Metabolic pathways in immune senescence and inflammaging: Novel therapeutic strategy for chronic inflammatory lung diseases. An EAACI position paper from the Task Force for Immunopharmacology.

The accumulation of senescent cells drives inflammaging and increases morbidity of chronic inflammatory lung diseases. Immune responses are built upon dynamic changes in cell metabolism that supply energy and substrates for cell proliferation, differentiation, and activation. Metabolic changes imposed by environmental stress and inflammation on immune cells and tissue microenvironment are thus chiefly involved in the pathophysiology of allergic and other immune-driven diseases. Altered cell metabolism is also a hallmark of cell senescence, a condition characterized by loss of proliferative activity in cells that remain metabolically active. Accelerated senescence can be triggered by acute or chronic stress and inflammatory responses. In contrast, replicative senescence occurs as part of the physiological aging process and has protective roles in cancer surveillance and wound healing. Importantly, cell senescence can also change or hamper response to diverse therapeutic treatments. Understanding the metabolic pathways of senescence in immune and structural cells is therefore critical to detect, prevent, or revert detrimental aspects of senescence-related immunopathology, by developing specific diagnostics and targeted therapies. In this paper, we review the main changes and metabolic alterations occurring in senescent immune cells (macrophages, B cells, T cells). Subsequently, we present the metabolic footprints described in translational studies in patients with chronic asthma and chronic obstructive pulmonary disease (COPD), and review the ongoing preclinical studies and clinical trials of therapeutic approaches aiming at targeting metabolic pathways to antagonize pathological senescence. Because this is a recently emerging field in allergy and clinical immunology, a better understanding of the metabolic profile of the complex landscape of cell senescence is needed. The progress achieved so far is already providing opportunities for new therapies, as well as for strategies aimed at disease prevention and supporting healthy aging.

AllergoOncology: Opposite outcomes of immune tolerance in allergy and cancer.

While desired for the cure of allergy, regulatory immune cell subsets and nonclassical Th2-biased inflammatory mediators in the tumour microenvironment can contribute to immune suppression and escape of tumours from immunological detection and clearance. A key aim in the cancer field is therefore to design interventions that can break immunological tolerance and halt cancer progression, whereas on the contrary allergen immunotherapy exactly aims to induce tolerance. In this position paper, we review insights on immune tolerance derived from allergy and from cancer inflammation, focusing on what is known about the roles of key immune cells and mediators. We propose that research in the field of AllergoOncology that aims to delineate these immunological mechanisms with juxtaposed clinical consequences in allergy and cancer may point to novel avenues for therapeutic interventions that stand to benefit both disciplines.

AllergoOncology - the impact of allergy in oncology: EAACI position paper.

Th2 immunity and allergic immune surveillance play critical roles in host responses to pathogens, parasites and allergens. Numerous studies have reported significant links between Th2 responses and cancer, including insights into the functions of IgE antibodies and associated effector cells in both antitumour immune surveillance and therapy. The interdisciplinary field of AllergoOncology was given Task Force status by the European Academy of Allergy and Clinical Immunology in 2014. Affiliated expert groups focus on the interface between allergic responses and cancer, applied to immune surveillance, immunomodulation and the functions of IgE-mediated immune responses against cancer, to derive novel insights into more effective treatments. Coincident with rapid expansion in clinical application of cancer immunotherapies, here we review the current state-of-the-art and future translational opportunities, as well as challenges in this relatively new field. Recent developments include improved understanding of Th2 antibodies, intratumoral innate allergy effector cells and mediators, IgE-mediated tumour antigen cross-presentation by dendritic cells, as well as immunotherapeutic strategies such as vaccines and recombinant antibodies, and finally, the management of allergy in daily clinical oncology. Shedding light on the crosstalk between allergic response and cancer is paving the way for new avenues of treatment.

Cigarette smoke suppresses the surface expression of c-kit and FcεRI on mast cells.

Chronic obstructive pulmonary disease (COPD) is a multicomponent disease characterized by emphysema and/or chronic bronchitis. COPD is mostly associated with cigarette smoking. Cigarette smoke contains over 4,700 chemical compounds, including free radicals and LPS (a Toll-Like Receptor 4 agonist) at concentrations which may contribute to the pathogenesis of diseases like COPD. We have previously shown that short-term exposure to cigarette smoke medium (CSM) can stimulate several inflammatory cells via TLR4 and that CSM reduces the degranulation of bone-marrow-derived mast cells (BMMCs). In the current study, the effect of CSM on mast cells maturation and function was investigated. Coculturing of BMMC with CSM during the development of bone marrow progenitor cells suppressed the granularity and the surface expression of c-kit and Fc ε RI receptors. Stimulation with IgE/antigen resulted in decreased degranulation and release of Th1 and Th2 cytokines. The effects of CSM exposure could not be mimicked by the addition of LPS to the culture medium. In conclusion, this study shows that CSM may affect mast cell development and subsequent response to allergic activation in a TLR4-independent manner.

