Cellular localization of interleukin 13 receptor alpha2 in human primary bronchial epithelial cells and fibroblasts.

BACKGROUND: Interleukin (IL) 13 is a key cytokine in asthma, regulating fibrosis, airway remodeling, induction of immunoglobulin E synthesis by B cells, bronchial hyperresponsiveness, and mucus production. IL-13 signals through the type II IL-4 receptor (IL-4R), which is composed of the IL-4Ralpha and the IL-13Ralpha1 chains. Another IL-13 binding chain, IL-13Ralpha2, binds IL-13 with high affinity but has no known signaling capability and is thought to serve as a decoy receptor providing tight regulation of IL-13 responses. METHODS: In this study, we investigated the cellular localization of IL-13Ralpha2 in human primary bronchial epithelial cells and fibroblasts using flow cytometry and confocal microscopy, as well as the in vivo expression of IL-13Ralpha2 in the human bronchial mucosa by means of immunohistochemistry. RESULTS: IL-13Ralpha2 is predominantly an intracellular rather than a membrane-bound molecule in both human primary bronchial epithelial cells and fibroblasts and displays a diffuse granular cytoplasmic distribution in both cell types. IL-13Ralpha2 protein is expressed in vivo in the human bronchial mucosa with its expression being higher in bronchial epithelial cells than bronchial fibroblasts both in vivo and in vitro. CONCLUSIONS: IL-13Ralpha2 is expressed by both human primary bronchial epithelial cells and fibroblasts as an intracellular protein with a diffuse cytoplasmic distribution. In vivo, IL-13Ralpha2 is expressed in the human airway mucosa mainly by bronchial epithelial cells.

Altered protein tyrosine phosphorylation in asthmatic bronchial epithelium.

A disease-related, corticosteroid-insensitive increase in the expression of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase in asthmatic bronchial epithelium has been shown previously by the current authors. To determine whether this is associated with enhanced intracellular signalling, the aim of this study was to evaluate epithelial tyrosine phosphorylation. Bronchial biopsies were analysed for the presence of phosphotyrosine by immunohistochemistry. Bronchial epithelial cells were exposed to EGF, hydrogen peroxide or tumour necrosis factor-alpha in vitro for measurement of tyrosine phosphorylated signalling intermediates and interleukin (IL)-8 release. Phosphotyrosine was increased significantly in the epithelium of severe asthmatics when compared with controls or mild asthmatics; however, in mild asthma, phosphotyrosine levels were significantly decreased when compared with controls. There was no significant difference between phosphotyrosine levels before or after 8 weeks of treatment with budesonide. Stimulation of bronchial epithelial cells resulted in tyrosine phosphorylation of several proteins, including EGFR, Shc and p42/p44 mitogen-activated protein kinase. In the presence of salbutamol, a transient partial suppression of EGFR phosphorylation occurred, whereas dexamethasone was without effect. Neither salbutamol nor dexamethasone inhibited EGF-stimulated IL-8 release. These data indicate that regulation of protein tyrosine kinase activity is abnormal in severe asthma. The epidermal growth factor receptor and/or other tyrosine kinase pathways may contribute to persistent, corticosteroid-unresponsive inflammation in severe asthma.

Non-bronchoscopic sampling and culture of bronchial epithelial cells in children.

