Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and ß-Arrestins 2.

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP). Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or ß-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes) allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/ß-arrestin 2 interactions (evaluated in whole cells). In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gß1 or NOP/ß-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified.

Comparative characterization of CHCl(3) metabolism and toxicokinetics in rodent strains differently susceptible to chloroform-induced carcinogenicity.

A comparative kinetic study in B6C3F1 mice, Osborne-Mendel (OM) and Sprague-Dawley (SD) rats has been undertaken with the major aim to determine the extent of covalent binding of chloroform reactive metabolites produced in vivo through oxidative and/or reductive metabolism in the target organs of chloroform carcinogenicity. Some additional kinetic observations of chloroform biotransformation were also collected comparatively. Expiration of [14C]-CO(2) showed that chloroform metabolism went to saturation in all tested rodent strains. In the B6C3F1 mouse maximal rates of approximately 135 µmol [14C]-CO(2)/kg b.w./h were reached at a dose of approximately 150 mg/kg, while in the two rat strains saturation occurred at a dose of approximately 60 mg/kg, with a maximal rate of approximately 40 µmol [14C]-CO(2)/kg b.w./h. At doses of 150-180 mg/kg b.w., limited differences were found in the distribution and elimination of [14C]-chloroform in the liver and kidney. Species differences have been found in the kinetics of alkali-extractable radioactivity in the blood. The levels of adducts of electrophilic intermediates with the polar heads (PH) of phospholipids (PL) showed a limited variability accross the rodents tested and did not correlate with the species and organ susceptibility to chloroform carcinogenicity. The levels of adducts of radical intermediates with the fatty acyl chains (FC) of PL were much lower than the PH adducts in all the samples analyzed; at the carcinogenicity bioassay doses, statistically significant levels of hepatic FC adducts were present only in the B6C3F1 mouse, where chloroform is hepatocarcinogenic. The observations in the rat kidney were suggestive of the formation of electrophilic reactive metabolites, presumably different from phosgene and associated with an initial chloroform reduction.

In vivo CHCl3 bioactivation, toxicokinetics, toxicity, and induced compensatory cell proliferation in B6C3F1 male mice.

Chloroform carcinogenicity has often been associated with acute tissue damage and consequent compensatory cell proliferation. However, available data do not fully support this hypothesis, and other biological factors may play a role in the tumor induction by chloroform. The purpose of this study was to characterize the in vivo CHCl3 metabolism and the time course of toxic effects and of cell proliferation in the liver and kidney of B6C3F1 male mice dosed i.p. or by gavage with 150 mg CHCl3/kg body wt. Microsomal phospholipid adducts attributed to (14)CHCl3 metabolism by both oxidative and reductive pathways were detected in both liver and kidney. The levels and composition of the adducts were similar in the liver and kidney of treated animals. In the liver, although no necrosis was histologically detectable, a transient cell proliferation was found starting at 24 and peaking at 48 hr post-treatment. Kidney toxicity was evident by biochemical and cytochemical methods at 5 hr after dosing and progressed to severe necrosis at 48 and 96 hr. An intense kidney cell regeneration began 48 hr after CHCl3 treatment, became maximal at 96 hr, and was sustained for at least the following 3 days. These observations raise questions about the purely epigenetic action of chloroform in tumor induction since bioassays have found tumors in liver but not kidneys of CHCl3-treated B6C3F1 mice.

In vitro quantitative determination of phospholipid adducts of chloroform intermediates in hepatic and renal microsomes from different rodent strains.

We have comparatively studied in vitro the oxidative and reductive pathways of chloroform metabolism in hepatic and renal microsomes of rodent strains used for carcinogenicity testing (B6C3F1 mice, Osborne Mendel and Sprague Dawley rats). To this aim we exploited the regioselective binding of phosgene to phospholipid (PL) polar heads and of dichloromethyl radical to PL fatty acyl chains, using a method based on the chemical transmethylation of PL adducts, followed by phase partitioning of the resulting products (De Biasi et al., 1992). The analysis of results let us to conclude at first that a (14)C label partitioning by 89.2 (±6.5)% or 13.7 (±5.0)% in the aqueous phase is typical of the PL adduct with phosgene (PL-PHOS) or with dichloromethyl radical (PL-RAD), respectively. Metabolism of 0.1 mM CHCl(3) was mainly oxidative in all the samples, being hepatic microsomes more active than renal ones by about one order of magnitude and levels of CHCl(3)-derived PL adducts in B6C3F1 mouse liver microsomes higher than in rat samples. At 5 mM CHCl(3), total levels of PL adducts in renal microsomes reached levels almost similar to those found in liver microsomes. However, while B6C3F1 mouse kidney microsomes produced both reactive metabolites, similarly as the hepatic samples, Osborne Mendel rat kidney microsomes bioactivated CHCl(3) only reductiveiy, producing the radical. The relevance of this finding depends on the fact that phosgene is known to be the major cause of CHCl(3) toxicity, based on data with the rat liver and mouse liver and kidney, while nephrotoxicity in rats occurs with minimal production of COCl(2). Chloroform reductive bioactivation may therefore provide a reasonable explanation for the toxicity of chloroform to the rat kidney. The same finding may be of interest in elucidating the metabolic reasons of the chloroform-induced kidney tumors in Osborne Mendel rats.

