Dichloromethane as an inhibitor of cytochrome c oxidase in different tissues of rats

 Based on the metabolism of dichloromethane (DCM) to carbon monoxide (CO), a process mediated by cytochrome P-4502E1 (CYP2E1), cytochrome c oxidase activity was determined in different tissues of rats after DCM exposure. It is likely that binding of CO to cytochrome c oxidase is significant at low carboxyhemoglobin levels, because intracellular effects of CO depend on CO partial pressures in the tissues. Two methods of exposure were used: (1) administration of DCM, 3.1, 6.2, and 12.4 mmol/kg p.o. in Oleum pedum tauri, 10% (v/v), producing a maximum of 10% COHb 6 h after gavage, and (2) accidental scenario, i.e. rats were exposed nose-only to DCM, 250 000 ppm for 20 s, producing 3–4% COHb after 2 h. Cytochrome c oxidase activity was reduced 6 h after the high oral DCM dose in brain, lung, and skeletal muscle by 28–42% and 20 min after inhalative uptake of DCM in the brain, liver, kidney, and skeletal muscle by 42–51%. COHb formation due to DCM, 6.2 mmol/kg p.o., was completely prevented after treatment of rats with the mechanism-based inhibitor of CYP2E1, diethyldithiocarbamate (DDTC), using an oral dose of 32 μmol/kg. The decrease in cytochrome c oxidase activity after exposure to DCM was not evident in rats treated with this dose of DDTC. Therefore, it seems that the effect of DCM is produced by the DCM metabolite CO. Received: 12 July 1994/Accepted: 5 September 1994     

Behavioural and neurochemical characteristics of phentermine and fenfluramine administered separately and as a mixture in rats

Clinical case studies suggest that combined administration of the serotonergic agent fenfluramine (FEN) and the weak amphetamine-like anorexic agent phentermine (PHEN) may be useful in the treatment of alcohol and cocaine addictions. The present experiment examined the nature of the interaction between the two agonists using the drug discrimination paradigm. In vivo microdialysis served to examine the neurochemical profile of dopamine and serotonin release in the nucleus accumbens. In conscious rats, acute injections of FEN (1.0–2.0 mg/kg IP) or PHEN (1.0–2.0 mg/kg IP) selectively elevated levels of serotonin and dopamine in the nucleus accumbens, respectively. A mixture (1 mg/kg of each) increased levels of both amines by similar magnitudes to those observed with each individually. Three groups of Sprague-Dawley rats were trained to discriminate (1) FEN (1.0 mg/kg IP) alone, (2) PHEN (1.0 mg/kg IP) alone or a mixture (3) PHEN+FEN (1 mg/kg of each, IP) from saline under a fixed ratio (FR-10) schedule of food reinforcement. Rats acquired the mixture discrimination rapidly, while for the other groups the training dose had to be increased to 2.0 mg/kg to attain stimulus control. The individual components of the mixture at the training dose generalized partially to the mixture, and complete generalisation was observed following 3.0 mg/kg FEN or PHEN. Rats trained to discriminate the individual components showed respective cross-generalisation profiles. Generalisation to cocaine (0.3–10.0 mg/kg IP), amphetamine (0.1–3.0 mg/kg IP) and nicotine (0.1–0.8 mg/kg SC) was greatest in the MIX-trained rats, while partial or no generalisation was observed in rats trained to discriminate the individual compounds. From the present results, it may be concluded that the two drugs given as a mixture do not produce a novel cue. Rather, these aminergics appear to interact additively. Furthermore, the dual stimulation of the amines by the mixture may be the basis for the cueing effects of the FEN+PHEN drug mixture, and its effectiveness in treating drug addictions. Received: 4 October 1996/Final version: 12 December 1996     

Formation of epoxide and quinone protein adducts in B6C3F1 mice treated with naphthalene,sulfate conjugate of 1,4-dihydroxynaphthalene and 1,4-naphthoquinone

