Long-term results of an intensive cognitive behavioral pain management program for patients with chronic low back pain: a concise report of an extended cohort with a minimum of 5-year follow-up.

PURPOSE: Treatment options for chronic low back pain (CLBP) include cognitive behavioral interventions. Most of these interventions only have small and short-lived effects. Using strict inclusion criteria for participation in an intensive combined physical and psychological program, encouraging effects were reported at 1-year follow-up. This study evaluates the long-term follow-up results of the same program. The hypothesis is that previously reported results are maintained. METHODS: Structured interviews were conducted in a prospective extended cohort with a minimum of 5-year follow-up in a similar fashion as in the 1-year follow-up report. The median follow-up in this cohort was 6.5 years. The extended cohort consisted of 277 patients (85% response). RESULTS: Outcomes include daily functioning, quality of life, current pain intensity, pain disturbance in daily activities and indicators of the use of pain medication and healthcare services. The previously reported positive 1-year follow-up results were maintained at a minimum of 5-year follow-up. Disability as measured with the Oswestry disability index (ODIv2.1a) decreased from 40 to 27 in the first year. This positive result was maintained at the 6.5-year follow-up with an ODI of 28. Pain intensity (NRS 0-100) improved from 60 to 39 in the first year, and at 6.5 years, this had further improved to 33. Improvement in quality of life (SF 36) at 1-year follow-up was maintained at 6.5-year follow-up, and healthcare consumption had decreased substantially as measured with doctor visits and analgesics used for CLBP. CONCLUSION: Selected and motivated patients with longstanding CLBP improve fast after an intensive combined physical and psychological program in daily functioning, pain and quality of life. Positive 1-year results are maintained, and healthcare utilization was still reduced at a minimum of 5-year follow-up. These slides can be retrieved under Electronic Supplementary Material.

Ibrutinib Dosing Strategies Based on Interaction Potential of CYP3A4 Perpetrators Using Physiologically Based Pharmacokinetic Modeling.

Based on ibrutinib pharmacokinetics and potential sensitivity towards CYP3A4-mediated drug-drug interactions (DDIs), a physiologically based pharmacokinetic approach was developed to mechanistically describe DDI with various CYP3A4 perpetrators in healthy men under fasting conditions. These models were verified using clinical data for ketoconazole (strong CYP3A4 inhibitor) and used to prospectively predict and confirm the inducing effect of rifampin (strong CYP3A4 inducer); DDIs with mild (fluvoxamine, azithromycin) and moderate inhibitors (diltiazem, voriconazole, clarithromycin, itraconazole, erythromycin), and moderate (efavirenz) and strong CYP3A4 inducers (carbamazepine), were also predicted. Ketoconazole increased ibrutinib area under the curve (AUC) by 24-fold, while rifampin decreased ibrutinib AUC by 10-fold; coadministration of ibrutinib with strong inhibitors or inducers should be avoided. The ibrutinib dose should be reduced to 140 mg (quarter of maximal prescribed dose) when coadministered with moderate CYP3A4 inhibitors so that exposures remain within observed ranges at therapeutic doses. Thus, dose recommendations for CYP3A4 perpetrator use during ibrutinib treatment were developed and approved for labeling.

Mechanistic understanding of the nonlinear pharmacokinetics and intersubject variability of simeprevir: A PBPK-guided drug development approach.

Simeprevir, a hepatitis C virus (HCV) NS3/4A protease inhibitor, displays nonlinear pharmacokinetics (PK) at therapeutic doses. Using physiologically based PK modeling, various drug-drug interactions were simulated with simeprevir as victim drug to identify whether saturation of the predominant metabolic enzyme (CYP3A4) or the active hepatic transporters (organic anion-transporting polypeptide (OATP)1B1/3) could account for the nonlinear PK. Interactions with ritonavir, a strong CYP3A4 inhibitor that does not affect OATP (at 100 mg dose), erythromycin, a moderate CYP3A4 inhibitor, and efavirenz, a moderate CYP3A inducer that does not affect OATP, demonstrated the involvement of CYP3A4. Interaction studies with low-dose cyclosporine confirmed the role of OATP. The interplay between hepatic uptake and CYP3A4 metabolism was verified by simulations with rifampicin, a potent CYP3A4 inducer and OATP1B1/3 inhibitor, and maintenance doses of cyclosporine. Saturation of gut and liver metabolism by CYP3A4, and saturation of hepatic uptake by OATP1B1/3, seem to account for the observed nonlinear PK of simeprevir.

