M.J. Garle

Inhibition of fatty acid amide hydrolase and cyclooxygenase-2 increases levels of endocannabinoid related molecules and produces analgesia via peroxisome proliferator-activated receptor-alpha in a model of inflammatory pain.

The antinociceptive effects of the endocannabinoids (ECs) are enhanced by inhibiting catabolic enzymes such as fatty acid amide hydrolase (FAAH). The physiological relevance of the metabolism of ECs by other pathways, such as cyclooxygenase-2 (COX2) is less clear. To address this question we compared the effects of local inhibition of FAAH versus COX2 (URB597 and nimesulide, respectively) on inflammatory hyperalgesia and levels of endocannabinoids and related molecules in the hindpaw. Inflammatory hyperalgesia was measured following intraplantar injection of carrageenan. Effects of intraplantar injection of URB597 (25 microg and 100 microg) or nimesulide (50 microg) on hyperalgesia and hindpaw levels of anandamide (AEA), 2-arachidonoylglycerol (2AG) and N-palmitoylethanolamine (PEA) were determined. Although both doses of URB597 increased levels of AEA and 2AG in the carrageenan inflamed hindpaw, only the lower dose of URB597 attenuated hyperalgesia (P<0.05). Nimesulide attenuated both hyperalgesia and hindpaw oedema (P<0.001, P<0.01, respectively) and increased levels of PEA (P<0.05) in the hindpaw. Since both AEA and PEA are ligands for peroxisome proliferator-activated receptor-alpha (PPARalpha), the effects of the PPARalpha antagonist GW6471 on nimesulide- and URB597-mediated effects were studied. GW6471, but not a PPARgamma antagonist, blocked the inhibitory effects of nimesulide and URB597 on hyperalgesia. Our data suggest that both COX2 and FAAH play a role in the metabolism of endocannabinoids and related molecules. The finding that PPARalpha antagonism blocked the inhibitory effects of nimesulide and URB597 suggests that PPARalpha contributes to their antinociceptive effects in the carrageenan model of inflammatory hyperalgesia.

Tonic Modulation of Spinal Hyperexcitability by the Endocannabinoid Receptor System in a Rat Model of Osteoarthritis Pain

Objective To investigate the impact of an experimental model of osteoarthritis (OA) on spinal nociceptive processing and the role of the inhibitory endocannabinoid system in regulating sensory processing at the spinal level. Methods Experimental OA was induced in rats by intraarticular injection of sodium mono-iodoacetate (MIA), and the development of pain behavior was assessed. Extracellular single-unit recordings of wide dynamic range (WDR) neurons in the dorsal horn were obtained in MIA-treated rats and saline-treated rats. The levels of endocannabinoids and the protein and messenger RNA levels of the main synthetic enzymes for the endocannabinoids (N-acyl phosphatidylethanolamine phospholipase D [NAPE-PLD] and diacylglycerol lipase α [DAGLα]) in the spinal cord were measured. Results Low-weight (10 gm) mechanically evoked responses of WDR neurons were significantly (P < 0.05) facilitated 28 days after MIA injection compared with the responses in saline-treated rats, and spinal cord levels of anandamide and 2-arachidonoyl glycerol (2-AG) were increased in MIA-treated rats. Protein levels of NAPE-PLD and DAGLα, which synthesize anandamide and 2-AG, respectively, were elevated in the spinal cords of MIA-treated rats. The functional role of endocannabinoids in the spinal cords of MIA-treated rats was increased via activation of cannabinoid 1 (CB1) and CB2 receptors, and blockade of the catabolism of anandamide had significantly greater inhibitory effects in MIA-treated rats compared with control rats. Conclusion Our findings provide new evidence for altered spinal nociceptive processing indicative of central sensitization and for adaptive changes in the spinal cord endocannabinoid system in an experimental model of OA. The novel control of spinal cord neuronal responses by spinal cord CB2 receptors suggests that this receptor system may be an important target for the modulation of pain in OA.

In vitro cytotoxicity tests for the prediction of acute toxicity in vivo

Investigations of the use of in vitro cytotoxicity tests for the prediction of acute toxicity in vivo have been reviewed with particular emphasis on those studies that have been published during the past 5 years. Numerous cell types, endpoints and exposure periods have been used in cytotoxicity tests, although these appear generally to have little effect on the resulting correlation between in vitro IC(50) values and in vivo LD(50) values. The in vitro data correlate better with rodent parenteral (ip or iv) LD(50) values than with oral LD(50) values due to kinetic considerations. For certain groups of related chemicals (e.g. antitumour compounds, metal salts), and for some sets of unrelated chemicals, the in vitro data correlate very well with LD(50) values. However, while cytotoxicity tests are useful for screening chemicals for their intrinsic and relative toxicities, it is impossible to tell whether predictions based on cytotoxicity data alone would be sufficiently accurate for labelling and classifying a new chemical according to its likely acute toxicity in vivo. The in vitro endpoints need to be of greater relevance to the possible mechanisms of chemically-induced acute toxicity in vivo than most of those that are used at present.