 Naphthalene (NA) is metabolically activated to the reactive intermediates, naphthalene oxide (NO) and naphthoquinones. To investigate the role of circulating reactive metabolites in NA toxicity, the half-life of NO was examined. The in vitro half-life of NO in both whole blood and plasma was 10 min. Detectable levels of NO were seen in perfusate leaving the isolated perfused liver of B6C3F₁ mice infused with 10 μmol/h NA. Identification of protein sulfhydryl adducts in NA-exposed mice (50 and 100 mg/kg, IP, 24 h) revealed a predominance of quinone adducts in liver, lung, kidney, red blood cells and brain. The epoxide adduct predominated in plasma protein. Administration of the sulfate conjugate of 1,4-dihydroxynaphthalene (NHQS) (100  mg/kg) resulted in formation of naphthoquinone protein sulfhydryl adducts in lung, liver and kidney. Administration of 1,4-naphthoquinone (NQ) (5 mg/kg) produced NQ adducts in liver, lung, kidney, plasma and brain. Received: 5 August 1994 / Accepted: 19 October 1994     

Effect of cytochrome P450 isozyme induction and glutathione depletion on the metabolism of CS2 to TTCA in rats

 Analysis of 2-thiothiazolidine-4-carboxylic acid (TTCA), a metabolite of carbon disulfide (CS₂), is used in the biological monitoring exposure to CS₂ at work. In order to clarify the metabolic reasons for individual variation in the urinary excretion of TTCA, the latter was studied in rats pretreated with model cytochrome P450 (CYP) enzyme inducers or glutathione (GSH) depletors. Ethanol, phenobarbital (PB) or 3-methylcholanthrene (MC) did not increase 24-h TTCA output following CS₂ inhalation (50 or 500 ppm, 6 h). After oral dosing (10 mg/rat), PB had an inhibiting effect on the excretion rate of TTCA. Tissue GSH depletors phorone, L-buthionine-(RS)-sulfoximine (BSO) and diethylmaleate (DEM) decreased TTCA excretion in rats given an oral dose (10 mg/rat) of CS₂. The initial inhibition by phorone and DEM was reversed after 6 h and from 12 h onward the TTCA in urine exceeded the control level, an effect not seen with BSO. The proportion of CS₂ excreted in urine as TTCA within 24 h was 1.7% in control rats and 1% after BSO treatment, 1.3% after PB, 1.7% after acetone, 1.8% after MC, 2.0% after phorone and 2.5% after DEM treatment. The amount of TTCA in urine increased with the CS₂ dose in a non-linear fashion: 1.6 μmol (50 ppm/6 h) vs. 4.9 μmol (500 ppm/6 h), and 0.2 μmol (1 mg/kg) versus 3.6 μmol (100 mg/kg). It is concluded that induction of different cytochrome P450 isoforms and transient glutathione depletion have only minor effects on the disposition of TTCA in rats following low-level CS₂ exposure persistently low glutathione level as achieved by E.G. BSO, markedly decreased the metabolism of CS₂ to TTCA; these metabolic effectors are unlikely to have a major role in the individual variation of CS₂ metabolism in exposed workers. Received: 14 June 1994/Accepted: 25 August 1994     

Glutamine contributes to ameliorate inflammation after renal ischemia/reperfusion injury in rats

The aim of this study was to investigate the effects of glutamine in an in vivo rat model of renal ischemia/reperfusion (I/R) injury. Male Wistar rats underwent bilateral renal pedicle clamping for 45 min followed by reperfusion for 6 h. Glutamine (1.5 mg/kg) was administered intraperitoneally (i.p.) 15 min prior to reperfusion. Plasma concentrations of urea, creatinine, γ-glutamyl transferase (γ-GT), and aspartate aminotransferase (AST) were measured for the assessment of renal function and reperfusion injury. Markers of oxidative stress, expression of the pro-inflammatory mediators inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), AT-1 expression, and changes in the oxidative stress-sensitive nuclear factor kappa B (NF-κB) signaling pathway were measured to investigate whether glutamine can reduce the renal dysfunction. Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of polymorphonuclear (PMN) cell infiltration and lipid peroxidation, respectively. Renal sections were used for histologic grading of renal injury and for immunohistochemical localization of nitrotyrosine and poly(ADP-ribose) synthetase (PARS). In vivo, glutamine significantly reduced the increase in urea, creatinine, γ-GT, AST, produced by renal ischemia/reperfusion (I/R), suggesting an improvement in both renal function and injury. Glutamine significantly reduced iNOS and NF-κB, kidney MPO activity and MDA levels, indicating a reduction in PMN infiltration and lipid peroxidation, respectively. Glutamine reduced the histological evidence of renal damage associated with I/R and caused a substantial reduction in the staining for nitrotyrosine and PARS, suggesting reduced nitrosative and oxidative stress. Moreover, glutamine attenuated the reduction of COX-2 expression and prevented the increased AT-1 expression after I/R. Our results suggest that glutamine reduces the renal dysfunction and injury associated with I/R of the kidney.     