Drug-drug interactions between rosuvastatin and oral antidiabetic drugs occurring at the level of OATP1B1.

Organic anion-transporting polypeptide 1B1 (OATP1B1) is an important hepatic uptake transporter, of which the polymorphic variant OATP1B1*15 (Asn130Asp and Val174Ala) has been associated with decreased transport activity. Rosuvastatin is an OATP1B1 substrate and often concomitantly prescribed with oral antidiabetics in the clinic. The aim of this study was to investigate possible drug-drug interactions between these drugs at the level of OATP1B1 and OATP1B1*15. We generated human embryonic kidney (HEK)293 cells stably overexpressing OATP1B1 or OATP1B1*15 that showed similar protein expression levels of OATP1B1 and OATP1B1*15 at the cell membrane as measured by liquid chromatography-tandem mass spectrometry. In HEK-OATP1B1*15 cells, the V(max) for OATP1B1-mediated transport of E(2)17ß-G (estradiol 17ß-d-glucuronide) was decreased >60%, whereas K(m) values (Michaelis constant) were comparable. Uptake of rosuvastatin in HEK-OATP1B1 cells (K(m) 13.1 ± 0.43 µM) was nearly absent in HEK-OATP1B1*15 cells. Interestingly, several oral antidiabetics (glyburide, glimepiride, troglitazone, pioglitazone, glipizide, gliclazide, and tolbutamide), but not metformin, were identified as significant inhibitors of the OATP1B1-mediated transport of rosuvastatin. The IC(50) values for inhibition of E(2)17ß-G uptake were similar between OATP1B1 and OATP1B1*15. In conclusion, these studies indicate that several oral antidiabetic drugs affect the OATP1B1-mediated uptake of rosuvastatin in vitro. The next step will be to translate these data to the clinical situation, as it remains to be established whether the studied oral antidiabetics indeed affect the clinical pharmacokinetic profile of rosuvastatin in patients.

In silico and in vitro pharmacogenetics: aldehyde oxidase rapidly metabolizes a p38 kinase inhibitor.

The clinical development of a candidate p38 kinase inhibitor was terminated because of its unexpectedly rapid clearance in human subjects. Its short half-life and metabolic profile in human beings were vastly different from that in rats, dogs, and monkeys characterized during routine pre-clinical studies. Mice generated the predominant drug (4-hydroxylated) metabolite produced in human beings, which was not found in other species. The data from a murine in vitro drug biotransformation assay that used liver extracts from 14 inbred mouse strains were analyzed by haplotype-based computational genetic analysis. This led to the identification of aldehyde oxidase-1 (AOX1) as the enzyme responsible for the rapid metabolism of this drug. Specific enzyme inhibitors and expressed recombinant enzymes were used to confirm that AOX catalyzed the formation of the 4-hydroxylated drug metabolite in mouse and man. Genetic variation within Aox1 regulated the level of hepatic Aox1 mRNA, AOX1 protein, and enzyme activity among the inbred strains. Thus, computational murine pharmacogenetic analysis can facilitate the identification and characterization of drug metabolism pathways that are differentially utilized by humans and other species.

Prediction of whole-body metabolic clearance of drugs through the combined use of slices from rat liver, lung, kidney, small intestine and colon.

1: The aim was to investigate whether precision-cut rat tissue slices could be used to predict metabolic drug clearance in vivo. To obtain a complete picture, slices not only from liver, but also from lung, kidney, small intestine and colon were included. 2: The metabolic clearances of 7-ethoxycoumarin, 7-hydroxycoumarin, testosterone, methyltestosterone and warfarin were determined by measuring the disappearance of these compounds during incubation with slices prepared from liver, lung, kidney, small intestine and colon. 3: The total in vitro metabolic clearance was determined by adding the individual in vitro organ clearances from the slices. Prediction based on the in vitro clearance was within an order of magnitude to the corresponding in vivo values. Interestingly, the relative contribution of extrahepatic metabolic clearance of the studied compounds to total clearance was remarkably high, ranging from 35 to 72% of the total metabolic clearance. 4: It is concluded that the model of multi-organ precision-cut slices is a useful in vitro tool for prediction of in vivo metabolic clearance. In addition, it provides information about the relative contribution of the liver, lung, kidney, small intestine and colon to the total metabolic clearance.

