[1] 12w Fenfluramine-induced serotonergic neurotoxicity in mice: lack of neuroprotection by inhibition/ablation of nNOS

[1] 184w Previous studies have implicated a role for nitric oxide (NO) and peroxynitrite in methamphetamine-induced dopaminergic neurotoxicity. The present study was undertaken to investigate whether NO is involved in serotonergic neurotoxicity caused by fenfluramine. In the first experiment, the effect of the neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI; 25 mg/ kg • 4) on fenfluramine (25 mg/kg • 4)-induced serotonergic neurotoxicity in Swiss Webster mice was investigated. In the second experiment, the effect of fenfluramine (25 mg/kg • 4) on nNOS (-/-) and wild-type (WT) mice was investigated. Fenfluramine induced hypothermia in all three mouse strains, and 7-NI had no thermoregulatory effect. Selective depletion of 5-HT and 5-HT transporter binding sites in the striatum, frontal cortex and hippocampus in all three mouse strains was observed, with no evidence of dopaminergic neurotoxicity. In the first experiment, 7-NI did not attenuate serotonergic neurotoxicity in Swiss Webster mice. In the second experiment, nNOS(-/-) and WT mice were equally sensitive to serotonergic neurotoxicity. These findings suggest that NO and peroxynitrite do not mediate fenfluramineinduced serotonergic neurotoxicity, and that NO is a selective mediator of amphetamines-induced dopaminergic neurotoxicity.

[1] 115w Repeated administration of fenfluramine (25 mg/kg) to Swiss Webster mice induced significant hypothermia ()1.8 ± 0.2°C) 30-60 min after each injection (Fig. 1a). Pre-treatment with 7-NI (25 mg/kg) prior to fenfluramine injection did not affect the hypothermic effect of the drug (Fig. 1a). Injections of either vehicle/saline or 7-NI (25 mg/kg)/saline had no effect on body temperature (Fig. 1a); the latter finding confirms our previous observations (Itzhak et al. 2000). Fenfluramine caused a greater decrease in body temperature in nNOS KO mice ()4.3 ± 0.2°C) than in WT mice ()2.4 ± 0.2°C) during the course of the four treatments (Fig. 1b). Saline injection had no effect on body temperature in nNOS KO and WT mice.

[2] 205w The effects of vehicle/fenfluramine and 7-NI/fenfluramine treatments on dopaminergic and serotonergic markers in various brain regions of Swiss Webster mice are summarized in Table 1. We observed a 39-72% reduction in 5-HT concentrations in the striatum, frontal cortex and hippocampus, as well as 56 and 61% reduction in the density of the 5-HTT in the hippocampus and frontal cortex, respectively. Pretreatment with 7-NI had no significant effect on fenfluramine-induced depletion of 5-HT and 5-HTT in any of the brain regions investigated (Table 1). Administration of vehicle/fenfluramine and 7-NI/fenfluramine had no significant effect on striatal DA, DOPAC, HVA and DAT levels (Table 1). In the second experiment, fenfluramine caused a 59-62% reduction in 5-HT concentrations in the striatum, frontal cortex and hippocampus, and 54 and 62% decrease in the density of 5-HTT in the frontal cortex and hippocampus, respectively, of WT mice (Table 1). The effect of fenfluramine in nNOS(-/-) mice was similar: a 73-88% decrease in the concentration of 5-HT in the striatum, frontal cortex and hippocampus, and 38 and 47% decrease in the density of 5-HTT in the hippocampus and frontal cortex, respectively, was observed (Table 1). Fenfluramine had no effect on striatal dopaminergic markers in either WT or nNOS KO mice (Table 1).

[1] 33w As summarized in the Introduction, several studies have indicated the role of NO/peroxynitrite in amphetamines-induced dopaminergic neu- NO and serotonergic neurotoxicity 269 Ó 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 268-271

[2] 46w rotoxicity. The major finding of the present study is that NO is not a mediator of fenfluramine-induced serotonergic neurotoxicity. This conclusion is supported by (i) the lack of protective effect of the nNOS inhibitor 7-NI, and (ii) the vulnerability of the nNOS(-/-) mice to serotonergic neurotoxicity.