Differential effects of diazepam on anxiety in streptozotocin induced diabetic and non-diabetic rats

 The anxiolytic activity of diazepam (DZP) (0.25–1 mg/kg) was investigated in streptozotocin (STZ)-induced diabetic adult Charles Foster albino rats of either sex. Diabetes was induced by injecting STZ IP (50 mg/kg; in citrate buffer, pH 4.5). Experiments were performed 72 h later. The rats were subjected to various anxiety paradigms, including the open-field exploratory behaviour, elevated plus maze and elevated zero maze behaviours and the social interaction tests. In addition, rat brain tribulin activity was also assessed as a biochemical marker of anxiety. The results indicate that diabetic rats showed significantly more anxiogenic activity in comparison to non-diabetic rats on open-field, elevated plus maze, zero maze and social interaction tests. In diabetic rats, brain tribulin activity (MAO-A inhibitory component) was significantly increased. DZP dose dependently produced anxiolytic activity on the various behavioural parameters in non-diabetic rats. DZP (0.5 and 1 mg/kg) partially reversed the anxiogenic behaviour of STZ diabetic rats in elevated plus maze and zero maze tests. However, in open field behaviour and social interaction tests significant anxiolytic activity was observed only at a higher dose of DZP (1 mg/kg). The findings indicate that STZ-induced diabetic rats exhibited augmented anxiety on various experimental paradigms and that the anxiolytic effect of diazepam was less marked in diabetic rats as compared to their euglycaemic counterparts. Received: 5 May 1997 / Final version: 14 August 1997     

Evidence for renal ischaemia as a cause of mercuric chloride nephrotoxicity

 The present study was undertaken to investigate if the source of oxidative stress and the renal injury produced by mercuric chloride could be renal ischaemia. Verapamil Vp was used because it was described that calcium channel blockers protect cells from nephrotoxicants and from ischaemia. Vp (75 μg/kg, i.v.; 30 min before HgCl₂ injection) prevented mercuric chloride renal injury observed 1 h post-HgCl₂ injection as measured by clearance techniques. Vp also prevented the diminution of non-protein-sulfhydryls (NPSH) and the increased lipid peroxidation (LPO) induced by HgCl₂ in renal tissue. Hg²⁺ toxicokinetic alterations were not observed in Vp plus HgCl₂ treated rats, nor was Vp ability found as a free radical scavenger in renal tissue homogenates. The results described in this study give some evidence for the role of renal ischaemia in the production of oxidative stress, generating LPO and functional and morphological renal injury described in mercuric chloride treated rats. Received: 17 October 1994/Accepted: 5 January 1995     

Impairment of schedule-controlled behavior by pre- and postnatal exposure to hexachlorobenzene in rats