BACKGROUND: The bronchial epithelium is likely to play a vital role in airway diseases in children, such as asthma and viral-associated wheeze. In adults, studies with primary bronchial epithelial cells cultured from samples obtained by fibre-optic bronchoscopy have provided key insights into the role of the epithelial cell. However, it is difficult to justify bronchoscopy in children to obtain epithelial cells for research purposes. OBJECTIVE: To examine the possibility of retrieving and culturing viable epithelial cells using a blind non-bronchoscopic method from children undergoing elective surgery. METHODS: Subjects were children undergoing elective surgery under general anaesthesia. Following intubation, non-bronchoscopic bronchoalveolar lavage and non-bronchoscopic bronchial brushing were performed. A sheathed bronchial cytology brush was advanced through the endotracheal tube, wedged and then withdrawn 2-3 cm before gentle sampling was used to collect bronchial epithelial cells. Initial samples were used to characterize the number, type and viability of epithelial cells recovered compared to a control group of adults undergoing standard bronchoscopic sampling. Subsequent samples were used to establish primary bronchial epithelial cell cultures in children both with and without wheezing illness. RESULTS: A total of 63 children underwent bronchial brushing [38 male; median age 7.1 years (1.0-14.2 years]. Initial samples (n=30) showed recovery of viable epithelial cells comparable to that from a single brush obtained via a bronchoscope in an adult control group (n=11). In 27 (82%) of the subsequent 33 samples obtained non-bronchoscopically from children, primary bronchial epithelial cell cultures were successfully established. There were no adverse effects attributable to sampling. CONCLUSION: We have shown that non-bronchoscopic bronchial brushing is a safe and effective technique for recovering viable bronchial epithelial cells that consistently yield primary cultures. This method will facilitate examination of the role of the epithelium in paediatric disease.

The role of the epidermal growth factor receptor in sustaining neutrophil inflammation in severe asthma.

BACKGROUND: The extent of epithelial injury in asthma is reflected by expression of the epidermal growth factor receptor (EGFR), which is increased in proportion to disease severity and is corticosteroid refractory. Although the EGFR is involved in epithelial growth and differentiation, it is unknown whether it also contributes to the inflammatory response in asthma. OBJECTIVES: Because severe asthma is characterized by neutrophilic inflammation, we investigated the relationship between EGFR activation and production of IL-8 and macrophage inhibitory protein-1 alpha (MIP-1alpha) using in vitro culture models and examined the association between epithelial expression of IL-8 and EGFR in bronchial biopsies from asthmatic subjects. METHODS: H292 or primary bronchial epithelial cells were exposed to EGF or H2O2 to achieve ligand-dependent and ligand-independent EGFR activation; IL-8 mRNA was measured by real-time PCR and IL-8 and MIP-1alpha protein measured by enzyme-linked immunosorbent assay (ELISA). Epithelial IL-8 and EGFR expression in bronchial biopsies from asthmatic subjects was examined by immunohistochemistry and quantified by image analysis. RESULTS: Using H292 cells, EGF and H2O2 increased IL-8 gene expression and release and this was completely suppressed by the EGFR-selective tyrosine kinase inhibitor, AG1478, but only partially by dexamethasone. MIP-1alpha release was not stimulated by EGF, whereas H2O2 caused a 1.8-fold increase and this was insensitive to AG1478. EGF also significantly stimulated IL-8 release from asthmatic or normal primary epithelial cell cultures established from bronchial brushings. In bronchial biopsies, epithelial IL-8, MIP-1alpha, EGFR and submucosal neutrophils were all significantly increased in severe compared to mild disease and there was a strong correlation between EGFR and IL-8 expression (r = 0.70, P < 0.001). CONCLUSIONS: These results suggest that in severe asthma, epithelial damage has the potential to contribute to neutrophilic inflammation through enhanced production of IL-8 via EGFR- dependent mechanisms.

Solution structure of the mEGF/TGFalpha44-50 chimeric growth factor.

The solution structure of the growth factor chimera mEGF/TGFalpha44-50 has been determined using an extended version of the dyana procedure for calculating structures from NMR data. The backbone fold and preferred orientation of the domains of the chimera are similar to those found in previous studies of EGF structures, and several H-bonds used as input constraints in those studies were found independently in the chimera. This shows that the modified activity of the chimera does not result from a major structural change. However, the improved precision of the structure presented here allows the origin of some unusual chemical shifts found in all of these compounds to be explained, as well as the results obtained from some site-specific mutants. Further studies of the properties of this chimeric growth factor should help to elucidate the mechanism(s) of hetero- and homodimerization of the c-erbB receptors.

Signal transducer and activator of transcription 6 (STAT-6) expression and function in asthmatic bronchial epithelium.

