochrome P450 (CYP) enzymes, in pigs is still very limited. Primary cultures of pig hepatocytes were used to characterize CYP enzymes. The characterization was performed at the level of enzymatic activities, apoprotein and mRNA analyses. Enzyme inducers investigated were beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) and rifampicin (RIF). After 48hr of BNF treatment, CYP1A protein and mRNA levels were increased, and ethoxyresorufin O-deethylation and caffeine 3-demethylation were strongly induced. PB and RIF increased the levels of CYP3A apoprotein and mRNA, whereas BNF down-regulated CYP3A and related activities. PB and RIF treatment resulted in increased ethylmorphine N-demethylation and testosterone hydroxylation, which appears to be the result of CYP3A induction. Hybridization of pig RNA with a human CYP2C9 cDNA probe showed a PB and RIF inducible CYP, which was down-regulated by BNF. Similar inducing effects were observed for tolbutamide, a marker substrate for CYP2C. DEX was not a potent inducer, although some induction of CYP3A mRNA was observed. The present results indicate the absence of CYP2B and probably CYP2D enzymes and activities in pig liver. Despite some dissimilarities, the results indicate that pigs, apart from their very human-like physiology, might represent a more appropriate model species for oxidative drug metabolism in humans than rats.

Current and future drug targets in weight management

ABSTRACTObesity will continue to be one of the leading causes of chronic disease unless the ongoing rise in the prevalence of this condition is reversed. Accumulating morbidity figures and a shortage of effective drugs have generated substantial research activity with several molecular targets being investigated. However, pharmacological modulation of body weight is extremely complex, since it is essentially a battle against one of the strongest human instincts and highly efficient mechanisms of energy uptake and storage. This review provides an overview of the different molecular strategies intended to lower body weight or adipose tissue mass. Weight-loss drugs in development include molecules intended to reduce the absorption of lipids from the GI tract, various ways to limit food intake, and compounds that increase energy expenditure or reduce adipose tissue size. A number of new preparations, including combinations of the existing drugs topiramate plus phentermine, bupropion plus naltrexone, and the selective 5-HT2C agonist lorcaserin have recently been filed for approval. Behind these leading candidates are several other potentially promising compounds and combinations currently undergoing phase II and III testing. Some interesting targets further on the horizon are also discussed.

Differential effect of pentoxifylline on lipopolysaccharide-induced downregulation of cytochrome p450☆

It is now established that inflammatory stimuli such as lipopolysaccharides (LPS) and polyinosinic acid:polycytidylic (polyIC) suppress hepatic expression of cytochrome P450 (P450) genes in rat liver. Previous studies have suggested that LPS- or polyIC-induced downregulation of P450 was due to endogenously released inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1, interleukin-6, and interferons (IFNs). To improve our understanding of the role of inflammatory cytokines in mediating P450 depression, we investigated the possibility of preventing P450 downregulation with pentoxifylline. Pentoxifylline has been shown to inhibit LPS-induced TNF-alpha production by suppression of TNF-alpha gene expression. The present study shows that in uninduced male rats pentoxifylline selectively prevents the downregulation of microsomal P4501A2 and P4502B caused by LPS. No protective effect of pentoxifylline on the downregulation of P4502E1 and P4503A1/2 was observed. PolyIC-induced downregulation of P4501A2, P4502B, P4502E1, and P4503A1/2 was not affected by pentoxifylline. These results suggest that the LPS-induced downregulation of P4501A2 and P4502B is mediated to a large extent by TNF-alpha. Other cytokines might be involved in the suppression of P4502E1 and P4503A1/2. The fact that polyIC-induced downregulation is not protected by pentoxifylline is further evidence that this agent acts via a selective induction of IFNs.

Phase I and phase II enzyme activities in Ringed seals (Phoca hispida): characterization of hepatic cytochrome P450 by activity patterns, inhibition studies, mRNA analyses, and western blotting

Abstract Hepatic phase I and phase II enzymes play an important role in contaminant metabolism in mammals. Knowledge of these enzymes is essential since their presence and activity determines the potential biological effects of contaminant exposure. In this study activities of hepatic phase I enzymes (cytochrome P450 (CYP)) and phase II enzymes (UDP glucuronosyl transferase (UDPGT) and glutathione S -transferase (GST)) in Ringed seals ( Phoca hispida ) were assessed. In addition, CYP enzymes were characterized using catalytic activities, selective inhibitors, mRNA analyses, as well as Western blotting. Both UDPGT and GST activities were present, indicating that these seals may form the reactive methylsulfonated PCB metabolites. The results from the CYP characterization showed ethoxyresorufin- O -deethylation (EROD) and caffeine demethylation activity, while the pentoxyresorufin- O -depenthylation activity was low. The activity towards testosterone resulted in several hydroxy-metabolites. Based on these activity studies the presence of CYP1A, CYP3A, but not CYP2B was insinuated. The inhibition of EROD and caffeine demethylation by α -naphthoflavone but not by furafylline suggested that in this seal species only one CYP1A enzyme was present. This was supported by the results from the mRNA measurements and Western blots. Only one mRNA band cross-hybridized with human CYP1A cDNA probes at the rat CYP1A1 position, while also one protein band, cross reacting with anti-rat CYP1A, was detected. The selective inhibition of the formation of the testosterone 2 β - and 6 β -hydroxy metabolites by ketoconazole supported the suggestion that the formation of these metabolites was mediated by CYP3A. The mRNA measurements and the results from the Western blots confirmed these results. The Northern blots showed cross hybridization with human CYP3A cDNA, while in the Western blots one protein band cross-reacting with anti-rat CYP3A was detected. No cross hybridization with rat CYP2B1/2 cDNA was observed. However, the Western blots showed a band cross-reacting with anti-rat CYP2B, suggesting the presence of a CYP2B-like protein. In conclusion, this study has shown that Ringed seal liver contains multiple forms of CYP as well as phase II enzymes, showing different catalytic activities, i.e. EROD, caffeine- N -demethylation, and testosterone hydroxylation at different positions. Only one CYP1A isoform seemed to be present as well as a CYP3A-like isoform. Although the catalytic activities and mRNA analyses did not indicate the presence of a CYP2B-like protein in Ringed seals, the Western blots suggested the presence of a CYP2B-like enzyme. However, its functional significance remains unclear.