Use of a novel carbon fibre composite material for the femoral stem component of a THR system: in vitro biological assessment.

A novel, low elastic modulus femoral component for THR has been developed using a composite of polyetheretherketone and carbon fibre. The objectives of this study were to investigate human osteoblast-like cell and macrophage responses to this material in vitro. Cells were grown on composite discs and controls. Osteoblast attachment and proliferation was not significantly different to that on Ti6Al4V. The levels of alkaline phosphatase activity, Type I collagen production and osteocalcin production were not significantly different to that on Ti6Al4V by the end of the experimental period. Hydrogen peroxide production by macrophages was significantly less than that detected for cells cultured on copper, but was still greater than that detected for cells cultured on tissue culture plastic and Ti6Al4V. Beta-glucoronidase activity was not significantly different to that detected for cells cultured on tissue culture plastic. The in vitro biocompatibility assessment of this composite undertaken in this study showed initial osteoblast attachment at least comparable to that of the tissue culture plastic and Ti6Al4V controls, with proliferation similar to the controls at all time points up to 11 days. Alkaline phosphatase activity was similar to that of Ti6Al4V but reduced compared to tissue culture plastic controls. Whilst hydrogen peroxide production by macrophages was raised on composite surfaces compared to controls, beta-glucoronidase activity and osteoblastic production of Type I collagen and osteocalcin were similar to levels detected on Ti6Al4V.

Stimulation of dichlorofluorescin oxidation by capsaicin and analogues in RAW 264 monocyte/macrophages: lack of involvement of the vanilloid receptor.

In studies into the oxidative burst in RAW 264 monocyte/macrophages, it was observed that capsaicin, a vanilloid receptor agonist, stimulated dichlorofluorescin (DCFH) oxidation in a concentration-dependent manner, which could be blocked by capsazepine, a vanilloid receptor antagonist. However, by use of a number of vanilloid agonists (including N-octyl-3-chloro-4-hydroxyphenylacetamide, 4m), we demonstrated that there was no relationship between vanilloid agonist potency and the capacity to stimulate DCFH oxidation. The oxidative burst stimulators Tween 20 and phorbol myristyl acetate (PMA) also stimulated reactive oxygen species generation, which again was inhibited by capsazepine. Use of the selective inhibitor diphenyliodonium iodide ruled out a role for plasma membrane NAD(P)H oxidase as the site of capsaicin- and 4m-stimulated DCFH oxidation. However, this DCFH oxidation was modulated by a number of inhibitors of mitochondrial respiration. Rotenone enhanced DCFH oxidation induced by capsaicin and 4m, whilst malonic acid and potassium cyanide inhibited this response. 2,4-Dinitrophenol, an inhibitor of oxidative phosphorylation, was without effect. The antioxidant trolox c inhibited DCFH oxidation stimulated by capsaicin, 4m, and PMA, whereas N-acetylcysteine, a precursor of glutathione, was without effect. Capsazepine inhibited DCFH oxidation in unstimulated cells and in cells treated with menadione, a redox-cycling quinone. Capsazepine was also a potent antioxidant when measured in a Fe3+ reduction assay. We concluded that DCFH oxidation stimulated by vanilloid analogues was not mediated via a vanilloid receptor, but rather by impairment of mitochondrial electron transport.

Human keratinocyte cultures in an in vitro approach for the assessment of surfactant-induced irritation.

A specific, mechanistic, in vitro approach for the assessment of human skin irritation potential is outlined for the evaluation of surfactants and the results compared with in vivo human patch test data. The level of free available surfactant monomer and the solubilization of the corn protein zein in vitro were confirmed to be related to surfactant in vivo human skin irritation potential. In vitro cytotoxicity to monolayer keratinocyte cultures could not discriminate between the moderate human skin irritant sodium dodecyl sulfate (SDS) and the mild irritants cocamidopropylbetaine (CA) and Polysorbate 20 (P20). An in vitro stratified differentiated human epidermal equivalent (HEE) exhibited reduced cytotoxicity to the test chemicals, compared with monolayer culture responses, and was able to discriminate between the toxic potential of SDS and CA. Stimulation of interleukin-1alpha release from the A431 human keratinocyte cell line reflected in vivo erythema scores more closely than cytotoxic potential, and coincided with nitric oxide production by macrophages upon exposure to A431-conditioned medium. Combination of these mechanistic assays has allowed a profile of likely in vivo human responses to be approximated. Additional knowledge of skin penetrability and rate of recovery from toxic damage would affirm these predictions.