Local free light chain expression is increased in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Free light chain (FLC) concentrations are demonstrated to be increased in different inflammatory disorders and are proposed to mediate mast cell-dependent immune responses. A role for mast cells is suggested in chronic rhinosinusitis with nasal polyposis (CRSwNP), which is characterized by a local Th2 inflammatory response. However, clear mast cell-activating factors are not always apparent. In this study, the presence of FLCs in CRS patients with or without nasal polyps (CRSw/sNP) was investigated and the effect of different treatments on FLC expression was analyzed. METHODS: Nasal tissue, nasal secretion, and serum of control patients, patients with CRSwNP, and CRSsNP were analyzed for the presence of kappa and lambda FLC. The expression of FLCs in nasal polyp tissue was investigated using immunohistochemistry. In addition, FLC was measured in serum and nasal secretion of nasal polyp patients treated with methylprednisolone, doxycycline, anti-IL-5, or placebo. RESULTS: Free light chain concentrations were increased in nasal secretion and mucosal tissue homogenates in patients with chronic rhinosinusitis, and this effect was most prominent in CRSwNP patients. Immunohistochemical analysis confirmed the increased FLC concentrations in nasal polyp tissue. In CRSwNP patients, treatment with methylprednisolone or anti-IL-5 resulted in the reduction in systemic or local FLC concentrations, respectively. CONCLUSION: The presence of FLC in CRSwNP and CRSsNP suggests a possible role in mediating the local immune reaction in the paranasal cavities. Furthermore, the decrease in local FLCs after treatment with anti-IL-5 presumes that IL-5 creates an environment that favors FLC production.

Decrease in immunoglobulin free light chains in patients with rheumatoid arthritis upon rituximab (anti-CD20) treatment correlates with decrease in disease activity.

OBJECTIVES: Immunoglobulin (Ig) free light chains (FLCs) are short-lived B cell products that contribute to inflammation in several experimental disease models. In this study, FLC concentrations in inflamed joints of patients with rheumatoid arthritis (RA) as compared to patients with osteoarthritis were investigated. In addition, the relationship of FLCs and disease activity upon B cell depletion (rituximab) in patients with RA was studied. METHODS: Synovial fluid (SF) and tissue from patients with RA were analysed for local presence of FLCs using ELISA and immunohistochemistry. In addition, FLC concentrations were measured (at baseline, 3 and 6 months after treatment) in 50 patients with RA with active disease who were treated with rituximab. Changes in FLCs were correlated to changes in disease activity and compared to alterations in IgM, IgG, IgA, IgM-rheumatoid factor (RF) and IgG-anti-citrullinated protein antibody (ACPA) concentrations. RESULTS: FLCs were detected in synovial tissue from patients with RA, and high FLC concentrations were found in SF from inflamed joints, which positively correlate with serum FLC concentrations. Serum FLC concentrations significantly correlated with disease activity score using 28 joint counts, erythrocyte sedimentation rate (ESR) and C reactive protein, and changes in FLC correlated with clinical improvement after rituximab treatment. Moreover, effect of treatment on FLC concentrations discriminated clinical responders from non-responders, whereas IgM-RF and IgG-ACPA significantly decreased in both patient groups. CONCLUSIONS: FLCs are abundantly present in inflamed joints and FLC levels correlate with disease activity. The correlation of FLC concentrations and disease activity indicates that FLCs may be relevant biomarkers for treatment response to rituximab in patients with RA and suggests that targeting FLC may be of importance in the therapy of RA.

Depletion of CD4+CD25+ T cells switches the whey-allergic response from immunoglobulin E- to immunoglobulin free light chain-dependent.