Precision-cut organ slices as a tool to study toxicity and metabolism of xenobiotics with special reference to non-hepatic tissues.

Metabolism of xenobiotics is often seen as an exclusive function of the liver, but some current findings support the notion that the lungs, kidneys and intestine may contribute considerably. After the establishment of the use of liver slices as a useful in vitro model to study metabolism and toxicity of xenobiotics, the same concept is currently being used for slices from lung, kidney and intestine. It is the aim of this review to discuss the use of organ slices in biotransformation research. The basic idea behind the use of tissue slices in biomedical research is the assumption that the cells under study will function optimally in vitro if they are cultivated in an environment that is most alike to their natural in vivo embedding, which is the case in tissue slices. Advantages in the use of organ slices are the relatively easy preparation as well as the potential standardization of both the preparation and use. Moreover, a direct interspecies comparison can be made between liver, lungs, kidneys and intestines, for example with respect to their metabolic capacity and their sensitivity for toxicants. Of major importance is that organ slices can be made with a similar procedure from organs/tissues originating from different species, including man. This latter aspect is useful in drug development in general but also for a better insight in the metabolic fate of compounds in man. Importantly the use of slices may largely contribute to a reduction in the use of experimental animals.

The use of genetically engineered cells for assessing CYP2D6-related polymorphic effects.

As an example of advanced testing in the field of metabolism in an industrial environment, the introduction of some novel approaches, including the use of genetically engineered cell lines for assessing CYP 2D6-related polymorphic effects is illustrated. In this paper, it is demonstrated that novel in vitro test systems can be developed by using these genetically engineered cell lines for evaluating the potential risks associated with proprietary drugs (especially if their metabolism depends to a high extent on CYP 2D6). Moreover, it is demonstrated that, by the use of these in vitro methods, issues such as polymorphism, for which no animal models are available, can be assessed in such a way that predictions can be made on adverse effects which, up to now, could only be detected during clinical trials. Through the use of these new biotechnological in vitro metabolism models, clinically relevant data can be obtained for a scientifically-based human risk assessment, and animal use can be reduced.

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.

Direct cell-to-cell contact between Kupffer cells and hepatocytes augments endotoxin-induced hepatic injury.

This study focuses on the importance of direct contact between Kupffer cells (KCs) and hepatocytes (HCs) during the hepatic inflammatory response using an in vitro approach. The lipopolysaccharide (LPS)-induced inflammatory response in monocultures of porcine HCs and KCs were compared with cocultures prepared either with direct contact between KCs and HCs (DC cocultures) or without direct contact using cell culture membrane inserts. Our data show that DC cocultures exhibited the highest production of tumor necrosis factor (TNF)-alpha, interleukin-6, and nitric oxide (NO) compared with the other cultures. Immunohistochemical studies revealed that TNF-alpha was exclusively produced by KCs, whereas HCs were responsible for NO production after LPS stimulation. Biotransformation capacity, as determined by cytochrome P-450 and UDP glucuronosyl transferase enzyme activities, was most significantly decreased in DC cocultures. These results provide evidence that direct contact between KCs and HCs favors the extensive TNF-alpha production by KCs but in turn affects HC functionality and viability. These findings suggest that direct contact between KCs and HCs plays a key role in the development of a fulminating hepatic inflammatory response.

Decrease in brain cytochrome P450 enzyme activities during infection and inflammation of the central nervous system.

