Mutagenic and genotoxic effects of carbosulfan in freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis

Carbosulfan insecticide is widely used in agriculture and was recently proposed for treatment against pyrethroid-resistant mosquitoes. The mutagenic and genotoxic effect of carbosulfan was carried out in fish Channa punctatus using micronucleus (MN) test and comet assay. The 96 h LC₅₀, estimated by probit analysis in a semi-static bioassay experiment, was 0.268 mg l⁻¹. Based on the LC₅₀ value, three sub-lethal concentrations of carbosulfan (1/4th LC₅₀ = ∼67 μg l⁻¹, 1/2nd LC₅₀ = ∼134 μg l⁻¹ and 3/4th LC₅₀ = ∼201 μg l⁻¹) were selected and fishes were exposed to the said concentrations for 96 h and the samplings were done at regular intervals of 24 h for assessment of the MN frequencies and DNA damage. In general, significant effects (P < 0.01) from both concentrations and time of exposure were observed in exposed fishes. The MN induction was highest on 96 h at all the concentrations in the peripheral blood. Similar trend was observed for the DNA damage measured in terms of the percentage of tail DNA in the erythrocyte and gill cells. This study confirmed that the comet and micronucleus assays are useful tools in determining potential genotoxicity of water pollutants and might be appropriate as a part of monitoring program.     

Assessment of genotoxic effects of benzyl derivatives by the comet assay

In this study, different concentrations of four benzyl derivatives (benzyl alcohol, benzyl acetate, benzoic acid and benzaldehyde) used as flavour ingredients were investigated for genotoxicity in in vitro. By taking blood from two healthy people comet assay was carried on to investigate the potential health damages of benzyl derivatives. For the evaluation of genotoxic effects, the tail moment and % tail DNA in the treated chemicals were compared to the solvent control, which is distilled water. The alkaline comet assay showed significantly increased tail moment and % tail DNA at 25 and 50 mM concentrations of benzyl alcohol. Benzyl acetate increased both % tail DNA and tail moment at 50 mM concentrations. While % tail DNA was statistically increased at 10 mM and higher concentrations, tail moment has significant difference at 10 and 25 mM concentrations of benzaldehyde. Benzoic acid has apoptotic effects at the concentrations higher than 5 mM, for this reason we tested concentrations less than 5 mM (0.05, 0.1, 0.5, 1 and 5 mM). Only the highest concentration of benzoic acid increased both tail moment and % tail DNA.     

Memory deficit,toxic effects and activity of Na+, K+-ATPase and NTPDase in brain of Wistar rats submitted to orally treatment with alpha-terpinene

The neurotoxic effects and activity of Na⁺, K⁺-ATPase and NTPDase in Wistar rats after treatment with α-terpinene (daily oral administration of 0.5, 0.75 and 1.0 mL kg⁻¹ for 10 days) were examined. Results of the inhibitory avoidance task showed a memory deficit (p < 0.05) in rats treated with all doses of α-terpinene. The evaluation of DNA damage in brain tissue revealed an increase (p < 0.05) on frequency of damage and damage index in all concentrations. According to the cytotoxicity assay, doses of 0.5, 0.75 and 1.0 mL kg⁻¹ increase the lactate dehydrogenase levels, and doses of 1.0 mL kg⁻¹ also decrease (p < 0.05) cell viability in brain cells. A decrease (p < 0.05) on Na⁺, K⁺-ATPase activity in brain tissue and on NTPDase activity in serum were observed in all concentrations of α-terpinene. These results suggest that the α-terpinene was cytotoxic and genotoxic to the brain cells by inducing loss of cell viability and DNA damage, as well as causing alterations in Na⁺, K⁺-ATPase and NTPDase activity, what may contribute to the memory deficit of treated animals. Thus, α-terpinene cannot be consumed by the population at the doses studied.     