BACKGROUND: Asthma is associated with increased production of IL-4 and IL-13. OBJECTIVE: Because many of the effects of these cytokines are mediated by activation of signal transducer and activator of transcription 6 (STAT-6), we investigated expression and function of this transcription factor in the airways. METHODS: STAT-6 expression was investigated through use of immunohistochemistry or RT-PCR applied to bronchial biopsy specimens or brushings from normal control or asthmatic subjects. STAT-6 function was investigated by means of Western blotting and ELISA applied to primary epithelial cell cultures. RESULTS: Immunohistochemistry revealed that the bronchial epithelium was the major site of STAT-6 expression, both cytoplasmic and nuclear staining being observed. The level of STAT-6 expression in subjects with mild asthma (median [range] percent epithelial staining, 3.4% [0% to 16.0%]; n = 14) did not differ significantly from that in normal controls (4.7% [0.0% to 20.0%]; n = 11); however, in subjects with severe asthma, epithelial STAT-6 expression (13.7% [4.8% to 25.7%]; n = 9) was increased in comparison with subjects with mild asthma and normal controls (P < .05). RT-PCR analysis showed that epithelial STAT-6 expression was heterogeneous and comprised both full-length STAT-6 and the dominant-negative variant that lacks the SH2 domain. Treatment of primary cultures of bronchial epithelial cells with IL-4 resulted in STAT-6 phosphorylation and stimulation of IL-8 secretion; however, no difference in the responses of epithelial cells was observed between normal (n = 12) and asthmatic (n = 14) donors. CONCLUSION: These data demonstrate expression and activation of STAT-6 in normal and asthmatic bronchial epithelium. The activity of this transcription factor is likely to play a key role in mediating the responses of the bronchial epithelium to T(H)2 cytokines that are characteristic of the asthmatic phenotype.

The contribution of interleukin (IL)-4 and IL-13 to the epithelial-mesenchymal trophic unit in asthma.

Interleukin (IL)-4 and IL-13 are key proinflammatory cytokines in asthma. Studies in transgenic mice show that both cytokines cause inflammation, but only IL-13 causes subepithelial fibrosis, a characteristic feature of asthma. We compared the in vitro profibrogenic effects of IL-4 and IL-13 using bronchial fibroblasts from asthmatic subjects. In the presence of transforming growth factor (TGF)-beta the cells transformed into contractile myofibroblasts and expressed alpha-smooth muscle actin and procollagen I. IL-4 and IL-13 also stimulated proliferation, but were relatively ineffective in promoting myofibroblast transformation. TGF-beta was more potent than the cytokines in stimulating release of endothelin-1 and vascular endothelial growth factor, whereas IL-4 and IL-13 were more potent stimuli for eotaxin release. Although neither IL-4 nor IL-13 induced profibrotic responses, both cytokines caused a corticosteroid-insensitive stimulation of TGF-beta2 release from primary bronchial epithelial cells. These data indicate that epithelial activation by IL-13 or IL-4 plays a critical role in initiating remodeling through release of TGF-beta2. TGF-beta2 then activates the underlying myofibroblasts to secrete matrix proteins and smooth muscle and vascular mitogens to propagate remodeling changes into the submucosa. In contrast, direct activation of submucosal fibroblasts by IL-4 and IL-13 has a proinflammatory effect via eotaxin release and recruitment of eosinophils into the airways.

Invited lecture: activation of the epithelial mesenchymal trophic unit in the pathogenesis of asthma.

BACKGROUND: A recent NIH Workshop and an ERS Task Force concluded that more work was needed to understand mechanisms of severe and chronic asthma. This report describes a series of studies that identify aberrant epithelial mesenchymal signalling in the airways as an important event in maintaining inflammation and driving remodelling in response to environmental injury. METHODS: Immunohistochemistry, genotyping and functional studies conducted on cultured asthmatic cells and mucosal biopsies were used to identify biochemical pathways involved in epithelial injury and repair in asthma and their relationship to disease severity. RESULTS: Our findings suggest that the asthmatic state results from an interaction between a susceptible epithelium and Th-2-mediated inflammation to alter the communication between the epithelium and the underlying mesenchyme - the epithelial mesenchymal trophic unit - leading to disease persistence, airway remodelling and refractoriness to corticosteroid treatment. CONCLUSIONS: Asthma is more than an inflammatory disorder, but requires engagement of important signalling pathways involved in epithelial repair and tissue remodelling. These pathways involving EGFRs and TGF-betaRs provide targets against which to develop novel therapies for chronic asthma.

