Priority Existing Chemical

j.9Neurotoxicity

The ability of MMT to induce neurotoxic effects was investigated in male CD-1 mice injected subcutaneously with MMT (10, 20 or 80 mg/kg bw in propylene glycol). The concentration of dopamine in the striatum of mice that received MMT at 20 and 80 mg/kg bw on alternate days for 3 weeks (total of 11 injections) was reduced by 10% and 23% respectively. The level of dopamine in the olfactory tubercle was also significantly reduced in mice receiving 80 mg/kg bw. MMT at 80 mg/kg bw increased 4-aminobutyric acid (GABA) levels in both the striatum and olfactory tubercle but not the cerebellum. MMT did not significantly alter the activity of choline acetyltransferase in the striatum, substantia nigra, hippocampus or cerebral cortex. There was no significant change in dopamine or GABA levels in mice that received a single 80 mg/kg bw MMT injection when examined 1 and 21 days post injection. Between 6 and 16 animals were used per treatment (Gianutsos and Murray 1982).

Yong et al. (1986) investigated the neurotoxic effects in female Wistar rats resulting from repeated subcutaneous injections of MMT in propylene glycol. Rats received either 24 injections over a 48-day period and were sacrificed 24 hours after the last injection or 75 injections over a 5-month period and sacrificed one month after the last injection. The first MMT injection was given at 5 mg/kg bw, the second at 10 mg/kg bw, the third 15 mg/kg bw, the fourth 25 mg/kg bw, and the remainder at 50 mg/kg bw. The concentration of Mn in the cerebellum of rats surviving to 24 hours after the last of 24 injections was approximately twice that of controls (4.59 verses 1.71 g/g dry weight). One month after the last of 75 MMT injections the level of Mn in rat cerebellum was only slightly increased above controls. Although 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the striatum were slightly depressed in rats that received 24 MMT injections, tyrosine hydroxylase activity and dopamine and homovanillic acid (HVA) levels were considered normal. All neurological markers examined in rats that received 75 injections were similar to controls. Histological analysis of the zona compacta of the substantia nigra indicated that the numbers of perikarya per unit area in rats that received 75 MMT injections were similar to controls. Between 8 and 13 rats were used per treatment.

The neurotoxicity of MMT was assessed in male CD-1 mice after a single IP injection of MMT (100-2000 mg/kg bw in propylene glycol or corn oil). Each treatment group contained between 4 and 6 animals. In animals that died, death was seizure-related and occurred within 2 hours of MMT administration. Seizure activity was observed within 0.5-1.5 min post MMT administration. MMT-induced seizure activity was accompanied by a 2.5-fold increase in brain Mn concentrations. The Mn brain content of control mice was 0.9 g/g. The brain Mn content of mice demonstrating seizure was 2.45 g/g when MMT was administered in propylene glycol and 3.25 g/g when MMT was administered in corn oil. Only a small increase in brain Mn concentrations was observed in MMT-treated mice that did not show seizure activity (1.14 g/g for propylene glycol and 1.63 g/g in corn oil). The synthesis and release of GABA was unaffected 30 min after animals were injected with MMT (25-50 mg/kg bw) and aminooxyacetic acid (AOAA) (20 mg/kg bw, ip) to inhibit GABA transaminase. MMT was found to inhibit the binding of t-butylbicycloorthobenzoate (TBOB), a ligand for the GABA-A receptor linked chloride channel, to mouse brain membranes (median inhibitory concentration (IC50) = 22.8 M) suggesting that seizure activity of MMT may be linked to inhibitory action at this site (Fishman et al., 1987).

The neurotoxicity of MMT was assessed in male ddY mice after chronic oral administration of MMT in food (0.5 g Mn/kg food) for 12 months. Between 4 and 6 animals were used per treatment. Daily food intake per mouse was reported as 3.6 ± 0.9 g for controls and 3.1 ± 0.7 g in the MMT-treated group. The dosage was therefore equivalent to approximately 51.7 mg Mn/kg bw/day (208.5 mg MMT/kg bw/day). MMT-treated mice showed significant weight loss compared to controls during the 12-month treatment period. Spontaneous motor activity was measured over a 30 minute period at intervals during the 12-month exposure period. The level of spontaneous motor activity was similar for the control and MMT-treated groups throughout the study, except on day 80, when the MMT-treated group showed significantly more motor activity compared to controls (Komura and Sakamoto 1992).

Although the MMT dose in this study are of similar magnitude to oral LD50 values for MMT in mice (Section 10.1), no deaths were reported in this study. This suggests a likely greater tolerance of a gradual MMT and Mn intake in food compared to a similar amount as a bolus dose.

In what appears to be a further study of the above animals, effects of MMT on various brain biogenic amines were reported by Komura and Sakamoto (1994). At the end of the exposure period the concentration of normetanephrine in the cerebellum of MMT-treated mice was significantly increased (46 ± 8 ng/g wet wt) when compared to the control group (6 ± 1 ng/g wet wt). This effect correlated with a significant increase in Mn levels in the cerebellum (0.29 g Mn/g ww versus 0.13 g Mn/g ww). Additional alterations were observed in brain biogenic amine levels but these effects did not correlate significantly with an increase in Mn. For example, the concentration of norepinephrine was significantly decreased and homovanillic acid was significantly increased in the corpus stratum; 3-methoxytyramine was significantly increased in the midbrain; 3,4-dihydroxyphenylacetic acid was significantly decreased in the cerebellum; 3,4-dihydroxyphenylacetic acid, homovanillic acid, and serotonin were significantly decreased in the medulla oblongata and metanephrine was significantly increased in the medulla oblongata. The concentration of Mn in the corpus striatum, hypothalamus, midbrain, cerebral cortex, hippocampus and medulla oblongata was similar in MMT-treated and control groups (Komura and Sakamoto, 1994).

Evidence of neurotoxicity has been reported in ICR mice treated with MMT. Mice were injected IP daily with 0.05 or 0.1 mg/g bw MMT in corn oil for 3 days. Between four and six animals were used per treatment. Each treatment group contained between 4 and 6 animals. Both doses of MMT were found to significantly decrease motor nerve conduction velocity and reduce ouabain-sensitive Na⁺K⁺-ATPase activity in the sciatic nerve in vivo. MMT (0.03-3 mM) did not affect the activity of sciatic nerve Na⁺, K⁺-ATPase in vitro. The reduced ATPase activity correlated with a 58% decrease in the amount of catalytic 1 subunit polypeptide. These effects were associated with significantly increased Mn concentrations in blood and sciatic nerve of MMT-treated mice. These results suggest that MMT-induced neuropathy is associated with reduced nerve Na⁺, K⁺-ATPase activity and motor nerve conduction velocity (Liu et al 2000).

The mechanisms of MMT neurotoxicity have also been studied in vitro. In a recent study of cultured dopamine-producing PC-12 cells and nondopaminergic striatal γ-aminobutyric acidergic M213-20 cells, MMT was shown to be acutely cytotoxic particularly to dopamine-producing cells and decreased intracellular dopamine levels. Generation of intracellular reactive oxygen species (ROS), an early effect in toxicant-induced apoptosis, was observed within 15 minutes of exposure. A hallmark of apoptosis, genomic DNA fragmentation, was induced in a concentration dependent fashion. Lastly, PC-12 cells overexpressing the apoptosis inhibitory molecule Bcl-2 were shown to be significantly refractory to MMT-induced ROS. These in vitro data indicate that oxidative stress plays an important role in mitochondrial-mediated apoptotic neuronal death after exposure to MMT (Kitazawa et al., 2002).