The contribution of electrophilic and radicalic intermediates to phospholipid adducts formed by halomethanes in vivo.

The different production of phosgene and free-radicals from CHCl3 and CCl4 was determined in vitro and in vivo, by measuring the regioselective binding to the two intermediates to phospholipid (PL) molecules. Results clearly indicated that this assay can be successfully used to selectively detect electrophilic and radicalic metabolites produced in vivo and selectively quantitate their adducts. The in vivo biotransformation of CCl4, similarly to the in vitro situation, resulted in the formation of radicals only, the contribution of phosgene to the structural damage of PL being negligible. These findings allowed us to rule out the hypothesis of substantial formation of radicalic intermediates from CHCl3 in phenobarbital (PB)-pretreated Sprague-Dawley (SD) rats, derived from in vitro data. While the role of reduced glutathione (GSH) in preventing COCl2-derived damages seems to be less important in vivo than in vitro, it is not possible to rule out the action of radical scavenging systems in decreasing the level of adducts with fatty acyl chains (FC) of PL measured in vivo.

In vivo production of different chloroform metabolites: effect of phenobarbital and buthionine sulfoximine pretreatment.

The regioselective attack on microsomal phospholipid (PL) polar heads (PH) and fatty acyl chains (FC) demonstrated in vitro has been exploited for the selective quantitation in vivo of the biochemical damages produced by the oxidation and reduction products of CHCl3 metabolism. Five hours after CHCl3 injection (60 mg/kg body weight, ip) to control Sprague-Dawley rats, most of the label covalently bound in the liver was associated to PH, indicating a predominant production of COCl2. The levels of radioactivity bound to both PL moieties increased proportionally when 180 mg/kg body weight 14CHCl3 was administered. Buthionine sulfoximine (BSO) pretreatment resulted in a further increase of binding either to PH or FC. The pretreatment of rats with phenobarbital (PB) reduced the PH/FC binding ratio to 3.4, still indicating the predominance of the oxidative metabolism, but giving some indication of the simultaneous presence of CHCl3 reduction. When reduced glutathione (GSH) was depleted by BSO in PB-induced animals prior to 14CHCl3 administration, only the level of radioactivity associated with oxidative intermediates was increased six times. The present results confirmed that GSH is able to exert an efficient protection mainly toward 14CHCl3 oxidation intermediates. Furthermore, they indicate that in the liver of the Sprague-Dawley rat the major pathway of CHCl3 biotransformation is its oxidation and that pretreatment of rats with a GSH-depleting agent (such as BSO) is more relevant than PB induction in enhancing the biochemical damages produced by CHCl3.

The regioselective binding of CHCl3 reactive intermediates to microsomal phospholipids.

Microsomal phospholipids (PL) are a good target for the reactive intermediates produced by either the oxidative or the reductive biotransformation of CHCl3 (Testai et al. (1990), Toxicol. Appl. Pharmacol. 104, 496-503). In order to preliminarily characterize the different PL with CHCl3 reactive intermediates, two common methods of PL breakdown have been exploited: the acid-catalyzed transmethylation and the enzymatic hydrolysis with phospholipase C. The results indicated that radioactivity derived from the adducts of PL with the oxidation metabolite, phosgene, partitioned preferentially in the aqueous phase (the ratio of aqueous to organic phase radioactivity contents was about 10); the opposite occurred (ratio about 0.1) when the PL adducts were produced by the reductive process metabolites (dichloromethyl radicals). Therefore, the two methods of PL adduct breakdown can be used to detect and quantitate selectively the two reactive intermediates of CHCl3 biotransformation. The use of phospholipase C, which specifically cleaves the bond between the glyceryl-oxygen and the phosphor atom of PL also gave some structural information. Indeed, the radioactivity partitioning in the aqueous phase after enzymatic hydrolysis of CHCl3 oxidation-associated PL adducts, indicated the selective covalent binding of phosgene residues with the PL polar heads. The clear-cut different partition of radioactivity observed after hydrolysis of PL adducts with CHCl3 reduction intermediates, analogously indicated that dichloromethyl radicals were selectively bound to the PL fatty acyl chains. Using this method we could confirm that in in vitro experimental conditions resembling the physiological status of the liver, both metabolic pathways were concurrently active in hepatic microsomes of B6C3F1 mice. Extents of reactive metabolites similar to those found in B6C3F1 mouse liver microsomes, could be measured in Sprague-Dawley rat liver microsomes only after pretreatment of the animals with PB and incubation with higher CHCl3 concentrations. The toxicological implications of these findings are discussed.