Involvement of the epidermal growth factor receptor in epithelial repair in asthma.

Epithelial damage and airway remodeling are consistent features of bronchial asthma and are correlated with disease chronicity, severity, and bronchial hyperreactivity. To examine the mechanisms that control bronchial epithelial repair, we investigated expression of the epidermal growth factor receptor (c-erbB1, EGFR) in asthmatic bronchial mucosa and studied repair responses in vitro. In biopsies from asthmatic subjects, areas of epithelial damage were frequently observed and exhibited strong EGFR immunostaining. EGFR expression was also high in morphologically intact asthmatic epithelium. Using image analysis, EGFR immunoreactivity (% of total epithelial area, median (range) was found to increase from 9.4 (4.1-20.4) in normal subjects (n=10) to 18.4 (9.3-28.9) in mild asthmatics (P<0.01, n=13) and 25.4 (15.4-31.8) in severe asthmatics (P<0.00, n=5). Epithelial EGFR immunoreactivity remained elevated in patients treated with corticosteroids and was positively correlated with subepithelial reticular membrane thickening. Using 16HBE 14o- bronchial epithelial cells, we found that EGF accelerated repair of scrape-wounded monolayers and that the EGFR-selective inhibitor, tyrphostin AG1478, inhibited both EGF-stimulated and basal wound closure whereas dexamethasone was without effect. Intrinsic activation of the EGFR was confirmed by analysis of tyrosine phosphorylated proteins, which revealed a rapid, damage-induced phosphorylation of the EGFR, irrespective of the presence of exogenous EGF. To assess the relationship between EGFR-mediated repair and tissue remodeling, release of the profibrogenic mediator TGF-beta2 was also measured. Scrape wounding increased release of TGF-beta2 from epithelial monolayers and EGF had no additional stimulatory effect. However, when repair was retarded with AG1478, the amount of TGF-beta2 increased significantly. These data indicate that the EGFR may play an important role in bronchial epithelial repair in asthma and that impairment of this function may augment airway remodeling.

Epithelial-mesenchymal interactions in the pathogenesis of asthma.

During lung development, repair, and inflammation, local production of cytokines (eg, transforming growth factor-beta) and growth factors (eg, epidermal growth factor) by epithelial and mesenchymal cells mediate bidirectional growth control effectively creating an epithelial-mesenchymal trophic unit. In asthma the bronchial epithelium is highly abnormal, with structural changes involving separation of columnar cells from their basal attachments and functional changes including increased expression and release of proinflammatory cytokines, growth factors, and mediator-generating enzymes. Beneath this damaged structure there is an increase in the number of subepithelial myofibroblasts that deposit interstitial collagens causing thickening and increased density of the subepithelial basement membrane. Our recent studies suggest that the extent of epithelial damage in asthma may be the result of impaired epidermal growth factor receptor-mediated repair. In view of the close spatial relationship between the damaged epithelium and the underlying myofibroblasts, we propose that impaired epithelial repair cooperates with the T(H)2 environment to shift the set point for communication within the trophic unit. This leads to myofibroblast activation, excessive matrix deposition, and production of mediators that propagate and amplify the remodeling responses throughout the airway wall.

The epidermal growth factor receptor and its ligand family: their potential role in repair and remodelling in asthma.