 Hexachlorobenzene (HCB) is still frequently found at elevated levels in human adipose tissue and breast milk. As intoxication with HCB causes neurological disturbance in human beings, the purpose of the present study was to examine neurobehavioral functions in rats after pre- and postnatal exposure. Female rats were fed diets with 0, 4, 8, or 16 mg HCB/kg diet. Exposure started 90 days prior to mating and was continued throughout mating, gestation, and lactation. Thereafter, the offspring were given the same diets as their respective mothers. HCB levels were determined in the brain, the liver, and in the adipose tissue from virgin rats, dams, and the offspring. Concentrations on a lipid basis were found to decline in the order adipose>liver>brain. The exposure levels chosen did not cause gross toxic effects in dams or offspring. There were dose-related increases in liver-to-body-weight ratios in exposed dams, but not in unmated females treated alike. Behavioral testing was conducted in the offspring. Examination of open-field activity on PND 21, and of active avoidance learning on PND 90 failed to reveal significant differences between groups. Training of operant behavior started at the age of 150 days in the offspring from the control, the 8-mg group, and the 16-mg group. Animals were trained on a fixed interval schedule of 1 min (FI-1). On this schedule, responses were reinforced by a food pellet every time 1 min had elapsed after the preceding reinforcement. There were dose-dependent reductions in the post-reinforcement pause, e.g. the time between each reinforcement and the first reaction emitted after it. In addition, the index of curvature, which describes the efficiency of performance on the FI-1 schedule, was decreased in a dose-dependent fashion. Received: 12 April 1994 / Accepted: 26 June 1995     

Influence of glucose on the toxicity of oxophenylarsine in MDCK cells

 Trivalent arsenicals like oxophenylarsine (PhAsO) inhibit cellular pyruvate dehydrogense, thus leading to a drop of acetylCoA formation and a slow-down of the citric acid cycle. Glucose may protect cells from arsenic toxicity, because increased glycolysis may prevent fatal shortage of ATP. On the other hand, PhAsO has been shown to inhibit glucose uptake in Madin-Darby canine kidney (MDCK) cells. We have investigated the effect of PhAsO on viability, ATP levels and glucose uptake of MDCK cells in the presence of normal (5 mmol/l) and low (0.01 mmol/l) glucose concentrations. At normal as well as at low glucose concentrations, cell viability as assessed by formazan formation was not affected by PhAsO concentrations up to 2 μmol/l within 3 h of observation. At higher PhAsO concentrations viability was diminished earlier and more pronounced in the presence of low glucose concentrations. 10 μmol/l PhAsO induced a drastic drop of ATP within 30 min which was followed by an almost complete loss of viable cells after 180 min in the presence of low glucose concentrations, while at normal glucose levels no influence on ATP contents or on cell viability was detected within 60 min of incubation. On the other hand, glucose uptake, determined as ¹⁴C accumulation by cells incubated for 10 min with D[6-¹⁴C]-glucose, was inhibited by PhAsO at low as well as at normal glucose concentrations in a dose dependent manner. At normal glucose concentrations, however, basal uptake was higher (57 nmol/mg protein) and half-maximum inhibition (IC₅₀) was shifted to higher PhAsO concentrations (2×10^-4 mol/l) than at low glucose levels (basal uptake: 1.6 nmol/mg protein; IC₅₀: 5×10^-5 mol/l). It is concluded that 1) in PhAsO-exposed MDCK cells different uptake mechanisms operate in high and low glucose states and 2) that glucose exerts a beneficial effect on the toxicity of PhAsO in these cells. Received: 22 August 1994 / Accepted: 6 December 1994     

Biotransformation of trichloroethene: dose-dependent excretion of 2,2,2-trichloro-metabolites and mercapturic acids in rats and humans after inhalation

 Chronic bioassays with trichloroethene (TRI) demonstrated carcinogenicity in mice (hepatocellular carcinomas) and rats (renal tubular cell adenomas and carcinomas). The chronic toxicity and carcinogenicity is due to bioactivation reactions. TRI is metabolized by cytochrome P450 and by conjugation with glutathione. Glutathione conjugation results in S-(dichlorovinyl) glutathione (DCVG) and is presumed to be the initial biotransformation step resulting in the formation of nephrotoxic metabolites. Enzymes of the mercapturic acid pathway cleave DCVG to the corresponding cysteine S-conjugate, which is, after translocation to the kidney, cleaved by renal cysteine S-conjugate β-lyase to the electrophile chlorothioketene. After N-acetylation, cysteine S-conjugates are also excreted as mercapturic acids in urine. The object of this study was the dose-dependent quantification of the two isomers of N-acetyl-S-(dichlorovinyl)-L-cysteine, trichloroethanol and trichloroacetic acid, as markers for the glutathione- and cytochrome P450-mediated metabolism, respectively, in the urine of humans and rats after exposure to TRI. Three male volunteers and four rats were exposed to 40, 80 and 160 ppm TRI for 6 h. A dose-dependent increase in the excretion of trichloroacetic acid, trichloroethanol and N-acetyl-S-(dichlorovinyl)-L-cysteine after exposure to TRI was found both in humans and rats. Amounts of 3100 μmol trichloroacetic acid+trichloroethanol and 0.45 μmol mercapturic acids were excreted in urine of humans over 48 h after exposure to 160 ppm TRI. The ratio of trichloroacetic acid+trichloroethanol/mercapturic acid excretion was comparable in rats and humans. A slow rate of elimination with urine of N-acetyl-S-(dichlorovinyl)-L-cysteine was observed both in humans and in rats. However, the ratio of the two isomers of N-acetyl-S-(dichlorovinyl)-L-cysteine was different in man and rat. The results confirm the finding of the urinary excretion of mercapturic acids in humans after TRI exposure and suggest the formation of reactive intermediates in the metabolism of TRI after bioactivation by glutathione also in humans. Received: 22 June 1995 / Accepted: 5 October 1995     