The effect of infection and inflammation of the central nervous system (CNS) on cytochrome-P450-dependent activities in brain, spinal cord and liver microsomes was determined. For this, two models were used: (1) the intracerebroventricularly injected lipopolysaccharide (LPS) model and (2) the experimental auto-immune encephalomyelitis (EAE) model. In the LPS model, aminopyrine N-demethylase (AMND) and ethoxycoumarin O-deethylase (ECOD) activities (both P450 dependent) were significantly decreased (35 and 20%, respectively) in brain microsomes. In the EAE model, only ECOD activity was significantly lower (18%). In the liver, a decrease in total P450, AMND and ECOD activities was only observed in the LPS model. In both models, tumour necrosis factor (TNF) was significantly elevated in brain and spinal cord tissues. In serum, TNF was only detectable in the LPS model. It is concluded that an infection or inflammation located in the CNS, which is accompanied by high TNF levels, results in a decrease in P450-dependent metabolism not only in the liver but in the brain as well.

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

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-alpha (TNF-alpha) 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-alpha production in monocytes/macrophages. In our laboratory it has been shown that the alternative way to increase cAMP levels, via stimulation of beta-adrenergic receptors, also provides an effective way, both in vitro and in vivo, to inhibit TNF-alpha 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-kappa B 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.

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.

Cocultures of porcine hepatocytes and Kupffer cells as an improved in vitro model for the study of hepatotoxic compounds.

In this study primary hepatocyte cultures (HC cultures) and cocultures comprised of hepatocytes and Kupffer cells (HC/KC cocultures) were compared to investigate the inflammatory response induced by lipopolysaccharide (LPS). In addition both culture types were compared to study the hepatotoxic effects of two frequently used drugs: tiamulin and chlorpromazine. The inflammatory response in both culture types was determined by measurement of tumour necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6) and nitric oxide (NO). The drug-induced hepatotoxic effects were determined by measuring production of intracellular reactive oxygen species (ROS) and cytotoxicity. Exposure of both cultures to LPS resulted in a significantly increased production of TNF-alpha, IL-6 and NO. However, the production of TNF-alpha, IL-6 and NO was substantially increased in culture supernatant of cocultures, compared to single HC-cultures. Both tiamulin and chlorpromazine were potent inducers of intracellular ROS production at concentrations > or = 50 microM. High ROS production was paralleled by increased cytotoxicity as observed in both culture types. Incubation of cocultures with chlorpromazine resulted in a significant increased ROS production as compared to HC cultures. In contrast, no significant differences between HC-cultures and HC/KC cocultures were observed for tiamulin induced ROS production or cytotoxicity. The present study demonstrates that cocultures between Kupffer cells and hepatocytes provide an excellent model for the study of hepatotoxic compounds which exert (part) of their toxic effects via the activation of Kupffer cells. Furthermore they offer a valuable tool to study increased susceptibility to intoxication from xenobiotic agents in case of a concurrent or pre-existing inflammation.

Suppression of the acute inflammatory response of porcine alveolar- and liver macrophages.

During infection and inflammation drug disposition and hepatic metabolism are markedly affected in mammals. Pro-inflammatory mediators play an important role in the suppression of (cytochrome-P450-mediated) drug metabolism. Inflammatory mediators like cytokines, nitric oxide (NO), reactive oxygen species (ROS) and eicosanoids are released by activated macrophages from various sources, including liver and lung. It was the aim of this study to investigate ways to suppress the activation of macrophages during the onset of the inflammatory cascade. Therefore porcine lung and liver macrophages were isolated, and incubated with lipopolysaccharide (LPS) to initiate an acute inflammatory response, represented by the release of high amounts of tumour necrosis factor-alpha (TNF-alpha) into the culture medium. Additionally the primary macrophages were coincubated with phosphodiesterase-IV-(PDE-IV)-inhibitors or beta-adrenoceptor agonists that in previous studies demonstrated strong suppressive effects on TNF-alpha release. Especially the beta-adrenoceptor agonists showed to be very potent TNF-alpha suppressants, which indicates that the beta-adrenoceptor might be an interesting target for suppression of activation of macrophages. This was strengthened by the observation that the beta-adrenoceptor expression was not altered during the onset of the inflammatory cascade.

The beta-adrenoceptor agonist clenbuterol is a potent inhibitor of the LPS-induced production of TNF-alpha and IL-6 in vitro and in vivo.