BACKGROUND: Symptoms of allergy are largely attributed to an IgE-mediated hypersensitivity response. However, a considerable number of patients also exhibit clinical features of allergy without detectable systemic IgE. Previous work showed that Ig-free light chains (IgLC) may act as an alternate mechanism to induce allergic responses. CD4+CD25+ T cells are crucial in the initiation and regulation of allergic responses and compromised function might affect the response to allergens. OBJECTIVE: To examine the contribution of CD4+CD25+ T cells and IgLC towards the whey-allergic response. METHODS: Mice were sensitized orally with whey using cholera toxin as an adjuvant. CD25+ T cells were depleted in vivo using a CD25 mAb. The acute allergic skin response to whey and ex vivo colon reactivity was measured in the presence or absence of F991, a specific inhibitor of IgLC. Serum whey-specific antibodies and IgLC in serum and mesenteric lymph node (MLN) supernatants were measured. Depletion of CD4+CD25+ T cells was confirmed in the spleen. RESULTS: Anti-CD25 treatment strongly reduced whey-specific antibody levels and resulted in a partial depletion of effector T cells and a major depletion of Foxp3(+) regulatory T cells. Surprisingly, despite the abolished specific IgE response, the acute allergic skin response to whey was not affected. IgLC levels were enhanced in the serum and MLN supernatants of CD25-depleted sensitized mice. F991 inhibited the acute skin response and colon hyperreactivity in anti-CD25-treated mice, indicating that these responses were mainly IgLC dependent. CONCLUSIONS: Depletion of CD4+CD25+ T cells resulted in a switch from an IgE- to an IgLC-dependent acute skin response and functional hyperresponsiveness of the colon. Our data suggest that CD25+ T cells play a crucial role in balancing cow's milk allergy between IgE and IgE-independent responses and both mechanisms might play a role in allergic responses to the same allergen.

Atopic and non-atopic allergic disorders: current insights into the possible involvement of free immunoglobulin light chains.

Allergic diseases have become a serious global health problem in the developed world. IgE interacting with its high-affinitiy receptor FcepsilonRI is considered a major contributing factor to most types of allergies, but depending on the type of allergy, however, a subgroup of patients displays common symptoms and yet lack elevated levels of total serum IgE and/or antigen-specific IgE. Novel therapeutic strategies such as anti-IgE therapy may therefore not be applicable to these patients. It is clear, however, that these patients do display activation of mast cells. In several patients suffering from immunological disorders, an increase in free immunoglobulin (IG) light chain levels can be detected. Previously, we have described the capability of free light chains to elicit immediate hypersensitivity responses. In this Opinion article, we will discuss the role of IgE- and non-IgE-mediated mechanisms in allergic disorders and point out a possible role of free IG light chains in the pathogenesis of the non-atopic types of these allergies.

Cigarette smoke suppresses in vitro allergic activation of mouse mast cells.

BACKGROUND: Mast cells are important effector cells in innate or acquired immunity that contribute to host defence. Excessive activation of mast cells can result in the development of allergic diseases, including atopic asthma. Mast cell activation by IgE and specific antigen induces the cells to release spasmogenic, vasoactive and pro-inflammatory mediators, which enhance airway smooth muscle contraction, vascular permeability and inflammatory cell recruitment. Recently, we have demonstrated that exposure of mast cells to cigarette smoke medium (CSM) triggered mast cells to produce chemokines. On the other hand, smoking may decrease the risk of allergic sensitization, which could be explained by a reduced IgE production or a diminished response of mast cells to activation of the IgE receptor. OBJECTIVE: In this study, we investigated the effect of CSM on the allergic activation of mast cells through IgE and antigen. METHODS: Primary cultured murine mast cells were exposed to CSM and activated with IgE and antigen or lipopolysaccharide (LPS). The release of granules, production of leukotrienes, chemokines and cytokines was determined in the supernatants by ELISA. The effect of CSM exposure on intracellular signalling, especially the nuclear factor (NF)-kappaB and extracellular signal-regulated kinase (Erk)1/2 pathways, was analysed by Western blotting. RESULTS: CSM suppressed IgE-mediated degranulation and cytokine release, but no effect was observed on leukotriene release. CSM induced phosphorylation of Erk1/2 in mast cells. In CSM-exposed mast cells, activating transcription factor (ATF)-1 was phosphorylated after stimulation with IgE/Ag. LPS-activated mast cells were not influenced by CSM. CONCLUSION: Our study suggests that exposure to cigarette smoke may lead to a reduced allergic activation of mast cells without affecting their response to activation via e.g. bacterial-derived LPS.

Cell-mediated cytotoxicity: contact and secreted factors.