Assessment of genotoxic and mutagenic effects of chlorpyrifos in freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis

Chlorpyrifos (CPF) is the single largest selling agrochemical that has been widely detected in surface waters in India. The studies on long-term genotoxic effects of CPF in different tissues of fish using genotoxic biomarkers are limited. Therefore, in the present study DNA damage by CPF in freshwater fish Channapunctatus using micronucleus (MN) and comet assays was investigated. The LC₅₀ – 96 h of CPF was estimated for the fish in a semi-static system. On this basis of LC₅₀ value sublethal and nonlethal concentrations were determined. The DNA damage was measured in lymphocytes and gill cells as the percentage of DNA in comet tails and micronuclei were scored in erythrocytes of fishes exposed to above concentrations of CPF. In general, significant effects for both the concentrations and time of exposure were observed in treated fish. It was found that MN induction in the blood was highest on day 14 at 203.0 μg/l of CPF. The highest DNA damage was observed on day 5, followed by a gradual non-linear decline in the lymphocytes and gill cells. The study indicated MN and comet assays to be sensitive and rapid methods to detect mutagenicity and genotoxicity of CPF and other pollutants in fishes.     

In vitro study on the genotoxicity of dichloromethane extracts of valerian (DEV) in human endothelial ECV304 cells and the effect of vitamins E and C in attenuating the DEV-induced DNA damages

To study the genotoxicity of valepotriates in vitro, the degree of DNA damage in human endothelial cell line ECV304 treated with 5-60 microg/mL of dichloromethane extracts of valerian (DEV) was analyzed by the Comet assay. No DNA damage was observed in ECV304 cells after culture for 48 h in the presence of 5,10, and 20 microg/mL of DEV. But a moderate degree of DNA damage was observed in the cells treated with 40 or 60 microg/mL of DEV. Quantitative analyses of DNA damage in the presence of antioxidants vitamin E (VE) and vitamin C (VC) were also carried out. The study revealed that both VE and VC exhibited a biphasic effect, reducing DEV-induced DNA damages at low concentrations but increasing them at high concentrations. We concluded that (1). the observed DNA damage in ECV304 cells induced by high concentrations of DEV was mainly through epigenetic mechanisms, i.e., reactive oxygen species mediated oxidative DNA damage (2). at the low doses, DEV did not appear to have any significant genotoxicity in ECV304 cells, and (3). VE and VC, at proper concentrations, can reduce or eliminate the adverse effects derived from high doses of DEV. This study should serve as scientific guidance for clinical therapy of valerian preparation.     

Genotoxic and cytotoxic effects and gene expression changes induced by fixed orthodontic appliances in oral mucosa cells of patients: a systematic review

Abstract

Context: The accumulation of chronic or severe acute DNA and cellular damage in oral mucosa cells is one of the main factors that help initiate a wide range of malignant lesions in the oral cavity. There has been considerable controversy in the literature about the effect of such sustained genotoxic and cytotoxic damage to oral mucosa cells.

Objective: The aim of this systematic review, reported in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, is to investigate the effects of such interventions.

Methods: Electronic and manual searches were performed (15 May 2015) for Randomized Clinical Trials/quasi-Randomized Clinical Trials that analyzed the genotoxic/cytotoxic effects of these types of oral appliances in humans. A primary outcome (cell/DNA damage) and a number of secondary outcomes were examined. Two reviewers carried out the study selection and performed a “risk of bias” assessment [Cochrane Collaboration's tool]. Wherever possible the meta-analysis was conducted on homogenous groups.

Results: From the electronic search (2797), 6 studies met the eligibility criteria. Most studies (5/6) observed significant differences in most comparisons at the short-term (1-3 months) and long-term (24–48 months) evaluations, with respect to critically acute genotoxic/cytotoxic effects. Some of the studies (2/3) concluded that the post-removable effects at DNA/cellular levels were not significant (p?>?0.05) with respect to the controls.

Conclusions: Acute DNA/cellular damage in oral mucosa cells is induced by orthodontic appliances. Nevertheless, even though these effects were no longer detected after removing the appliances, more rigorous RCTs are needed to explore the extent to which acquired damage can be observed in the oral mucosa.     

