Four Weeks' Inhalation Exposure of Long Evans Rats to 4‐tert‐Butyltoluene: Effect on Evoked Potentials,Behaviour, and Brain Neurochemistry

Long‐lasting central nervous system (CNS) neurotoxicity of 4‐tert‐butyltoluene (TBT) has been investigated using electrophysiology, behaviour, and neurochemistry in Long Evans rats exposed by inhalation to 0, 20, or 40 p.p.m. TBT 6 hr/day, 7 days/week for 4 weeks. Flash evoked potentials and somatosensory evoked potentials were not affected by TBT. In Auditory Brain Stem Response there was no shift in hearing threshold, but the amplitude of the first wave was increased in both exposed groups at high stimulus levels. Three to four months after the end of exposure, behavioural studies in Morris water maze and eight‐arm maze failed to demonstrate any TBT induced effects. Exposure was followed by a 5 months exposure‐free period prior to gross regional and subcellular (synaptosomal) neurochemical investigations of the brain. TBT reduced the NA concentration in whole brain minus cerebellum. Synaptosomal choline acetyltransferase activity increased and acetylcholinesterase activity was unchanged suggesting increased synaptosomal ability for acetylcholine synthesis. The relative and total yield of synaptosomal protein was reduced suggesting reduced density and total number of synapses in situ, respectively. We hypothesise that a reduced yield of synaptosomal protein reflects a more general effect of organic solvent exposure on the software of the brain. The synaptosomal concentration per mg synaptosomal protein and the total amount of 5‐hydroxytryptamine were not affected whereas the total amount of synaptosomal noradrenaline decreased. The concentration and the total amount of synaptosomal dopamine decreased. The noradrenergic and dopaminergic parts of CNS may be more vulnerable to TBT than the serotonergic, and these long‐lasting effects may cause or reflect TBT‐compromised CNS function.     

Counteraction of the Rapid Tolerance to 8‐Hydroxy‐2‐(di‐n‐propylamino)tetralin‐Induced Hypothermia in Rats by Activation of the GABAA Receptor‐Chloride Channel Complex

Abstract: The effects of compounds that open the GABA_A receptor‐chloride channel complex on the rapidly developed tolerance to the 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin hydrobromide(8‐OH‐DPAT)‐induced hypothermia in rats were examined. The test compound was injected 15 min. before 1 mg/kg subcutaneous 8‐OH‐DPAT or saline and 24 hr later a challenge dose of 0.1 mg/kg subcutaneous 8‐OH‐DPAT was given. The rectal temperature was measured before the challenge dose and 30, 60, 90 and 120 min. thereafter. The hypothermic effect was calculated as the area under the curve. It was found that all the GABAergic compounds examined significantly counteracted the 8‐OH‐DPAT‐induced tolerance to the hypothermic response: muscimol at 3 mg/kg subcutaneous, diazepam at 1 – 3 mg/kg subcutaneous, pentobarbitone sodium at 20 mg/kg subcutaneous, and chlormethiazole at 40 mg/kg subcutaneous. Combined treatment of the rats with the GABA_A receptor antagonist, bicucculine, or the GABA_A receptor‐chloride channel blocker, picrotoxin and diazepam, pentoparbitone sodium or chlormethiazole significantly antagonised this counteraction of the 8‐OH‐DPAT‐induced tolerance. Depletion of 5‐HT by pretreatment of the rats with the tryptophan hydroxylase inhibitor p‐chlorophenylalanine did not counteract the 8‐OH‐DPAT‐induced tolerance to the hypothermic response. Pretreatment of the rats with dexamethazone did not change the development of the tolerance to 8‐OH‐DPAT‐induced hypothermic effect which seems to exclude the involvement of the hypothalamo‐pituitary‐adrenocortical axis in the tolerance development. It is concluded that the results support the hypothesis that GABA neurones beyond the 5‐HT neurones are involved in the mechanism causing tolerance to the 5‐HT_1A receptor‐mediated hypothermia in rats.     

Investigational New Drug‐Directed, 5‐Day Repeat Dose Toxicity Study of 4‐[3‐(2‐Nitro‐1‐Imidazolyl)‐Propylamino]‐7‐Chloroquinoline Hydrochloride (NLCQ‐1, NSC 709257) Administered with or without Taxol® in Sprague–Dawley Rats