Effect of glutathione (GSH) depletion on DNA damage and blood chemistry in aged and young rats

DNA is damaged by reactive oxygen species (ROS) and such damage is age-dependent. Blood chemical parameters also change age-dependently. Glutathione (GSH) plays an important role as an antioxidant. However, the effects of GSH on DNA damage and blood chemistry are unclear. Therefore, this study was aimed to evaluate GSH contribution to DNA damage and changes of blood chemical parameters in aged and young rats. The GSH content in the livers and kidneys of aged rats (20 months) were lower than that in young rats (9 weeks of age) with higher DNA damage detected by a comet assay. There was a negative correlation between the GSH content and the DNA damage in the liver and kidney. L-buthionine (S,R)-sulfoximine (BSO; 0, 5, 20 mM), which inhibits GSH synthesis, was administered in drinking water for 28 days to young and aged rats (8 weeks and 19 months of age at the start of the administration). The treatment significantly decreased GSH levels in the heart, liver, lung and kidney of either the young or aged rats without causing DNA damage in those organs. When compared with young rats, aged rats showed higher levels in aspartate aminotransferase, alanine aminotransferase, total bilirubin, total cholesterol, globulin, creatinine, sodium and chloride and lower levels in alkaline phosphatase, triglyceride, albumin/globulin and inorganic phosphorus. However, BSO did not change these parameters in young or aged rats. These results showed that there was a negative correlation between GSH and DNA damage during aging, but the BSO-induced GSH depletion did not affect DNA damage or blood chemistry levels in young and aged rats under these study conditions.     

Distribution after repeated oral administration of different dose levels of risperidone and 9-hydroxy-risperidone in the brain and other tissues of rat

Rats were treated with daily oral doses of 1, 4, and 6 mg/kg risperidone (RSP) and its metabolite, 9-hydroxy-risperidone (9-OH-RSP), for 15 consecutive days. Concentrations of RSP and 9-OH-RSP were measured in plasma, brain, liver, kidney, lungs and fat tissue by high-performance liquid chromatography with electrochemical detection. Non-specific distribution of RSP and 9-OH-RSP in various brain regions was also studied after administration of 6 mg/kg per day oral dose for 15 days. After RSP treatment, concentrations of 9-OH-RSP were higher than those of RSP in plasma and tissues except in brain, where both compounds were present in nearly equal concentrations. Similarly, after 9-OH-RSP treatment, levels of 9-OH-RSP were higher than levels of either RSP or 9-OH-RSP or the sum of RSP and 9-OH-RSP levels measured after treatment with RSP. There was a moderate relationship between RSP dose and tissue levels of RSP and 9-OH-RSP (all r _s ≥ 0.62, P < 0.01), except in fat. There was also a strong relationship between the dose and tissue levels of 9-OH-RSP (all r _s≥ 0.68, P < 0.005). A significant relationship was found between plasma levels of RSP and brain levels of RSP and 9-OH-RSP (all r _s ≥ 0.57, P < 0.03) after treatment with RSP. After 9-OH-RSP treatment, a much stronger relationship was observed between plasma and brain 9-OH-RSP levels (r _s ≥ 0.90, P < 0.005). The plasma concentrations of RSP and 9-OH-RSP appear to reflect their concentrations in brain. The tissue-to-plasma ratios of RSP and 9-OH-RSP were relatively low compared to other antipsychotics. In liver, kidney and lung the tissue to plasma ratio for RSP and 9-OH-RSP after treating with RSP ranged from 0.85 to 3.4. The brain to plasma ratio for RSP and 9-OH-RSP was several-fold lower than that in peripheral tissues. After RSP administration, the mean brain to plasma level ratio for RSP was 0.22, and for 9-OH-RSP to it was 0.04. The brain to plasma ratio of 9-OH-RSP after giving 9-OH-RSP was similarly low (0.04). The low brain/plasma ratio of high potency RSP and 9-OH-RSP may in part be due to their low lipophilicity, log P = 3.04 and 2.32, respectively, resulting in limited non-specific accumulation in brain tissue. Received: 11 September 1997/Final version: 4 February 1998     