Structural Requirements for the Direct and Cytochrome P450-dependent Reaction of Cyclic α,β-Unsaturated Carbonyl Compounds with Glutathione: a Study with Coumarin and Related Compounds

Abstract— The interaction of glutathione (GSH) with coumarin, or one of a series of compounds related to coumarin, was assessed in the absence and presence of liver microsomes (direct reaction and indirect reaction, respectively) to determine the structural requirements for direct and mono‐oxygenase‐mediated reaction of cyclic α,β‐unsaturated carbonyls with GSH. Acrolein was used as a positive control for the direct reaction, and produced complete or nearly complete depletion of GSH under all assay conditions. 5,6‐Dihydro‐2H‐pyran‐2‐one and 2‐cyclohexen‐1‐one also produced substantial depletion of GSH in the direct reaction, which was not increased by the addition of liver microsomes. Coumarin, 2H‐pyran‐2‐one and precocene I (a substituted pyran lacking the 2‐one structure) were not substrates for the direct reaction but did cause depletion of GSH when incubated in the presence of rat or human liver microsomes. These depletions were dependent on a functioning mono‐oxygenase system as judged by the effects of omission of cofactors, addition of competitive or inactivating inhibitors of cytochrome P450, and induction. Dihydrocoumarin, ∂‐valerolactone, cyclohexanone and 4H‐pyran‐4‐one were not substrates for either the direct or indirect reaction. These findings are rationalized on the basis of a direct nucleophilic attack of GSH on the α,β‐centre of the α,β‐unsaturated carbonyl compounds, which is hindered by benzenoid resonance in coumarin and 2H‐pyran‐2‐one, for which enzyme‐mediated reaction with GSH, probably via a 3,4‐epoxide, is the favoured mechanism.

Fluorescein cadaverine incorporation as a novel technique for the characterization of terminal differentiation in keratinocytes.

A novel technique for detecting transglutaminase activity and the production of cornified envelopes in keratinocytes has been devised. This was based on the enzymatic incorporation of fluorescein-labelled cadaverine (FC) into cornified envelopes. The addition of FC (20mum) to the incubation medium served as an amine donor for transglutaminase reactions in place of protein lysine residues. Cells incorporating the label became visible with fluorescence microscopy and were quantified by fluorimetry. There was a significant difference in the level of FC incorporation into cornified envelopes under the various media conditions and time points employed. The greatest incorporation was observed by keratinocytes cultured in Greens medium and fluorescent intensity decreased in the order: Greens> KGM with calcium>KGM. Confocal imaging of keratinocytes dual stained with FC and propidium iodide revealed the presence of distinct layers and demonstrated how FC was incorporated into differentiating cells and not the basal layer. FC incorporation has the potential to serve as a rapid assessment of terminal differentiation in keratinocytes. It is simple and less time-consuming than currently available alternative techniques. This approach also has the advantage of combining microscopic and quantitative data.

Depletion of glutathione by the hepatotoxins paracetamol and bromobenzene, and their non-hepatotoxic analogues, in a fortified liver microsomal system

The proposal that liver microsome mixed-function oxidase (MFO)-mediated generation of reactive metabolites may be detected by depletion of reduced glutathione (GSH) added to a fortified microsome incubation containing substrate has been investigated. Paracetamol, 3-hydroxyacetanilide, bromobenzene and p-bromophenol were used as substrates for a MFO-mediated depletion of GSH in vitro. Studies involving modulation of the extent of GSH depletion in vitro and comparison with published findings indicated that metabolism-mediated depletion of GSH in a fortified liver microsome incubation correlated with covalent binding of reactive metabolites but did not correlate well with the ability of compounds to cause GSH depletion in vivo or liver damage.

Imipramine for Cytochrome P450 Activity Determination: a Multiple-species Metabolic Probe

Imipramine is metabolized in vitro by rat, mouse or human microsomes to several metabolites including 2-hydroxyimipramine, 2-hydroxydesipramine, desipramine, didesmethylimipramine and imipramine-N-oxide. Heat treatment (which inhibits FMO), verified the role of this group of enzymes in the oxidation of imipramine to imipramine N-oxide. These studies demonstrate that while rats and humans showed similar metabolic profiles, producing several minor metabolites in addition to the 2-hydroxyimipramne and desipramine, differences were seen in the mouse microsomes. In these samples, few minor metabolites were produced. In addition, imipramine N-oxide was the major metabolite in mouse, whereas none was detected in human samples. To conclude, the metabolic profile of imipramine can be used to reveal a number of cytochrome P450 enzymes active in microsomal fractions.