The bronchial epithelium acts as a physical barrier to the ingress of airborne irritants and plays an active role in the airways immune response, reacting to a range of environmental stimuli to enable interaction with immune and inflammatory cells. It is well established that mucosal damage and epithelial cell shedding are important features of inflammatory airways diseases such as asthma. Evidence now suggests that the repairing epithelium has the potential to contribute to disease chronicity through production of an array of soluble mediators and adhesion molecules. However, the biochemical mechanisms that control epithelial maintenance and repair are poorly understood, even though avoiding or reversing the changes in the airways which are likely to result from chronic inflammation and epithelial restitution remains a significant challenge in asthma therapy. The purpose of this review is to highlight the potential of the epidermal growth factor receptor (EGFR/c-erbB) and its ligands in restitution of the bronchial epithelium. This receptor tyrosine kinase plays a pivotal role in regulation of epithelial cell behaviour, having the capacity to elicit a broad spectrum of cellular responses ranging from migration or proliferation to differentiation and enhanced survival; it also has the ability to regulate expression of various inflammatory mediators, mucins, adhesion molecules, matrix proteins, and metalloproteinases which are relevant to the chronic disease phenotype.

Structure-function studies of ligand-induced epidermal growth factor receptor dimerization.

We present a novel 96-well assay which we have applied to a structure-function study of epidermal growth factor receptor dimerization. The basis of the assay lies in the increased probability of EGFRs being captured as dimers by a bivalent antibody when they are immobilized in the presence of a cognate ligand. Once immobilized, the antibody acts as a tether, retaining the receptor in its dimeric state with a resultant 5-7-fold increase in binding of a radiolabeled ligand probe. When the assay was applied to members of the EGF ligand family, murine EGF, transforming growth factor alpha, and heparin-binding EGF-like growth factor were comparable with human EGF (EC50 = 2nM); betacellulin, which has a broader receptor specificity, was slightly less effective. In contrast, amphiregulin (AR1-84), which has a truncated C-tail and lacks a conserved leucine residue, was ineffective unless used at >1 microM. We further probed the involvement of the C-tail and the conserved leucine residue in receptor dimerization by comparing the activities of two genetically modified EGFs (the chimera mEGF/TGFalpha44-50 and the EGF point mutant L47A) and a C-terminally extended form of AR (AR1-90) with those of two other unrelated EGF mutants (I23T and L15A). The potency of these ligands was in the order EGF > I23T > mEGF/TGFalpha44-50 > L47A = L15A >> AR1-90 > AR1-84. Although AR was much worse than predicted from its affinity, this defect could be partially rectified by co-localization of the immobilizing antibody with heparin. Thus, it seems likely that AR cannot dimerize the EGFR unless other accessory molecules are present to stabilize its functional association with the EGFR.

The interaction of an epidermal growth factor/transforming growth factor alpha tail chimera with the human epidermal growth factor receptor reveals unexpected complexities.

It has been assumed that substitution of homologous regions of transforming growth factor alpha (TGF-alpha) into epidermal growth factor (EGF) can be used to probe ligand-receptor recognition without detrimental effects on ligand characteristics for the human EGF receptor (EGFR). We show that a chimera of murine (m) EGF in which the carboxyl-terminal tail is substituted for that of TGF-alpha (mEGF/TGF-alpha44-50) results in complex features that belie this initial simplistic assumption. Comparison of EGF and mEGF/TGF-alpha44-50 in equilibrium binding assays showed that although the relative binding affinity of the chimera was reduced 80-200-fold, it was more potent than EGF in mitogenesis assays using NR6/HER cells. This superagonist activity could not be attributed to differences in ligand processing or to binding to other members of the c-erbB family. It appeared to be due, in part, to choice of an EGFR-overexpressing target cell where high receptor number compensated for the low affinity of the ligand; it also appeared to be related to the ability of the chimera to activate the EGFR tyrosine kinase. Thus, when EGFR autophosphorylation was measured, mEGF/TGF-alpha44-50 was more potent than EGF, despite its low affinity. When tested using chicken embryo fibroblasts, substitution of the TGF-alpha carboxyl-terminal tail into mEGF failed to enhance its binding affinity for chicken EGFRs; however, the chimera was intermediate in potency between TGF-alpha and mEGF in mitogenesis assays. Our results suggest a contextual requirement for EGFR recognition which is ligand-specific. Further, the unpredictable responses to chimeric ligands underline the complex nature of the processes of ligand recognition, receptor activation, and the ensuing cellular response.