Effect of age on cadmium-induced metallothionein synthesis in the rat

Cadmium-induced metallothionein (MT) synthesis was investigated in male rats of three ages, 3, 12 and 24 months. Physiological levels of MT in kidney, liver and lung measured simultaneously in untreated animals were found to be within the same range in all three age groups. Following 3-day treatment with CdCl₂ equivalent to 1 mg/kg/day Cd, renal MT increased 10–15-fold in all animals. However, hepatic MT became elevated to 36 times the original value in the youngest (3-month-old) rats and 90 and 74 times, respectively, in the 12- and 24-month-old groups. Lung MT, which responded rather poorly to the cadmium inducer, increased 2-fold in the young group, but about 10 and 8 times, respectively, in the two older groups. High mortality of 75% occurred in the aged (24-month-old) group following cadmium administration, indicating age to be an important sensitizing factor in the toxic hazard of heavy metal exposure. The possible role in this connection of MT is discussed.     

Behavioural and pharmacological characterisation of the canopy stretched attend posture test as a model of anxiety in mice and rats

The behavioural element, stretched attend posture (SAP), is an important component of the “risk-assessment” repertoire of defensive behaviour in rodents. The present experimental paradigm was devised as a novel and simple method of eliciting high levels of SAP in mice and rats. The SAP test apparatus comprised an elevated black Perspex circular platform. A smaller clear red Perspex circular “Canopy” was supported directly above the platform by a central pillar, thus dividing the platform into an inner, dimly lit covered zone and an outer, brightly lit exposed zone. In both the rat and mouse version of this model, vehicle-treated animals exhibited a marked preference for exploring the covered zone and also exhibited high baseline levels of SAP, particularly at the covered zone boundary whilst they investigated the exposed zone. In the mouse SAP test, the benzodiazepine receptor agonists, diazepam (0.5 mg/kg SC) and chlordiazepoxide (2 mg/kg SC), and the 5-HT_1A receptor agonists, buspirone (1 and 3 mg/kg SC), ipsapirone (3 mg/kg SC) and 8-OH-DPAT (0.2 mg/kg SC), all significantly decreased the frequency of SAP without impairing motor activity. In the rat SAP test, diazepam (0.5 mg/kg SC) significantly decreased, whilst the anxiogenic 5-HT_2C/1B receptor agonist, mCPP (0.25 and 0.5 mg/kg SC), significantly increased, the frequency of SAP. Ipsapirone (3 mg/kg SC) induced a non-specific behavioural inhibition. These data suggest that the “Canopy” SAP test is a useful paradigm to investigate risk assessment behaviour in both rats and mice, and may provide a sensitive novel rodent model of anxiety. Received: 11 October 1996/Final version: 18 February 1997     

The α-2a noradrenergic agonist, guanfacine, improves delayed response performance in young adult rhesus monkeys