The list of cells with cytotoxic potential now may include small resting T cells, but the exact nature of 'lethal hit delivery' by cytotoxic T lymphocytes remains elusive. Cell-mediated cytotoxicity by cytotoxic T lymphocytes is a complex, multistep process which seems likely to be mediated by several different pathways. Recent experimental evidence for the functioning of a novel cytotoxic mechanism through a target cell's surface receptor illustrates and emphasizes the necessity to study the interactions of cytotoxic T lymphocytes and target cells as a whole. Progress is evident in the description of molecular requirements for triggering cytotoxicity, cell-cell contacts and the regulation of the effector responses of cytotoxic T lymphocytes by extracellular, intracellular and granular proteins. Extracellular Ca(2+)-dependent secretion of perforin and protease(s) may explain several aspects of cellular cytotoxicity, whereas the apoptosis-mediating cell surface Fas protein is now implicated in Ca(2+)-independent cytotoxicity.

Phosphorylation of T-lymphocyte plasma membrane-associated proteins by ectoprotein kinases: implications for a possible role for ectophosphorylation in T-cell effector functions.

Extracellular adenosine triphosphate (ATPo) has been suggested to play a role in lymphocyte effector functions. Recently, it has been suggested that MgATP2- may be the molecular species which is involved in modulating the lytic interaction between cytotoxic T-lymphocytes (CTL) and their target cells. In this study, we provide evidence that ATPo mediates the phosphorylation of extracellular proteins on T-lymphocytes through the action of ectoprotein kinases. The ectophosphorylation is temperature-dependent, supported by Mg2+ and Mn2+, and both ATP and GTP, whereas kinase activity and/or substrates were removed by pretreatment of intact lymphocytes with trypsin. We show the presence of extracellular ATP/GTP-binding sites, indicating the presence of ectoenzymes on intact lymphocytes. The major ectoprotein kinase was identified as a casein kinase II-like protein kinase and could be inhibited by heparin, whereas its activity was enhanced by spermine. The ectoprotein kinase showed remarkable substrate specificity, phosphorylating the serum protein vitronectin, but not fibronectin. In experiments with the cell-impermeable protein kinase inhibitor K-252b, we demonstrate the possible functional importance of ectoprotein kinase in CTL-mediated cytotoxicity, i.e., target cell death was completely blocked by K-252b without affecting intracellular phosphorylation. These results suggest that ectoprotein phosphorylation may possibly be an important event in immunologically relevant cell-cell interactions.

Ecto-protein kinases: ecto-domain phosphorylation as a novel target for pharmacological manipulation?

An increasing number of studies document the presence of protein kinases facing outwards at the cell surface of a diverse array of cells. These ecto-protein kinases phosphorylate cell-surface proteins and soluble extracellular substrates, and thus could affect many physiological processes involving cell-cell contacts, cellular differentiation and proliferation, ion fluxes and cellular activation. To date, only limited attention has been paid to exploring ecto-protein kinases as possible pharmacological targets. Here, the identification and physiological role of ecto-protein kinases in different biological systems is described; it is suggested that ecto-protein kinases are attractive and novel candidates for pharmacological manipulation under various (patho)physiological conditions.

Stem cell factor and interleukin-4 increase responsiveness of mast cells to substance P.

The response of mast cells (MC) to non-IgE-mediated stimulation is critically dependent on the population of MC examined. The neuropeptide Substance P (SP) has been reported to activate connective tissue-type MC (CTMC), while mucosal MC (MMC) are not activated by SP. We examined the effect of stem cell factor (SCF) plus interleukin-4 (IL-4) on SP-initiated activation of bone marrow-derived MC (BMMC). Mouse MC, derived from a culture of BM cells with IL-3, were subsequently treated with recombinant SCF plus IL-4 for 6 days. Responsiveness to SP was monitored measuring beta-hexosaminidase and lipid mediator release. Histochemical staining, histamine analysis, and granule protease expression were achieved to characterize the cells. In contrast to IL-3 grown cells, SCF/IL-4-exposed cells showed functional responsiveness to release beta-hexosaminidase (42.25% +/- 1.46% at SP concentration of 100 microM) and produce leukotriene C(4) (LTC(4)) (7.4 +/- 1.5 ng/10(6) cells)/prostaglandin D(2) (PGD(2)) (2.0 +/- 0.3 ng/10(6) cells) upon stimulation by SP. The increase in sensitivity of the cells to SP was not due to differentiation into CTMC, as the cells remained heparin negative. Both SCF and IL-4 were needed because SCF or IL-4 alone were insufficient to keep cells viable after 3 to 4 days post coculture. SP-induced secretion from BMMC cultured in medium containing SCF plus IL-4 (25.76% +/- 1.83%) was higher in comparison with cells cultured with SCF plus IL-3 (8.85% +/- 0.68%).These findings indicate that temporal changes in cytokine expression can influence the sensitivity of MC to non-immunologic stimuli. Local cytokine production leading to an increase in MC responsiveness to SP and inducing secretion of granule content and lipid generation may, therefore, propagate and worsen inflammatory conditions.