Effects of 2,5-hexanedione on the ovary and fertility. An experimental study in mice.

Sixty-day-old virgin female Swiss CD1 mice were treated with 1.5% 2,5-hexanedione in their drinking water; control mice received tap water; duration of treatment was either 4 or 6 weeks. Under these conditions the treated mice did not show any clinical symptoms although electromyography revealed some signs of polyneuropathy. Protein and DNA content per mg of ovarian tissue in treated mice were not significantly different from controls. Histological examination of ovarian sections at the light microscope level showed no significant alterations after exposure. A morphometric study revealed a statistically significant reduction in the number of growing oocytes after 6 weeks of treatment. For fertility studies three groups of 15 female mice each were treated for 0, 4 or 6 weeks as above and then permanently housed with untreated proven breeder male mice (one male per female); cages were checked daily for newly born mice. All litters appeared normal by gross examination. During the first 14 weeks of continuous mating the mean litter size (number of newborns per litter) remained about 11.4 in all groups; this number subsequently began to decrease. Control and 4-week treatment regression curves did not differ statistically, while the slope of the 6-week line was significantly steeper, indicating a faster decrease in litter size over time and a shortening of fertile life in the latter group of treated females.

Heterogeneous visceral nerve changes in acrylamide intoxication.

A variety of visceral nerves were studied by intermediate filament immunocytochemistry in rats intoxicated with acrylamide. In such animals, oesophageal and diaphragmatic motor end-plates were invaded and deformed by neurofilament protein-like material, while afferent fibres of diaphragmatic neuromuscular spindles and myelinated sensory fibres of the iris showed striking terminal accumulation of similar material. Conversely, the rich population of thin afferent fibres of the iris showed no obvious abnormality, while pre-terminal changes were seen along the extrinsic nerve fibres supplying the cornea and myenteric ganglia. Multiple lesions were demonstrated in gut nerves of acrylamide-treated rats, while scattered "enteric glial cells" showed abnormally coarse morphology and a striking increase in glial fibrillary acidic protein immunoreactivity. A distinct, delicately varicose appearance was revealed by neurofilament protein-immunostaining in bladder nerve fibres of normal rats, which was changed to one of coarse dilations by acrylamide. In conclusion, apparently selective changes were found along different types of axons, indicating marked heterogeneity in cytoskeletal organisation among visceral nerves. Taken together with the proposed inhibition by acrylamide of neurofilament proteins degradation, the above findings may suggest a non-uniform distribution of neurofilament degradation sites along distal regions of different axons.

2,5-Hexanedione-induced accumulations of neurofilament-immunoreactive material throughout the rat autonomic nervous system.

In rats intoxicated with 2,5-hexanedione, nerve fibres supplying virtually all visceral organs showed large numbers of densely immunoreactive accumulations of neurofilament-like material, of fusiform, elongated, smoothly tapering morphology. In the gut, round to oval, morphologically different lesions were also present, and abnormal neurofilament-immunoreactive accumulations were revealed in oesophageal terminal end-plates. An extensive damage to autonomic nerve fibres, which are largely non-myelinated, was thus revealed in 2,5-hexanedione intoxication. The observed diversity in lesion morphology may suggest heterogeneity in cytoskeletal and/or associated proteins among autonomic neurons.

Cholinesterases in blood plasma and tissues of rats treated with n-hexane or with its neurotoxic metabolite 2,5-hexanedione.

Adult male rats were subjected to 4 weeks' respiratory treatment with n-hexane (5000 ppm, 16h/day, 6 days/week); motor conduction velocity was significantly decreased in tail nerves at all weekly intervals and did not approach normal values in the 4 weeks following interruption of treatment. Plasma acetylcholinesterase (AChE) levels were significantly increased at all weekly intervals during treatment (25-40%); 2 weeks after the end of treatment they had returned to baseline. Oral treatment with 2,5-hexanedione (HD) (1% in drinking water) caused a similar increase in plasma levels; this increase was statistically significant also when compared with pair-fed (PF) control rats. A sucrose density gradient analysis showed only one peak of AChE activity at approximately 10 S (as in normal plasma). The levels of butyrylcholinesterase were unaltered in plasma of both n-hexane-and HD-treated rats. Both the fast-contracting EDL and the slow-contracting soleus muscles lost weight in HD-treated rats with respect to free-fed (AL) and PF controls. AChE levels responded differently to HD treatment in the two muscle types: in EDL total extracts, AChE activity increased considerably with respect to AL controls (+ 70%, p less than 0.001), while the levels of the 16 S and 4 S molecular forms were unaltered. The increased levels of AChE found in plasma of rats intoxicated with n-hexane or with its metabolite HD may originate from muscle and correspond to an increased secretion of this molecular form.