[3] 75w We previously reported that 25 mg/kg 7-NI administered 30 min before METH prevented the depletion of striatal dopaminergic markers (Itzhak and Ali 1996). The protective effects of 7-NI and other nNOS inhibitors against METH-induced dopaminergic neurotoxicity were independent of METH-induced hyperthermia (Itzhak and Ali 1996;Itzhak et al. 2000). Likewise, another selective nNOS inhibitor, AR-R17477AR, attenuated the depletion of DA and the neurofilament protein NF68, but not the hyperthermia, caused by METH (Sanchez et al. 2003).

[4] 158w Fewer studies have focused on the mechanisms mediating druginduced serotonergic neurotoxicity. Fenfluramine induced rather selective depletion of 5-HT and 5-HTT in the striatum, frontal cortex and hippocampus of Swiss Webster, nNOS KO and WT mice, with no evidence of striatal dopaminergic marker depletion (Table 1). Selective depletion of serotonergic markers by fenfluramine in Swiss Webster (McCann et al. 1998) andC57BL/6 mice (O'Callaghan andMiller 1994) has been previously reported. Thus, we chose to investigate whether NO mediates serotonergic neurotoxicity caused by fenfluramine, but not other substituted amphetamines such as MDMA and p-chloroamphetamine, which induce both serotonergic and dopaminergic neurotoxicity in mice (e.g. Itzhak et al. 2003). If serotonergic neurotoxicity is dependent upon dopaminergic neurotoxicity (Kuhn and Arthur 1998), it may be more difficult to determine to what extent NO is involved in 5-HT neurotoxicity. Nevertheless, it has been reported that the NOS inhibitor L-nitro-arginine methyl ester attenuated METH-induced dopaminergic but not serotonergic neurotoxicity in rats (Abekawa et al. 1996).

[5] 184w Our results indicate that the nNOS inhibitor 7-NI did not attenuate the hypothermic and neurotoxic effects of fenfluramine in Swiss Webster mice. Additionally, fenfluramine caused depletion of 5-HT and 5-HTT in both WT and nNOS KO mice. Although marked differences in basal levels of 5-HT in WT and KO mice were observed, fenfluramine caused significant depletion of 5-HT in both WT and KO mice, and the depletion of 5-HT in the KO mice was even greater than in WT mice (Table 1). The extent to which there is correlation between fenfluramine-induced hypothermia and neurotoxicity is unclear, but, notably, fenfluramine caused greater hypothermia in nNOS KO than in WT mice (Fig. 1b). The variation in basal 5-HT levels in WT and KO mice may be due to developmental differences as a result of deletion of the nNOS gene. However, the density of basal 5-HTT binding sites was similar in WT and KO mice. Although fenfluramine caused greater depletion of hippocampal 5-HTT in WT (62%) than in KO (38%) mice, the depletion of frontocortical 5-HTT in WT and KO mice was similar (54 and 47%, respectively).

[6] 38w In summary, results of the present study suggest that NO/peroxynitrite, which are thought to mediate dopaminergic neurotoxicity, are not involved in fenfluramine-induced serotonergic neurotoxicity. These findings also point to neurotransmitter-specific interactions, e.g. NO/ DA as opposed to NO/5-HT.

[1] 147w Male Swiss Webster mice (8-9 weeks old; Charles River, Wilmington, MA, USA), male nNOS (-/-) mice and their wild-type B6129SF2/J counterparts (8-9 weeks old; Jackson Laboratories, Bar Harbor, ME, USA) were maintained on a 12-h light/dark cycle at a room temperature of 23°C, with free access to food and water. Animal care was in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, 1996) and approved by the University of Miami Animal Care and Use Committee. (±)Fenfluramine-HCl and 7-NI (Sigma, St Louis, MO, USA) were dissolved in saline and dimethyl sulfoxide/propylene glycol/water (1 : 3 : 6; vehicle), respectively. [ 3 H]Citalopram (81 Ci/mmole) and [ 3 H]mazindol (24 Ci/mmol) were purchased from New England Nuclear, Wilmington, DE, USA. Drugs and vehicle solutions were administered by intraperitoneal (i.p.) injections in a volume of 0.1 mL/10 g of weight.