Evaluation of the protective effect of Ilex paraguariensis and Camellia sinensis extracts on the prevention of oxidative damage caused by ultraviolet radiation

We evaluated the effects green and mate teas on oxidative and DNA damages in rats exposed to ultraviolet radiation. Were utilized 70 adult male Wistar rats that received daily oral or topic green or mate tea treatment during exposed to radiation by seven days. After, animals were killed by decapitation. Thiobarbituric acid-reactive species levels, protein oxidative damage were evaluated in skin and DNA damage in blood. Our results show that the rats exposed to ultraviolet radiation presented DNA damage in blood and increased protein carbonylation and lipid peroxidation in skin. Oral and topic treatment with green tea and mate tea prevented lipid peroxidation, both treatments with mate tea also prevented DNA damage. However, only topic treatment with green tea and mate tea prevented increases in protein carbonylation. Our findings contribute to elucidate the beneficial effects of green tea and mate tea, here in demonstrated by the antioxidant and antigenotoxic properties presented by these teas.     

Assessment of the DNA damaging potency and chemopreventive effects towards BaP-induced genotoxicity in human derived cells by Monimiastrum globosum, an endemic Mauritian plant

Naturally occurring compounds have protective effects towards mutagens and carcinogens. The leaf extract of Monimiastrum globosum (Bois de Clous), a Mauritian endemic plant from the Myrtaceae family, was studied for its potency to induce DNA damage in human HepG2 hepatoma cells using DNA migration as a biological endpoint in the alkaline single cell gel electrophoresis (SCGE) assay. This was contrasted with the ability to modulate the benzo[a]pyrene (BaP)-dependent DNA damage in human hepatoma cells. M. globosum caused genotoxicity in HepG2 cells at concentrations exceeding 3 mg fresh weight (FW) per ml cell culture in the absence of cytotoxicity. Pre-treatment of the cells with 12.2 μg FW/ml to 1.56 mg FW/ml led to a pronounced antigenotoxic effect towards BaP-induced DNA damage. DNA migration (OTM) was reduced by 66%, 81.5% and 74% for 49, 98 and 195 μg FW/ml, respectively. A U-shaped dose-response curve was derived for M. globosum indicating genotoxic effects in high doses and antigenotoxic effects in low doses. M. globosum extract had total phenolics (15 mg/g FW) with flavonoids (aglycones and conjugates: 8 mg/g FW) and proanthocyanidins (3 mg/g FW) as major phenolic subclasses. The hydrolysis of conjugated flavonoids yielded the aglycones quercetin (606 μg/g FW) and kaempferol (117.8 μg/g FW) while HPLC-MS/MS analysis of the total extract revealed free flavonoids such as quercetin (19.2 μg/g FW) and myricetin (2.5 μg/g FW). The antioxidant activity of the extract of M. globosum, assessed by the FRAP and TEAC assays yielded values of 275 ± 3.82 μmol/g FW and 346 ± 4.2 μmol/g FW, respectively.     

The inhibitory effects of garlic and <Emphasis Type="Italic">Panax ginseng</Emphasis> extract standardized with ginsenoside Rg3 on the genotoxicity,biochemical, and histological changes induced by ethylenediaminetetraacetic acid in male rats