The significance of valine 33 as a ligand-specific epitope of transforming growth factor alpha.

Although binding of epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) to the EGF receptor (EGFR) is mutually competitive, their binding is not identical, and their biological activities are not always equivalent. To probe for ligand-specific interactions, we have synthesized analogues of TGFalpha with modifications to the residue lying between the fourth and fifth cysteines (the "hinge"). Although this residue lies in a structurally conserved region of the protein, it is not conserved within the EGFR ligand family. Our results show that in TGFalpha there is a preference for a bulky hydrophobic hinge residue; this contrasts with EGF, for which a hydrogen bond donor functionality is preferred. Sequence analysis of the human EGFR ligands revealed that the nature of the hinge residue correlated with the sequence in the B-loop beta-sheet. As this region is an important determinant in recognition of TGFalpha by the chicken EGFR, we assessed the mitogenicity of the TGFalpha hinge mutants, as well as the other EGFR ligands, using chicken embryo fibroblasts. The preference of the chicken EGFR for TGFalpha hinge mutants with hydrophobic side chains paralleled that of the human EGFR. Betacellulin and heparin-binding EGF-like growth factor also possess an hydrophobic hinge; both were at least as potent as TGFalpha for chicken embryo fibroblasts. EGF and amphiregulin, both with hydrogen bond donor functionalities at their hinge, displayed markedly decreased in potency by comparison with TGFalpha. We propose that EGFR ligands can be subclassified into TGFalpha-like and EGF-like and that this is of functional significance, identifying a potential mechanism whereby EGFR can discriminate between its ligands.

Investigations into the actions of glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1(7-36)amide on lipoprotein lipase activity in explants of rat adipose tissue.

The direct actions of glucose-dependent insulinotropic polypeptide, glucagon-like peptide-1(7-36)amide and insulin on lipoprotein lipase activity in explants of rat epididymal adipose tissues were investigated. Lipoprotein lipase was extracted into the incubation medium by heparin release of lipoprotein lipase and measured by fatty acid release from a glyceroltriolein emulsion. Insulin and glucose-dependent insulinotropic polypeptide caused a significant stimulation of lipoprotein lipase activity over a dose range of 0.25-4 nmol/L and 4-8 nmol/L, respectively. Explants incubated in the presence of both insulin and glucose-dependent insulinotropic polypeptide (at 0.5 and 4 nmol/L, respectively) showed levels of lipoprotein lipase activity significantly greater than that seen with either hormone alone. Neither insulin- nor glucose-dependent insulinotropic polypeptide-stimulated lipoprotein lipase was modified by the presence of the antibiotic actinomycin-D in the incubation medium, indicating that these two hormones exert their actions on the pre-existing cellular pool of lipoprotein lipase. Glucagon-like polypeptide-1(7-36)amide, over a dose range of 1-8 nmol/L, did not stimulate lipoprotein lipase activity. This study indicates that glucose-dependent insulinotropic polypeptide, in addition to stimulating insulin secretion, has a direct biological action on adipose tissue and in vivo, together with insulin, may promote lipoprotein lipase activity postprandially.

Contribution of the transforming growth factor alpha B-loop beta-sheet to binding and activation of the epidermal growth factor receptor.