[2] 150w In the first experiment, Swiss Webster mice (n ¼ 10-12 per group) received one of the following treatments twice a day for two consecutive days: (i) Vehicle/saline, (ii) vehicle/fenfluramine (25 mg/kg), (iii) 7-NI (25 mg/kg)/saline and (iv) 7-NI/fenfluramine. The interval between paired injections was 30 min, and each pair of injections was separated by 4-5 h. The dose of the nNOS inhibitor was based on our previous studies which showed that 25 mg/kg 7-NI inhibited about 80% of mouse brain NOS activity (Itzhak 1996) and attenuated METH-induced dopaminergic neurotoxicity in Swiss Webster mice (Itzhak and Ali 1996). In the second experiment, nNOS(-/-) and wild-type (WT) mice (n ¼ 10-12 per group) received saline or fenfluramine (25 mg/kg), twice a day for 2 days. Body temperature was measured for 2 h following each treatment by a small flexible rectal probe (1 mm diameter; VWR, USA) with an accuracy of ± 0.2°C.

[3] 162w Mice were killed 4 days following the last treatment, and the striatum, frontal cortex and hippocampus were freshly dissected and frozen at ) 80°C. Routinely, tissue from one hemisphere was used for monoamine assays and tissue from the other hemisphere was used for [ 3 H]mazindol binding to the DAT and [ 3 H]citalopram binding to the 5-HTT. High performance liquid chromatography (HPLC) combined with electrochemical detection (Itzhak et al. 1998) were used to determine the concentrations of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-HT. The densities of DAT and 5-HTT binding sites were determined by saturation binding assays using [ 3 H]mazindol (1.0-20.0 nM; Itzhak et al. 1998) and [ 3 H]citalopram (0.5-5.0 nM; Itzhak et al. 2003), respectively. Changes in rectal temperature were analyzed by a paired Student's t-test, and changes in the concentrations of neurotransmitters and transporters were analyzed by one-way ANOVA followed by post-hoc Newman-Keuls' test. A p-value of less than 0.05 was considered statistically significant.

[1] 116w Fenfluramine is an amphetamine analog with anorectic properties that inhibits the reuptake and increases the release of 5-hydroxytryptamine (5-HT; serotonin). High doses of fenfluramine cause long-lasting depletion of 5-HT and 5-HT transporter (5-HTT) binding sites (Kleven and Seiden 1989;O'Callaghan and Miller 1994), deficits that could be consistent with neurotoxic insult in rodents and non-human primates (Schuster et al. 1986;Ricaurte et al. 1991;McCann et al. 1994). The mechanism underlying the development of serotonergic neurotoxicity is unclear. Endogenous formation of 5,6-dihydroxytryptamine due to 5-HT excess release following the administration of substituted amphetamines (Commins et al. 1987), and the generation of other products of 5-HT oxidation (Wrona et al. 1995) may be involved in degeneration of 5-HT nerve terminals.

[2] 239w Other amphetamine analogs, such as methamphetamine (METH) and 3,4-methylenedioxy-methamphetamine (MDMA) cause varying degrees of dopaminergic and serotonergic neurotoxicity, depending on the species examined. Evidence suggests the involvement of free radicals and, specifically, reactive oxygen species in amphetamines-induced dopaminergic neurotoxicity (Cadet and Brannock 1998;Hanson et al. 1998). Peroxynitrite, formed from the interaction between nitric oxide (NO) and superoxide radicals, is considered a major neurotoxin (Beckman et al. 1990). Inactivation of tyrosine hydroxylase (Kuhn et al. 1999), the dopamine transporter (DAT;Park et al. 2002), and oxidation of dopamine (LaVoie and Hastings 1999) by peroxynitrite are thought to be involved in METH-induced dopaminergic neurotoxicity. Evidence for the role of NO in dopaminergic neurotoxicity stems from several in vivo studies. METH-induced dopaminergic neurotoxicity in mice (i) caused a marked increased striatal 3-nitortyrosine which is a marker of peroxynitrite formation in vivo (Imam et al. 2001), (ii) was attenuated by selective neuronal nitric oxide synthase (nNOS) inhibitors such as 7-nitroindazole (7-NI) (Di Monte et al. 1996;Itzhak and Ali 1996;Itzhak et al. 2000), and (iii) was attenuated in mice deficient in the nNOS gene (nNOS knockout; KO) (Itzhak et al. 1998). In addition, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity in mice was blocked by 7-NI (Schulz et al. 1995), and attenuated in nNOS KO mice (Przedborski et al. 1996). As there is compelling evidence suggesting the involvement of NO/peroxynitrite in amphetamines-induced dopaminergic neurotoxicity, the present study sought to investigate if NO is involved in fenfluramine-induced serotonergic neurotoxicity.