Ethylenediaminetetraacetic acid (EDTA) is widely used in food and other industries to sequester metal ions and to prevent their disadvantageous effects. The objective of the current study was to evaluate the protective effect of Panax ginseng extract standardized with ginsenoside Rg3 (ginsenoside Rg3 content was 3.6% w/w, i.e., 36 μg/mg P. ginseng extract) and garlic against EDTA-induced biochemical, genotoxic, and histological changes in rats. Forty male rats were divided into eight treatment groups and treated for 7 days as follows: the control group, the group treated with EDTA (20 mg/kg b.w) and the groups treated with P. ginseng extract (20 mg/kg b.w), garlic (5 mg/kg b.w), P. ginseng plus garlic alone or in combination with EDTA. In vivo bone marrow micronucleus test and random amplified polymorphism DNA-PCR (RAPD-PCR) method were performed to assess the antigenotoxic effect of both protective agents. The results indicated that EDTA administration caused a significant decrease in the serum biochemical parameters and antioxidant enzymes activity. The administration also increased lipid peroxidation and the incidence of micronucleated polychromatic erythrocytes (MnPCEs), caused appearance of some changes in polymorphism band patterns, and induced different histopathological lesions in the livers, kidneys, and testis. Treatment with P. ginseng, garlic alone or plus EDTA significantly improved all the tested parameters. Moreover, P. ginseng extract was found to be more effective than garlic in restoring the parameters that were altered by EDTA.     

Dietary administration of sodium arsenite to rats: Relations between dose and urinary concentrations of methylated and thio-metabolites and effects on the rat urinary bladder epithelium

Based on epidemiological data, chronic exposure to high levels of inorganic arsenic in drinking water is carcinogenic to humans, inducing skin, urinary bladder and lung tumors. In vivo, inorganic arsenic is metabolized to organic methylated arsenicals including the highly toxic dimethylarsinous acid (DMA^III) and monomethylarsonous acid (MMA^III). Short-term treatment of rats with 100 μg/g trivalent arsenic (As^III) as sodium arsenite in the diet or in drinking water induced cytotoxicity and necrosis of the urothelial superficial layer, with increased cell proliferation and hyperplasia. The objectives of this study were to determine if these arsenic-induced urothelial effects are dose responsive, the dose of arsenic at which urothelial effects are not detected, and the urinary concentrations of the arsenical metabolites. We treated female F344 rats for 5 weeks with sodium arsenite at dietary doses of 0, 1, 10, 25, 50, and 100 ppm. Cytotoxicity, cell proliferation and hyperplasia of urothelial superficial cells were increased in a dose-responsive manner, with maximum effects found at 50 ppm As^III. There were no effects at 1 ppm As^III. The main urinary arsenical in As^III-treated rats was the organic arsenical dimethylarsinic acid (DMA^V). The thio-metabolites dimethylmonothioarsinic acid (DMMTA^V) and monomethylmonothioarsinic acid (MMMTA^V) were also found in the urine of As^III-treated rats. The LC₅₀ concentrations of DMMTA^V for rat and human urothelial cells in vitro were similar to trivalent oxygen-containing arsenicals. These data suggest that dietary As^III-induced urothelial cytotoxicity and proliferation are dose responsive, and the urothelial effects have a threshold corresponding to the urinary excretion of measurable reactive metabolites.     

Prenatal treatment with clomipramine: effects on the behaviour of male and female adolescent rats

The tricyclic anti-depressant clomipramine (7.5 or 15 mg/kg/day) was administered to pregnant rats between days 8 and 21 of gestation. Between postnatal days 31 and 47, both male and female offspring received three behavioural tests. Prenatal clomipramine (15 mg/kg/day) increased baseline acoustic startle in females, but not in males; both sexes showed greater between-day response decrements if they had received clompramine. In the social interaction test of anxiety, males prenatally exposed to clomipramine (both dosages), and females prenatally exposed to 7.5 mg/kg/day, revealed a similar profile to that seen after chronic administration of benzodiazepines in the adult. The likelihood that differences in within-session habituation could underlie the changes in social interaction that have been found in this and other studies is assessed.     