We have exploited the differences in binding affinities of the chicken epidermal growth factor (EGF) receptor for EGF and transforming growth factor alpha (TGF alpha) to study the role of the B-loop beta-sheet of these ligands in receptor recognition and activation. Although EGF and TGF alpha share similar secondary and tertiary structures imposed by three highly conserved intramolecular disulfide bonds, they have only 30-40% overall sequence identity. The B-loop beta-sheet is the major structural element in EGF and TGF alpha, but sequence similarity in this region is low. To investigate its role in receptor binding, we constructed two chimeric growth factors (mEGF/hTGF alpha 21-30 and mEGF/hTGF alpha 21-32) composed of the murine EGF (mEGF) amino acid sequence with residues 21-30 of the B-loop beta-sheet replaced by the equivalent residues of human TGF alpha (hTGF alpha); in chimera mEGF/hTGF alpha 21-32, asparagine 32, which lies at the boundary of the amino and carboxyl domains of mEGF, was also replaced by its hTGF alpha counterpart (valine). In initial studies using unpurified medium, it was found that the recombinant growth factors exhibited differing mitogenic potencies (mEGF/hTGF alpha 21-32 > mEGF/hTGF alpha 21-30 > mEGF) when assayed on chicken fibroblasts, even though they were equivalent in mitogenesis assays using cells expressing the human EGF receptor. After purification, mEGF/hTGF alpha 21-32 was found to be 50 times more potent than mEGF in the chick fibroblast mitogenesis assay and exhibited a 10-fold increase in relative affinity for the chicken EGF receptor; both growth factors still exhibited equivalent mitogenic and receptor binding activity when tested on cells expressing human EGF receptors. We conclude that the B-loop beta-sheet of hTGF alpha is an important determinant of EGF receptor binding affinity and biological activity.

Mutagenicity and CYP1A induction by azobenzenes correlates with their carcinogenicity.

The genotoxicity of six azobenzenes was evaluated in the Ames test, in the presence of an activation system derived from Aroclor 1254-treated rats. Moreover, the ability of these azobenzenes to induce rat hepatic CYP1A activities and apoprotein levels, and stimulate their own bioactivation to mutagens, was also determined. In the presence of the Aroclor 1254-activation system, o-aminoazotoluene and 3-methoxy-4-aminoazobenzene were potent mutagens, whereas 4-amino-azobenzene and 4-diethylaminoazobenzene failed to elicit a positive mutagenic response. A very weak mutagenic response was induced by 2-methyl-4-dimethylaminoazobenzene and by azobenzene. o-Aminoazotoluene and 3-methoxy-4-aminoazobenzene were potent inducers of CYP1A activities and apoprotein levels, whereas the remaining four compounds displayed either very weak or no induction capability. None of the azobenzenes studied could induce its own activation to mutagens in the Ames test. All six azobenzenes displaced [3H]tetrachlorodibenzo-p-dioxin from the cytosolic Ah receptor, with o-aminoazotoluene and 3-methoxy-4-aminoazobenzene being the most effective. A correlation appears to exist between carcinogenic activity of azobenzenes in the rat on one hand, and of their mutagenic potential and hepatic CYP1 induction on the other. Possible mechanisms accounting for this relationship are discussed.

Pretranslational regulation of the expression of the lipoprotein lipase (EC 3.1.1.34) gene by dietary fatty acids in the rat.

Although there have been a number of studies of effects of diet and hormones on lipoprotein lipase (EC 3.1.1.34; LPL) activity and levels of LPL mRNA (Raynolds et al. 1990), there have been no studies which have investigated effects of different dietary fatty acids on LPL gene expression. In the present study male Wistar Albino rats were pair-fed diets containing 50 g fat/kg of different fatty acid composition for 2 weeks. The diets fed were (1) a mixed oil (450 g saturated fatty acids, 420 g monounsaturated fatty acids, 130 g polyunsaturated fatty acids/kg; n 8), (2) maize oil (n 8), or (3) fish oil (n 8). Animals were killed, RNA was extracted from liver and perirenal and epididymal fat pads, and analysed by 'Northern methodology'. Samples were hybridized to a human cDNA probe for LPL (Gotoda et al. 1989). Two transcripts were identified in epididymal and perirenal adipose tissue which were approximately 3.7 and 1.7 kb in size. The results suggested that (1) fish oil-fed animals had significantly greater production of LPL mRNA in epididymal adipose tissue compared with maize oil-fed animals (P < 0.05), (2) maize oil-fed animals had significantly greater production of LPL mRNA in perirenal fat compared with the other dietary groups (P < 0.05), (3) expression in the liver was not significant. Rats fed on a fish oil diet had significantly reduced plasma triacylglycerol concentrations compared with the mixed-oil group (P < 0.05), but there were no significant differences in plasma cholesterol. The differences in LPL could not be explained directly by the changes in plasma immunoreactive-insulin and glucose-dependent insulinotrophic polypeptide levels in the three groups.