 In aged monkeys with naturally occurring catecholamine depletion, α-2 adrenergic agonists such as guanfacine have repeatedly been shown to improve dorsolateral prefrontal cortical function, as assessed by the spatial delayed response task. Both low (0.0001–0.001 mg/kg) and high (0.5 mg/kg) but not intermediate (0.01–0.05 mg/kg) doses of guanfacine improve spatial working memory performance in aged animals. However, it is not known whether guanfacine would similarly improve performance in young animals. In the present study, the effects of guanfacine on delayed response performance were characterized in seven young adult rhesus monkeys. Low doses of guanfacine (0.0001–0.01 mg/kg) had no effect on task performance, while high doses of guanfacine (0.1–0.7 mg/kg) significantly improved task performance. The highest doses produced mild sedation that was independent of drug effects on delayed response. The most effective dose of guanfacine was challenged with the α-2 antagonist idazoxan (0.1 mg/kg). This dose of idazoxan had no effect on task performance when given alone. Consistent with an α-2 mechanism, idazoxan significantly decreased delayed response performance in guanfacine-treated animals. These results support the hypothesis that delayed response performance in young intact animals can be improved through actions at α-2 adrenergic receptors. Received: 25 January 1997 / Final version: 15 October 1997     

Difference in rates of the reaction of various mammalian oxyhemoglobins with phenylhydrazine

 Second order rate constants for the initial reaction of 12 mammalian oxyhemoglobins (Hb) with equimolar phenylhydrazine (PHZ), a compound inducing Heinz body hemolytic anemia, were determined by recording continuous changes in absorbance with time at 577 nm. The rate constants were varied in a range from 43 m^-1 ⋅ s^-1 with pig Hb to 255 m^-1 ⋅ s^-1 with dog Hb. On the other hand, isosbestic points at 526 and 587 nm were common to all the reaction processes. The aerobic reaction of Hb with PHZ resulted in denaturation of hemoprotein, and final reaction products were determined to be β-meso-phenylbiliverdin IXα and N-phenylprotoporphyrin IX. These results suggest that the reactivity of PHZ to Hb is influenced by the globin molecule, and the oxidative cleavage of the porphyrin ring causes the denaturation of hemoprotein. Received: 25 May 1994/Accepted: 10 August 1994     

Effects of stimulation of alpha1-adrenergic and NMDA/glycine-B receptors on learning defects in aged rats

The present study was designed to investigate the efficacy of stimulation of alpha₁-adrenoceptors and the strychnine insensitive glycine-B binding sites of the N-methyl-D-aspartate (NMDA) receptor complex to alleviate the age-related defect in water maze (WM) spatial (hidden platform) navigation. We found that daily pretraining IP treatment with 2-(2-chloro-5-trifluoromethylphenylamino) imidazole nitrate (ST 587), an alpha₁-adrenoceptor agonist, at 3000 μg/kg, but not at 1000 μg/kg, facilitated acquisition of water maze spatial navigation in aged rats. However, ST 587 3000 μg/kg (IP) did not stimulate WM spatial reversal learning or cue navigation to a visible platform in aged rats. A partial strychnine insensitive glycine-B binding site agonist, D-cycloserine (DCS) at 10000 μg/kg stimulated acquisition of WM navigation, but had no effect on reversal learning or cue navigation. DCS at 1000 or 3000 μg/kg (IP) had no marked effect on WM spatial navigation, and did not enhance the WM performance improving effect of ST 587 in aged rats. A subthreshold dose of ST 587 1000 μg/kg did not enhance the therapeutic effect of DCS 10000 μg/kg. The present results indicate that activation of alpha₁-adrenoceptors and glycine binding sites of NMDA receptor may to some extent alleviate the age-related defect in spatial navigation. DCS treatment does not enhance the therapeutic effects of ST 587 and vice versa. Received: 20 August 1996/Final version: 21 November 1996     

Assessment of primary neuronal culture as a model for soman-induced neurotoxicity and effectiveness of memantine as a neuroprotective drug