淫羊藿和三七提取物组合活血化瘀作用及对东莨菪碱造模小鼠被动回避的影响

目的考察淫羊藿(Herba Ep im ed ii,HE)和三七(Rad ix Notoginseng,RN)乙醇提取物组合对大鼠的活血化瘀作用及对东莨菪碱造模智障小鼠被动回避的影响,为该组合物治疗老年性痴呆提供实验依据。方法大鼠连续ig HE和RN提取物组合(1,3.1g/kg)7d,采用试剂盒测定该组合物对大鼠血清凝血酶原时间(PT)、部分凝血活酶时间(KPTT)的影响,用下腔静脉结扎法考察其对大鼠下腔静脉血栓形成的影响。此外,采用相同剂量及给药方法考察了HE和RN提取物组合对东莨菪碱造模智障小鼠被动回避的影响。结果连续ig HE和RN提取物组合(1,3.1g/kg)可显著延长KPTT(P<0.05),而对PT没显著性影响(P>0.05);能显著预防下腔静脉血栓的形成(P<0.01);在活血化瘀作用剂量下HE和RN提取物组合对东莨菪碱造模小鼠被动回避没显著性影响(P>0.05)。结论HE和RN提取物组合具有显著的活血化瘀作用,但对东莨菪碱造模智障小鼠被动回避无显著影响。     

Influence of stressors on the rewarding effects of alcohol in Wistar rats: studies with alcohol deprivation and place conditioning

Rationale and objectives Studies on laboratory animals have provided conflicting results regarding the actions of stressors on the rewarding effects of alcohol. In the present study, we first examined the effects of footshock or social defeat, given during deprivation, on the alcohol deprivation effect (ADE). We then tested the effects of stressors on place conditioning to alcohol, another technique used to measure drug reward.Methods Male Wistar rats were trained to drink 10% alcohol in a 24 h access, free-choice design and received intermittent footshock or defeat 5 times during a 2-week alcohol deprivation period, followed by 2 weeks of free access to alcohol. There were three such cycles. In the place conditioning studies, animals received footshock, defeat, or no stress immediately prior to conditioning sessions where they received alcohol (0.6 or 1.0 g/kg, IP) or vehicle injections.Results Alcohol intake of footshock-treated animals was significantly higher than that of controls following the first and second, but not the third period of alcohol deprivation and stress exposure. Defeat caused a smaller increase in alcohol intake that was significant only after the first deprivation and stress cycle. In the place conditioning studies, we found that either stressor blocked the place aversion induced by 1.0 g/kg alcohol.Conclusions These results demonstrate that stressors can modify the rewarding and aversive properties of alcohol, measured using two different paradigms. Footshock and defeat produced transient, but significant increases in the magnitude of ADE, while exposure to either stressor reduced the aversive effects of a high dose of alcohol measured using the place conditioning paradigm.     

Differential effects of the new antipsychotic risperidone on large and small motor movements in rats: a comparison with haloperidol

Risperidone, a new antipsychotic agent, was studied for its effects on spontaneous motor activity in rats in comparison with haloperidol. Motor activity was recorded via the optical scanning technique (horizontal and vertical activity) and via a recently developed technique based on the piezo-electric principle which, in contrast to optical scanning, is very sensitive to small, stationary movements (piezo activity). Risperidone and haloperidol at low doses depressed both vertical activity (ED₅₀s: 0.062 and 0.038 mg/kg, respectively) and horizontal activity (0.18 and 0.060 mg/kg, respectively). With increase of dose, motor activity decline was significantly faster with haloperidol than with risperidone. Moreover, haloperidol also rapidly depressed piezo activity (ED₅₀: 0.085 mg/kg) whereas risperidone depressed this component of motor behaviour at much higher doses only (ED₅₀: 2.80 mg/kg). Visual inspection did not reveal abnormal behavioural movements following the test compounds. Risperidone, therefore, preserves normal small movements over a much larger dose interval than haloperidol; this effect may be related to its relatively low cataleptogenic activity and potentially also to a reduced EPS liability. The present results further confirm that the piezo technique may complement the optical scanning method, and thereby enhance the information on the extent that test compounds modify behaviour.     