Abstract: In pre‐clinical studies, 4‐[3‐(2‐nitro‐1‐imidazolyl)‐propylamino]‐7‐chloroquinoline hydrochloride (NLCQ‐1, NSC 709257) is a weak DNA‐intercalating, hypoxia‐selective cytotoxin with a promising profile as an adjuvant to radio/chemotherapy and it is about to enter phase I clinical trials. The present investigation was undertaken to further evaluate potential systemic toxicity induced by i.v. doses of NLCQ‐1 alone or in combination with Taxol ^® in Sprague–Dawley rats, in support of an investigational new drug application. Doses of NLCQ‐1 were based on previous range‐finding studies. In the present study, NLCQ‐1 was administered either alone, at 0, 6, 9 or 12 mg/kg/dose to male rats and 8, 12 or 16 mg/kg/dose to female rats or, at 9 (male rats) and 12 (female rats) mg/kg/dose, in combination with Taxol^®, on a qd × 5 schedule. Taxol^® was administered i.v. at 3.5 mg/kg/dose 1 hr before NLCQ‐1. Observations were recorded for mortality/moribundity, clinical signs of toxicity, body weights, food consumption, haematology, clinical chemistry, gross lesions at necropsy and histopathology. Blood samples were taken from 10 animals from each dose group on each of 2 days (days 8 and prior to scheduled necropsy on day 33). Administration of i.v. doses of NLCQ‐1 alone, on a qdx5 schedule, resulted in no signs of toxicity over the 33‐day study. Taxol^®‐induced toxicity included minimal decreases in the group mean RBC, haemoglobin and haematocrit values, minimal increases in group mean reticulocyte counts (females), marked decreases in group mean neutrophil counts and minimal decreases in group mean monocyte and eosinophil counts. Lymphoid atrophy of thymus, atrophy of bone marrow and atrophy of the germinal epithelium of the testis were also associated with the administration of Taxol^®. There was no additional toxicity associated with the co‐administration of NLCQ‐1 and Taxol^®. In the present study, the ‘no observable adverse effect level’ for NLCQ‐1, when administered on a qdx5 schedule, was >12 and >16 mg/kg/dose in male and female rats respectively. Daily administration of 9 (male rats) or 12 (female rats) mg/kg of NLCQ‐1 1 hr after i.v. administration of Taxol^® (3.5 mg/kg) had no effect on Taxol^®‐induced toxicity.     

Binding of agonists,antagonists and inverse agonists to the human δ‐opioid receptor produces distinctly different conformational states distinguishable by plasmon‐waveguide resonance spectroscopy

Abstract: Structural changes induced by the binding of agonists, antagonists and inverse agonists to the cloned δ‐opioid receptor from human brain immobilized in a solid‐supported lipid bilayer were monitored using plasmon‐waveguide resonance (PWR) spectroscopy. Agonist (e.g. deltorphin II) binding causes an increase in membrane thickness because of receptor elongation, a mass density increase due to an influx of lipid molecules into the bilayer, and an increase in refractive index anisotropy due to transmembrane helix and fatty acyl chain ordering. In contrast, antagonist (e.g. TIPPpsi) binding produces no measurable change in either membrane thickness or mass density, and a significantly larger increase in refractive index anisotropy, the latter thought to be due to a greater extent of helix and acyl chain ordering within the membrane interior. These results are closely similar to those reported earlier for another agonist (DPDPE) and antagonist (naltrindol) [Salamon et al. (2000) Biophys. J. 79, 2463–2474]. In addition, we now find that an inverse agonist (TMT‐Tic) produces membrane thickness, mass density and refractive index anisotropy increases which are similar to, but considerably smaller than, those generated by agonists. Thus, a third conformational state is produced by this ligand, different from those formed by agonists and antagonists. These results shed new light on the mechanisms of ligand‐induced G‐protein‐coupled receptor functioning. The potential utilization of this new biophysical method to examine structural changes both parallel and perpendicular to the membrane normal for GPCRs is emphasized.     

Exposure to cadmium and mono‐(2‐ethylhexyl) phthalate induce biochemical changes in rat liver,spleen, lung and kidney as determined by attenuated total reflection‐Fourier transform infrared spectroscopy

Attenuated total reflection‐Fourier transform infrared (ATR‐FTIR) spectroscopy is a label‐free, non‐destructive analytical technique for biochemical analysis of macromolecular components within tissue samples. Cadmium (Cd) and mono‐(2‐ethylhexyl) phthalate (MEHP), a primary metabolite of di‐(2‐ethylhexyl) phthalate, are present ubiquitously in the environment and in organisms, and have adverse impacts on ecosystems and human health. Herein we employed ATR‐FTIR analysis to identify biomolecular changes in rat liver, spleen, lung and kidney after prepubertal exposure to Cd and MEHP. Our results showed clear segregations between the 3 mg/kg Cd‐, 10 mg/kg, 50 mg/kg, 250 mg/kg MEHP‐ and binary mixture‐treated groups vs. the solvent control group. Following principal components analysis coupled with linear discriminant analysis, biochemical alterations associated with different doses of Cd and MEHP were attributed mainly to lipids, proteins, phosphates and carbohydrates. In addition, the ratios of lipid/protein, C=O stretching/CH₂ methylene (lipid oxidation level), amide I/amide II, α‐helix/β‐sheet and CH₃ methyl/CH₂ methylene (acetylation level) in target organs were affected by these toxicants. There seems to be no dose‐response effect of Cd and MEHP on target organs. We observed hardly any joint toxic action of these toxicants. This is the first study showing the application of ATR‐FTIR spectroscopy to the assessment of toxicity of Cd and MEHP. Possibly, destruction of cell membrane structure and integrity could be the common mechanism of Cd and MEHP toxicity in liver, spleen, lung and kidney.