 An in vitro mammalian model neuronal system to evaluate the intrinsic toxicity of soman and other neurotoxicants as well as the efficacy of potential countermeasures was investigated. The link between soman toxicity, glutamate hyperactivity and neuronal death in the central nervous system was investigated in primary dissociated cell cultures from rat hippocampus and cerebral neocortex. Exposure of cortical or hippocampal neurons to glutamate for 30 min produced neuronal death in almost 80% of the cells examined at 24 h. Hippocampal neurons exposed to soman for 15–120 min at 0.1 μM concentration caused almost complete inhibition (≥90%) of acetylcholinesterase but failed to show any evidence of effects on cell viability, indicating a lack of direct cytotoxicity by this agent. Acetylcholine (ACh, 0.1 mM), alone or in combination with soman, did not potentiate glutamate toxicity in hippocampal neurons. Memantine, a drug used for the therapy of Parkinson’s disease, spasticity and other brain disorders, significantly protected hippocampal and cortical neurons in culture against glutamate and N-methyl-D-aspartate (NMDA) excitotoxicity. In rats a single dose of memantine (18 mg/kg) administered 1 h prior to a s.c. injection of a 0.9 LD₅₀ dose of soman reduced the severity of convulsions and increased survival. Survival, however, was accompanied by neuronal loss in the frontal cortex, piriform cortex and hippocampus. Received: 2 May 1994 / Accepted: 9 November 1994     

Chemical form of selenium-containing metabolite in small intestine and liver of mice following orally administered selenocystine

 The chemical form of a selenium-containing metabolite in the small intestine following a single oral administration of selenocystine was investigated with ICR male mice. Selenium content in the small intestine of animals treated with 50 mg/kg selenocystine significantly increased 15 min, 1 h and 6 h after treatment. In contrast, selenocystine significantly depressed the intestinal reduced glutathione (GSH) level at 1 h after administration. A significant negative correlation between the selenium level and the level of GSH in the small intestine was observed (r=−0.83, p<0.001). Analysis of the intestinal metabolite of selenocystine showed that selenium-containing metabolites elute in two fractions from a Sephadex G-25 column: the low-molecular fraction (peak I) contained the selenocystine, while the high-molecular fraction (peak II) contained selenocysteine-containing metabolite. An in vitro experiment was performed to gain insight into the mechanism for selenocysteine-containing metabolite production in the intestinal cytosol. When selenocystine or selenocysteine reacted with excess GSH in the presence of intestinal homogenate, the peak II fraction which involved the selenocysteine-containing metabolite was recognized in the Sephadex G-25 chromatogram. From an examination of the distribution of the selenocysteine-containing metabolite, it was recognized that this metabolite exists in plasma and liver cytosol of mice after oral administration of selenocystine. These results suggested that the mice treated with selenocystine produce selenocysteine-containing metabolite by reaction of selenocystine with excess GSH in the small intestine, and the metabolite is then transported to the liver through blood plasma. Received: 29 August 1994/Accepted: 14 November 1994     

Behavioral sensitization to nicotine is associated with behavioral disinhibition; counteraction by citalopram

This study investigated the effects of repeated nicotine treatment on locomotor activity and behavioral inhibition, and the influence of citalopram on the behavioral effects obtained. Male rats received daily subcutaneous injections of vehicle + vehicle (veh + veh), citalopram (5.0 mg/kg) + vehicle (cit + veh), vehicle + nicotine (1.0 mg/kg; veh + nic) or citalopram+nicotine (cit + nic). Acutely, nicotine stimulated locomotor activity, and repeated daily nicotine injections sensitized veh+nic rats to the nicotine-induced locomotor stimulation after 5, 10 and 15 treatment days, whereas in cit+nic rats, the enhancement of nicotine-induced locomotion was suppressed. However, when challenged with nicotine after citalopram withdrawal (−36 h), the cit + nic treated animals were also observed to be sensitized. In the elevated plus-maze, repeated nicotine treatment produced behavioral disinhibition, measured as an increase of time spent in and entries made into open arms (%), and chronic citalopram treatment attenuated the expression of behavioral disinhibition. Moreover, the degree of nicotine sensitization correlated to the behavioral disinhibition observed. In summary, these findings suggest that behavioral sensitization to nicotine is associated with behavioral disinhibition and that chronic citalopram treatment counteracts the expression of both phenomena. Since citalopram is a highly selective serotonin reuptake inhibitor, the effects of citalopram may be due to a facilitation of serotonin neurotransmission caused by the chronic citalopram treatment. Received: 2 December 1997/Final version: 16 August 1998