The pharmacology of impulsive behaviour in rats II: the effects of amphetamine, haloperidol, imipramine, chlordiazepoxide and other drugs on fixed consecutive number schedules (FCN 8 and FCN 32)

 The effects of drugs on one aspect of impulsive behaviour were evaluated using a schedule in which rats were trained to complete a fixed consecutive number of responses on one of two levers before pressing the second to obtain a reinforcer (FCN). Terminating the chain before completing the FCN resulted in the omission of the food, and can be considered an impulsive decision. Two groups of food-deprived rats were trained to press either 8 or 32 times on the left lever (FCN lever) of a two lever operant chamber before pressing the right lever (Reinforcement lever) to deliver a food pellet. Responding on the Reinforcement lever before completion of the sequence resulted in a short time-out and the rat had to begin the sequence again. After responding had stabilised, the rats were treated with a range of doses of a number of drugs. Impulsivity was assessed by several measures, including the mean chain length and the proportion of chains terminating in food delivery, and the distribution of chain lengths was analysed. The efficiency of the rats was similar under both FCN 8 and FCN 32, although it was more difficult to maintain a consistent baseline under FCN 32. Under the FCN 8 schedule, significant decreases in chain length were obtained with d-amphetamine (0.8–2.4 mg/kg), haloperidol (0.1 mg/kg), ethanol (1 and 3 g/kg) and chlordiazepoxide (10.0 mg/kg), and there were alterations in other measures consistent with an increase in impulsivity. Imipramine (1–10 mg/kg), citalopram (1–10 mg/kg) and metergoline (0.3–3.0 mg/kg) had no effect on mean chain length, although the first two drugs shifted the chain length distribution to the left. d-Amphetamine (0.4–1.2 mg/kg) and PCPA (100 mg/kg) reduced chain length and had other effects consistent with increased impulsivity under FCN 32 schedule, whereas imipramine had little, and citalopram no, effect. Taken generally, effect of the active drugs was relatively non-specific, including both a reduction in response rate and alterations in choice measures proposed to reflect an increase in impulsivity. Detailed analysis of the effect of amphetamine revealed that three processes were at work: chain shortening, an increased preference for the lever most closely associated with food delivery, and a gradual shift in the control over responding from the response sequence (pattern) to the individual lever press (act). Received: 24 April 1997 / Final version: 14 January 1998     

Water-soluble and organic extracts of ambient PM2.5 in Tehran air: assessment of genotoxic effects on human lung epithelial cells (A549) by the Comet assay

The main aim of the present study is to investigate the physicochemical characterization of water-soluble extract (WS) and organic extract (OE) of PM_2.5 ambient in Tehran city air in order to evaluate the genotoxicity effects and the potential attribution to these effects. The lung epithelial cells (A549) were exposed to WS and OE, while Comet assays were conducted to analyze the genotoxicity. The results show that the amount of DNA damage in WS fraction and solvent-extractable organic samples is significantly higher than the control group and the increase in concentration significantly contributes to increase in the amount of DNA damage.     

The pharmacology of impulsive behaviour in rats VI: the effects of ethanol and selective serotonergic drugs on response choice with varying delays of reinforcement

Rationale: Tolerance to delay of reinforcement has been proposed as an important facet of self-control in both animals and man. Poor self-control, leading to impulsive behaviour, can be a major problem if it reaches pathological levels. Objectives: The effects of five serotonergic drugs were compared to those of ethanol on a procedure for measuring tolerance to delay of reinforcement in rats in order to elucidate further the role of the serotonin systems in the regulation of impulsive behaviour. Methods: Rats were trained to choose between a single food pellet (small reinforcer) delivered immediately or five food pellets (large reinforcer) delivered after programmed delays. At the start of each session, there was no delay between the response and delivery of the large reinforcer, but this was increased stepwise during the session to delays of 10, 20, 40 and 60 s. Results: The rats showed consistent preference for the larger reinforcer when it was not delayed but showed a shift in preference as the session continued, so that they preferred the small reinforcer when the large was delayed by 40 or 60 s. Ethanol at a dose of 1.0 g/kg produced a significance increase in preference for the small, immediate reinforcer throughout the session, although there were marked individual differences in the size of the effect. A similar, but somewhat smaller effect was seen with the 5-HT₂ agonist, DOI, at a dose of 1.0 mg/kg. In contrast, the 5-HT_1A agonist, 8-OH-DPAT (0.3 mg/kg) reduced preference for the large reinforcer at the start of the session, and reduced preference for the small reinforcer at the end of the session, i.e. produced a regression to indifference. Lower doses of these three drugs, and treatment with the 5-HT receptor subtype selective antagonists WAY-100635 (5-HT_1A: 0.01–0.1 mg/kg), ritanserin (5-HT₂: 0.1 and 0.3 mg/kg) and MDL-72222 (5-HT₃: 1.0 and 3.0 mg/kg) had no significant effects on reinforcer choice. Conclusion: These data show that ethanol and DOI increase preference for the immediate reinforcer, which can be construed as evidence of an increase in impulsive behaviour (reduction in self control), whereas selective blockade of the 5-HT_1A, 5-HT₂ or 5-HT₃ receptors using selective antagonists does not affect self-control. Received: 24 October 1998 / Final version: 16 February 1999     

