M. Monshouwer

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.

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.

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.

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

Cytochromes and cytokines: changes in drug disposition in animals during an acute phase response: a mini-review.

Summary Diseases are a major cause of variation in drug response. Although many different diseases are known that have an effect on the pharmacokinetics or sometimes the pharmacodynamics of a drug, disorders associated with a so‐called acute phase response (APR) are the most important in this respect. During APR, for example caused by tissue damage or invasion of a pathogen, a group of symptoms can be observed that often include fever, lassitude, inhibition of gastric function and synthesis of acute phase proteins. All phases that together determine the pharmacokinetic profile of a drug, absorption, distribution, metabolism and excretion, can be affected during APR. From a clinical point of view however, the effects on absorption and metabolism are the most relevant. For drugs that are given orally, a slower absorption rate is often observed during APR due to a delayed gastric emptying. Even more important from a clinical point of view is the depression of biotransformation capacity in the liver during APR, especially affecting the enzymes of the cytochrome P450 (CYP450) complex. Although much has become known about the mechanism of this effect, a number of questions remain. Cytokines, nitric oxide and possibly the enzyme heme oxygenase are playing a role in a complex process that depends on a mutual interaction between Kupffer cells (macrophages) and hepatocytes in the liver. The clinician should be aware of unexpected changes in drug effects or residue levels due to cumulation of the compound during disease or after vaccination. In these situations, drugs that are excreted unchanged may be better alternatives.

Selective effects of a bacterial infection (Actinobacillus pleuropneumoniae) on the hepatic clearances of caffeine, antipyrine, paracetamol, and indocyanine green in the pig

1. In order to investigate the effect of a bacterial acute phase response model on drug disposition in vivo, plasma clearances of antipyrine, caffeine, paracetamol and indocyanine green were investigated in the healthy and Actinobacillus pleuropneumoniae-infected pig. 2. Indocyanine green plasma and endogenous creatinine clearance were not changed during the infection, which indicates that hepatic blood flow and renal function were not significantly affected. 3. In the A. pleuropneumoniae-infected pig, plasma clearances of antipyrine and caffeine, both marker substrates for hepatic oxidative biotransformation, were decreased by 72 and 68% respectively. The clearance of paracetamol, a drug mainly glucuronidated in the pig, was reduced by 39%. 4. It is concluded that the most important change in drug elimination during an acute phase response induced by A. pleuropneumoniae is a suppression of oxidative hepatic biotransformation.

Cytochrome P450-mediated enzyme activities and polychlorinated biphenyl accumulation in harp seal (Phoca groenlandica)

The presence and activities of hepatic cytochrome P450 (CYP) isoforms in Barents Sea harp seals (n=13) were studied using catalytic activities, selective inhibitors, and western blots. In addition the polychlorinated biphenyl (PCB) burden was measured in both the blubber and the food of the seals. The CYP activities and CYP isoforms present were related to the blubber PCB load and the relative presence of each PCB congener in seals relative to their food (metabolic index). The CYP activities measured were 2‐7 times higher than in ringed and harp seals from other studies, suggesting induction due to contaminant exposure. However, PCB burdens were surprisingly low and were not related to the CYP activities. Probably these high activities were due to exposure to other contaminants or due to the diet consisting mainly of crustaceans, which are rich in carotenes. Carotenes have been shown to be potent CYP inducers in several laboratory animals. The CYP studies indicated the presence of CYP1A, -2B and -3A isoforms. A decrease in the relative presence of a number of PCB congeners in seal blubber relative to their food, supposed to be due to CYP-mediated metabolism, corresponded with the metabolic capabilities of the CYP isoforms found. # 1999 Elsevier Science Ltd. All rights reserved.

Differential effects of pentoxifylline on the hepatic inflammatory response in porcine liver cell cultures. Increase in inducible nitric oxide synthase expression

Pentoxifylline (PTX) has been shown to exert hepatoprotective effects in various liver injury models. However, little information is available about the effect of PTX on the hepatic acute phase response. In the present study, the effect of PTX on a lipopolysaccharide (LPS)-induced acute phase response in primary porcine liver cell cultures was examined. During 72 hr of incubation with or without LPS, the ability of PTX to influence the secretion of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), acute phase proteins, and nitric oxide (NO) was assessed. PTX completely inhibited LPS-induced TNF-alpha production and attenuated IL-6 only after 48 hr of incubation. In contrast, PTX potentiated NO production and the expression of inducible nitric oxide synthase (iNOS) in hepatocytes after stimulation with LPS. The increased expression of iNOS and concurrent production of NO was also observed when liver cell cultures were incubated with dibutyryl cyclic adenosine monophosphate. No effect of PTX on acute phase protein secretion was observed during 72 hr of incubation. The present results show that PTX differentially affects the endotoxin-induced inflammatory response in primary porcine liver cell cultures by suppressing TNF-alpha and IL-6 while potentiating NO production.