Stem cell factor and interleukin-4 induce murine bone marrow cells to develop into mast cells with connective tissue type characteristics in vitro.

In this study, we have developed a method to obtain mast cells with connective tissue type mast cell (CTMC) characteristics directly from mouse bone marrow (BM) cells. BM cells were grown for 3 weeks in presence of interleukin-4 (IL-4) plus stem cell factor (SCF). SCF alone poorly supported growth and development of mast cells. IL-4 dose-dependently enhanced the expression of c-kit and high-affinity receptor for IgE (Fc(epsilon)RI) on the cell surface of SCF-cultured BM cells. Furthermore, cytoplasmic granulation and histamine synthesis of BM-derived mast cells were increased in presence of IL-4 and SCF. Histochemical staining demonstrated that granules were safranin positive. BM-derived mast cells could be activated for granule exocytosis (beta-hexosaminidase release) and lipid mediator generation (LTC4 production) via Fc(epsilon)RI after sensitization with IgE and subsequent crosslinking with multivalent antigen. In addition, mast cells derived from BM cells cultured with SCF plus IL-4 could be activated by substance P, a nonimmunologic stimulus, to release beta-hexosaminidase. The results presented indicate that IL-4 and SCF both have a prominent role in the development of mast cells from murine BM cells in vitro. Mast cells can directly be derived from BM cells in presence of SCF and IL-4 and the cultured cells show typical hallmarks of CTMC, indicating that precursor cells for CTMC may be present in BM. The described culture procedure may be useful to investigate the molecular aspects of the development of committed mast cell lineages.

Nitric oxide production by macrophages stimulated by antigen-binding T-cell factors.

Contact sensitivity to small molecular weight compounds is accompanied by the production of antigen-specific T-cell factors (TCF) shortly after skin application of the sensitizing agents. In this study, we show that macrophages can be activated by these TCF to generate large amounts of nitric oxide (NO). Incubation of the murine macrophage cell line J774 for 24 h with TCF raised against dinitrofluorobenzene (DNFB) or picryl chloride (PCL) resulted in a nitrite accumulation in the culture medium. Priming of J774 with rIFN-gamma synergistically enhanced stimulation of NO synthesis by DNFB-F and PCL-F. A possible contribution of lipopolysaccharide (LPS) as a contaminant of the TCF was excluded. The enhanced production of NO after stimulation with TCF was accompanied with an increased expression of inducible NO synthase. Inclusion of inhibitors of protein tyrosine kinase and protein kinase C inhibited the TCF-induced NO production by macrophages, indicating the involvement of both protein kinases in the signaling pathway activated by TCF. Since NO is an important biological mediator with many immunoregulatory properties, our results suggest a potential role for increased NO production by macrophages in the elicitation of contact sensitivity to small molecular weight compounds.

Depletion of ATP but not of GSH affects viability of rat hepatocytes.

The purpose of this study was to examine the role of glutathione depletion and alterations in the energy status in the induction of acute cytotoxicity to freshly isolated rat hepatocytes. Depletion of intracellular glutathione by diethyl maleate and phorone to levels below 5% of control did not induce loss of viability nor loss of intracellular ATP. Ethacrynic acid, a compound known to deplete mitochondrial GSH in addition to cytosolic GSH, induced cell killing after a depletion of ATP, next to GSH depletion. The results confirmed that depletion of intracellular glutathione alone does not necessarily result in cell killing. Only when glutathione depletion is succeeded by reduction in ATP levels, loss of cell viability is observed. The relationship between alterations in the energy status and the induction of cell death was further substantiated by inhibition of glycolytic and mitochondrial ATP generation. Treatment of hepatocytes either with iodoacetic acid to inhibit glycolysis (in hepatocytes from fed rats) or with potassium cyanide to inhibit mitochondrial respiration (in hepatocytes from both fed and fasted rats) revealed that depletion of intracellular ATP could lead to lethal cell injury. The susceptibility of cells to metabolic inhibition was better reflected by the rate of reduction in the energy charge than by the reduction of ATP alone. In conclusion, our results suggest that alterations of the energy status may be a critical event in the induction of irreversible cell injury. Depletion of cellular GSH is only cytotoxic when followed by a reduction of the energy charge.