A behavioural analysis of the delayed non-matching to position task: the effects of scopolamine, lesions of the fornix and of the prelimbic region on mediating behaviours by rats

The delayed non-matching to position task (DNMP) is a widely used automated test of spatial memory, yet its validity has been challenged by suggestions that animals use motor mediating behaviours which facilitate correct responding. This possibility was systematically studied by analysing video recordings of rats displaying delay-dependent and delay-independent deficits following lesions or drug manipulations. Rats were first trained to perform the DNMP task and whilst untreated, a number of potential mediating behaviours were identified from the video recorded behaviour. Two independent raters recorded any apparent motor strategies and attempted to predict the response the animals made during the choice phase of the task by viewing only behaviour during the delay periods. Subsequently, the behaviour of the same animals was examined following scopolamine treatment and following lesions of the prelimbic cortex or of the fornix. The experiment confirmed previous reports of delay-dependent and delay-independent deficits under the varying conditions (drug, lesions), but also revealed that rats use clearly identifiable mediating behaviours that appear to facilitate correct responding in the DNMP task. Consequently, apparent “memory” impairments in the DNMP task, may reflect a disruption of behavioural strategies used by the animal to assist in performing the task. Received: 19 December 1996/Final version: 14 April 1997     

Involvement of free radicals followed by the activation of phospholipase A2 in the mechanism that underlies the combined effects of methamphetamine and morphine on subacute toxicity or lethality in mice: comparison of the therapeutic potential of fullerene, mepacrine, and cooling

An increase in polydrug abuse is a major problem worldwide. The coadministration of methamphetamine and morphine increased subacute toxicity or lethality in rodents. However, the underlying mechanisms by which lethality is increased by the coadministration of methamphetamine and morphine are not yet fully understood. Coadministered methamphetamine and morphine induced lethality by more than 80% in BALB/c mice, accompanied by the rupture of cells in the kidney and liver, and an increase in poly (ADP-ribose) polymerase (PARP)-immunoreactive cells in the heart, kidney and liver. The lethal effect and the increase in the incidence of rupture or PARP-immunoreactive cells induced by the coadministration of methamphetamine and morphine was significantly attenuated by pretreatment with mepacrine (phospholipase A(2) inhibitor) or fullerene (a radical scavenger), or by cooling from 30 to 90 min after drug administration. Furthermore, based on the results of the electron spin resonance spin-trapping technique, hydroxyl radicals were increased by the administration of methamphetamine and morphine, and these increased hydroxyl radicals were potently attenuated by fullerene and cooling. These results suggest that hydroxyl radicals plays an important role in the increased lethality induced by the coadministration of methamphetamine plus morphine. The potency of cooling or drugs for decreasing the subacute toxicity or lethality induced by the coadministration of methamphetamine and morphine was in the order fullerene=cooling>mepacrine. These results indicate that fullerene and cooling are beneficial for preventing death that is induced by the coadministration of methamphetamine and morphine.