OBJECTIVE AND DESIGN: To investigate the suppressive effects of the beta-agonist clenbuterol on the release of TNF-alpha and IL-6 in a lipopolysaccharide (LPS)-model of inflammation, both in vitro and in vivo. MATERIAL AND SUBJECTS: Human U-937 cell line (monocyte-derived macrophages), and male Wistar rats (200-250 g). TREATMENT: U-937 macrophages were incubated with LPS at 1 microg/ml, with or without 1.0 mM-0.1 nM test drugs (clenbuterol and other cAMP elevating agents) for 1-24 h. Rats were administered either 1 or 10 microg/kg clenbuterol (or saline) orally, 1 h before intraperitoneal administration of 2 mg/kg LPS. METHODS AND RESULTS: TNF-alpha and IL-6 time-concentration profiles were determined both in culture media and plasma, using ELISA' s and bioassays. LPS-mediated release of both cytokines was significantly suppressed by clenbuterol. CONCLUSIONS: The beta-agonist clenbuterol very potently suppresses the LPS-induced release of the pro-inflammatory cytokines TNF-alpha and IL-6 both in vitro and in vivo.

Participation of beta-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.

Novobiocin inhibits both UDP-glucuronosyltransferase and cytochrome P450-mediated enzyme activities in pig liver microsomes.

The effects of novobiocin (range 0.0125-2 mmol/L) on the hydroxylation of testosterone, the N-demethylation of erythromycin, and the glucuronidation of alpha-naphthol and paracetamol were studied using pig hepatic microsomes, pooled from five animals. The final concentrations of these substrates in the incubation mixtures were selected to meet Vmax conditions. Novobiocin caused a concentration-dependent inhibition of the glucuronidation of paracetamol; the formation of alpha-naphthol-glucuronide was reduced to a lesser degree. These results confirm and extend earlier findings in laboratory animal species that novobiocin inhibits UDP-glucuronosyltransferases (UDPGTs). Moreover, novobiocin strongly inhibited 6 beta-hydroxylation of testosterone. The microsomal N-demethylation of erythromycin and hydroxylation of testosterone at the 15 alpha position were less affected by novobiocin. These results suggest that novobiocin inhibits not only UDPGTs, but also cytochrome P450 (CYP) enzyme activities, probably those belonging to the CYP3A subfamily. More research is needed to reveal which CYPs and UDPGTs are affected by novobiocin in vivo, in order to improve the understanding the probably the predictability of potential drug interactions with this antibiotic.

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.

Kupffer cell depletion partially prevents hepatic heme oxygenase 1 messenger RNA accumulation in systemic inflammation in mice: role of interleukin 1beta.

The heme oxygenase 1 (HO-1) gene is rapidly activated in the liver after lipopolysaccharide (LPS) treatment. Ninety minutes after LPS treatment (0.1 mg/kg, intraperitoneally) hepatic HO-1 messenger RNA (mRNA) of mice was 40 times the control value. To investigate the hepatic cellular source of the increased HO-1 transcript, we treated mice with LPS and galactosamine (700 mg/kg, intraperitoneally), a selective transcriptional inhibitor of hepatocytes. Galactosamine prevented the LPS-mediated increase of HO-1 mRNA in the liver, indicating that hepatocytes are the main cell type in which HO-1 mRNA accumulates after LPS treatment. We then tested in vitro and in vivo the hypothesis that LPS-mediated hepatic accumulation of HO-1 mRNA is caused by intercellular communication between Kupffer cells and hepatocytes. Isolated rat hepatocytes showed an increase in HO-1 mRNA compared with controls after 90 minutes of exposure to a LPS stimulated Kupffer cell-conditioned medium. This suggests that soluble mediators from Kupffer cells were responsible for this effect. To study the role of Kupffer cells in vivo, we treated mice with Kupffer cell-inactivating or -depleting agents and LPS. Gadolinium chloride and liposome-encapsulated dichloromethylene diphosphonate lowered LPS-mediated HO-1 mRNA accumulation (by about 50%); in these groups hepatic levels of interleukin (IL)-1beta were decreased, by more than 75%. Methylpalmitate hardly affected hepatic HO-1 mRNA accumulation or IL-1beta content after LPS treatment. There was no relationship between HO-1 mRNA and serum TNF or IL-6 levels. These results suggest that LPS-mediated hepatic HO-1 mRNA accumulation is a hepatocyte response partly caused by soluble mediators, particularly IL-1beta, released from Kupffer cells.