Flow-dependent extraction of 1-naphthol by the rat isolated perfused kidney.

The influence of variation of perfusion flow rate on the renal clearance of p-aminohippuric acid and 1-naphthol was studied with an isolated perfused rat kidney preparation. Kidney functions were well maintained at low perfusion flow rates by the use of a fluorocarbon emulsion to increase the oxygen capacity of the perfusion buffer. Renal extraction of p-aminohippuric acid decreased with increasing perfusion flow. Our data show that at high perfusion flow rates maximal extractable perfusion flow forms only a small part of the total perfusion flow. 1-Naphthol is rapidly metabolized to its glucuronide and sulfate conjugate in the isolated perfused rat kidney. Using PAH as a marker for the maximal extractable perfusion flow, 1-naphthol could be regarded as a high-extraction compound even at high perfusion flow rates. Our results suggest that p-aminohippuric acid clearance, rather than total perfusion flow rate, should be used as the measure of maximal extractable blood flow for the estimation of extraction ratio in the isolated perfused kidney of compounds excreted or metabolized by the proximal tubules.

Hepatic, intestinal and renal transport of 1-naphthol-beta-D-glucuronide in mutant rats with hereditary-conjugated hyperbilirubinemia.

Recently, a mutant rat strain was described with a genetic defect for the biliary excretion of organic anions (TR-rats). To determine the possible heterogeneity of the transport systems in liver, intestine and kidney we investigated the transport of the anion 1-naphthol-beta-D-glucuronide (1-NG) in isolated vascularly perfused organ preparations of the rat liver, intestine and kidney of both Wistar rats and TR- rats. 1-NG was administered as such (liver and kidney experiments) or formed intracellularly from 1-naphthol (1-N) (liver and gut experiments). Independent of the type of exposure to 1-NG, the biliary excretion was considerably impaired in TR- rats. In the intestine the total appearance and the vascular/luminal distribution pattern of 1-NG were not significantly different from the values in control rats. Furthermore, no significant disturbance was found with respect to the renal clearance of 1-NG in the TR- rat when compared with the Wistar rat. Thus, the genetic defect in the TR- rat is restricted to an impaired hepatobiliary excretion of 1-NG and does not affect the excretory systems of the intestine and kidney. These results suggest that the excretion of 1-NG by the liver, intestine and kidney involves distinct organ-specific transport systems.

The 3-methylcholanthrene-mimetic effect of 4,4'-dichlorobiphenyl-treatment on phenacetin-induced hepatic glutathione depletion and liver microsomal phenacetin O-deethylation in rats.

Both 4,4'-dichlorobiphenyl (4,4'-DCB) and 3-methylcholanthrene (3-MC) caused a substantial increase of phenacetin-induced hepatic glutathione (GSH) depletion, whereas phenobarbital (PB) had no effect, suggesting that 4,4'-DCB possesses cytochrome P-448 inducing activity. The O-deethylation of phenacetin by liver microsomes from control and PB- and 4,4'-DCB-treated rats showed biphasic Michaelis-Menten kinetics, in contrast to the monophasic course after pretreatment with 3-MC. Hepatic phenacetin levels indicated that in vivo interaction with only a high affinity site is involved in the O-deethylation of phenacetin. 4,4'-DCB and 3-MC caused marked increases in intrinsic clearance and extraction ratio of phenacetin, whereas control values were obtained after PB-treatment. Because of an absence of a spectral change at low phenacetin concentrations, it could not be demonstrated whether the observed differences in metabolism should be ascribed to a change in binding of phenacetin to cytochrome P-450. The results of this study indicate that after pretreatment with various enzyme inducers the phenacetin-induced hepatic GSH depletion strongly correlates with microsomal phenacetin O-deethylation. Further, these findings suggest a discrepancy between 4,4'-DCB and PB in cytochrome P-450 inducing activity, as 4,4'-DCB mimics 3-MC in the induction of phenacetin O-deethylase. The difference between 4,4'-DCB and PB is discussed in relation to the multiplicity and induction of cytochrome P-450 isoenzymes.