Time-dependent induction of two distinct hepatic cytochrome P4501A catalytic activities at low temperatures in Arctic charr (Salvelinus alpinus) after oral exposure to benzo(a)pyrene

Due to increased industrial and other anthropogenic activities during the last decades, the arctic environment faces increasing levels of organic pollution. The presence of polycyclic aromatic hydrocarbons in the arctic environment is of major concern because of their carcinogenic potential and their effect on the health and reproductive performance of animals. There is an increasing demand for sensitive and relatively inexpensive diagnostic biomarkers, applicable for environmental monitoring in the Arctic seas to assess the human impact on the Arctic. As a first step to validate the use of enzymatic assays in fish as an indicator for environmental pollution, the hepatic cytochrome P450 induction in the anadromous Spitsbergen Arctic charr was studied at several time intervals during 4 days following oral exposure to benzo(a)pyrene. Three different enzyme activities were studied, i.e. testosterone-6β-hydroxylation, the ethoxyresorufin-O-deethylation and the caffeine-N3-demethylation. Both the ethoxyresorufin-O-deethylation and the caffeine-N3-demethylation activity (only the 1,7-dimethylxanthine metabolite was formed) showed a strong and time-dependent induction after exposure. Western immunoblotting revealed an increase in the amount of protein. In the exposed groups a clear time-dependent increase of protein reacting with anti-cod CYP1A was observed. Between the ethoxyresorufin-O-deethylation and the caffeine-N3-demethylation activity in the exposed animals there was a strong and linear correlation. There was no effect of benzo(a)pyrene exposure on the testosterone-6β-hydroxylation. The slow and long lasting induction, probably due to the low water temperatures 5 °C, can be regarded as advantageous for biomonitoring. Differences in inhibition (α-naphtaflavone, furafylline) characteristics between ethoxyresorufin-O-deethylation rate and caffeine-N3-demethylation rate could be interpreted in terms of possible presence of more than one P4501A isoform in this fish species.

Presence, formation and putative biological activities of N-acyl serotonins, a novel class of fatty-acid derived mediators, in the intestinal tract

Following the discovery of the endocannabinoid arachidonoyl ethanolamide (anandamide) and other N-acyl-ethanolamines, several other compounds have been found in which amino acids or neurotransmitters rather than ethanolamide are linked to fatty acids. Studies have shown that the local availability of fatty acid precursors, which in turn is modulated by dietary intake of lipids, determines the pattern of conjugates formed. Less information is available whether the same might be true for the amines or neurotransmitters involved. We hypothesized that N-arachidonoyl-serotonin (AA-5-HT) and its analogs could be endogenously present in those tissues that have high contents of serotonin. We investigated the endogenous presence of N-acyl serotonins in different parts of the gastro-intestinal tract of pigs and mice. We discovered that AA-5-HT, oleoyl-serotonin, palmitoyl-serotonin, and stearoyl-serotonin were endogenously present, particularly in the jejunum and ileum. Their formation in vitro was stimulated by the addition of serotonin to intestinal tissue incubations. Furthermore, in a mouse study we showed that the pattern of formation is dependent on the relative amount of fatty acids in the diet. The formation of docosahexaenoyl-serotonin and eicosapentaenoyl-serotonin was elevated in mice fed with a diet containing fish oil. Preliminary data showed that several of the serotonin conjugates are able to inhibit glucagon-like peptide-1 secretion and FAAH activity in vitro. Taken together, our data suggest that N-acyl serotonins are a novel class of lipid mediators present in the gut with highly promising biological properties.

Signal transduction in inflammatory processes, current and future therapeutic targets: A mini review

Summary The selective control of inflammatory reactions will continue to be a major issue in the development of new drugs. Many new molecular targets are coming up. This paper highlights a few key mediators that are nowadays considered as interesting therapeutic intervention points. Cytokines play an important regulatory role in the initiation, maintenance and termination of inflammatory reactions. More than 50 cytokines have been identified, and more and more has become known about their receptors and signal transduction pathways. Tumour necrosis factor‐α (TNF‐α) is still regarded as one of the initial cytokines of the cascade, and different approaches are followed to control its synthesis, release or effects. Lipopolysaccharide (LPS) is a one of the triggers that is able to induce a strong TNF‐response. Inhibitors of cyclic nucleotide phosphodiesterases (PDEs), including rolipram and pentoxifylline suppress the LPS‐induced TNF‐α production in monocytes/ macrophages. In our laboratory it has been shown that the alternative way to increase cAMP levels, via stimulation of ß‐adrenergic receptors, also provides an effective way, both in vitro and in vivo, to inhibit TNF‐α release. Other therapeutic ways include the use of antibodies directed to cytokines, TNF receptor fused to IgG, antibody therapy against TNF, the use of MAP kinase inhibitors. The different signal transduction pathways, including the NF‐κB activation route may provide alternative pharmacological tools. We may surely expect anti‐inflammatory drugs of much greater specificity to be developed in the next decade. Despite the relative limited investments in veterinary drug development this will also have consequences for veterinary therapy