Garcia RCT et al. (2019), Anhydroecgonine methyl ester (AEME), a cocaine ...

ECB-ART-48781

Toxicol Rep 2019 Nov 09;6:1223-1229. doi: 10.1016/j.toxrep.2019.11.001.

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Anhydroecgonine methyl ester (AEME), a cocaine pyrolysis product, impairs glutathione-related enzymes response and increases lipid peroxidation in the hippocampal cell culture.

Garcia RCT , Torres LL , Dati LMM , Loureiro APM , Afeche SC , Sandoval MRL , Marcourakis T .

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Crack cocaine smokers inhale, alongside with cocaine, its pyrolysis product, anhydroecgonine methyl ester (AEME). We have previously described AEME neurotoxic effect and its additive effect when co-incubated with cocaine. Our aim was to evaluate, the effect of AEME, cocaine and AEME-cocaine combination on glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) activities after 3 and 6 h of exposure, periods previous to neuronal death. Lipid peroxidation was evaluated through malonaldehyde (MDA) levels at 3, 6, 24 and 48 h of exposure. All treated groups reduced neuronal viability after 24 h of exposure. AEME and cocaine decreased GPx, GR and GST activities after 3 and 6 h, with an increase in MDA levels after 48 h. AEME-cocaine combination decreased the enzymes activities after 3 and 6 h, showing an additive effect in MDA levels after 48 h. These data show that the glutathione-related enzymes imbalance caused by AEME, cocaine or AEME-cocaine combination exposure preceded neuronal death and lipid peroxidation. Moreover, the additive effect on lipid peroxidation observed with AEME-cocaine exposure after 48 h, suggest a higher neurotoxic effect after crack cocaine use when compared to cocaine alone.

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Genes referenced: gsr LOC115919910 LOC594349 LOC752853

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	Fig. 1. Neuronal cell viability results (n = 3), expressed in percentage of control group, after 3, 6, 24 and 48 h of exposure to 1 mM AEME (☐), 2 mM COC (▲) and AEME + COC (○). The positive control of neuronal death was 250 mM KCl (•). Dotted line represents the control group (error bar was omitted). AEME, anhydroecgonine methyl ester; COC, cocaine; and AEME + COC, AEME-cocaine combination. p < 0.05, p < 0.001 and p < 0.0001 for all groups when compared with control group. After 48 h of exposure, p < 0.01 for the AEME+COC when compared with either 1 mM AEME or 2 mM COC (ANOVA and Newman-Keuls multiple comparisons test). Results are presented as mean ± SEM.
	Fig. 2. Glutathione peroxidase (GPx) activity, expressed in percentage of control group, after 3 (A) and 6 h (B) of exposure to 1 mM AEME, 2 mM COC and AEME + COC (n = 3). CTRL, control; AEME, anhydroecgonine methyl ester; COC, cocaine; and AEME + COC, AEME-cocaine combination. p < 0.05 and p < 0.01 for all groups when compared with control group. After 6 h of exposure (B), p < 0.05 for the AEME+COC when compared with 2 mM COC (ANOVA and Newman-Keuls multiple comparisons test). Results are presented as mean ± SEM.
	Fig. 3. Glutathione reductase (GR) activity, expressed in percentage of control group, after 3 (A) and 6 h (B) of exposure to 1 mM AEME, 2 mM COC and AEME + COC (n = 3). CTRL, control; AEME, anhydroecgonine methyl ester; COC, cocaine; and AEME + COC, AEME-cocaine combination. p < 0.05, p < 0.01 and *p < 0.001 for all groups when compared with control group. After 6 h of exposure (B), p < 0.05 for the 1 mM AEME when compared with either 2 mM COC or AEME+COC (ANOVA and Newman-Keuls multiple comparisons test). Results are presented as mean ± SEM.
	Fig. 4. Glutathione S-transferase (GST) activity, expressed in percentage of control group, after 3 (A) and 6 h (B) of exposure to 1 mM AEME, 2 mM COC and AEME + COC (n = 3). CTRL, control; AEME, anhydroecgonine methyl ester; COC, cocaine; and AEME + COC, AEME-cocaine combination. p < 0.05 and *p < 0.01 for all groups when compared with control group (ANOVA and Newman-Keuls multiple comparisons test). Results are presented as mean ± SEM.
	Fig. 5. Malondialdehyde (MDA) levels, expressed in percentage of control group, after 3 (A), 6 (B), 24 (C) and 48 h (D) of exposure to 1 mM AEME, 2 mM COC and AEME + COC (n = 3). CTRL, control; AEME, anhydroecgonine methyl ester; COC, cocaine; and AEME + COC, AEME-cocaine combination. p < 0.01 and p < 0.0001 for all groups when compared with control group. After 48 h of exposure (C), *p < 0.001 for the AEME+COC when compared with either 1 mM AEME or 2 mM COC (ANOVA and Newman-Keuls multiple comparisons test). Results are presented as mean ± SEM.

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	Fig. 1. Neuronal cell viability results (n = 3), expressed in percentage of control group, after 3, 6, 24 and 48 h of exposure to 1 mM AEME (☐), 2 mM COC (▲) and AEME + COC (○). The positive control of neuronal death was 250 mM KCl (•). Dotted line represents the control group (error bar was omitted). AEME, anhydroecgonine methyl ester; COC, cocaine; and AEME + COC, AEME-cocaine combination. p < 0.05, p < 0.001 and p < 0.0001 for all groups when compared with control group. After 48 h of exposure, p < 0.01 for the AEME+COC when compared with either 1 mM AEME or 2 mM COC (ANOVA and Newman-Keuls multiple comparisons test). Results are presented as mean ± SEM.
	Fig. 2. Glutathione peroxidase (GPx) activity, expressed in percentage of control group, after 3 (A) and 6 h (B) of exposure to 1 mM AEME, 2 mM COC and AEME + COC (n = 3). CTRL, control; AEME, anhydroecgonine methyl ester; COC, cocaine; and AEME + COC, AEME-cocaine combination. p < 0.05 and p < 0.01 for all groups when compared with control group. After 6 h of exposure (B), p < 0.05 for the AEME+COC when compared with 2 mM COC (ANOVA and Newman-Keuls multiple comparisons test). Results are presented as mean ± SEM.
	Fig. 3. Glutathione reductase (GR) activity, expressed in percentage of control group, after 3 (A) and 6 h (B) of exposure to 1 mM AEME, 2 mM COC and AEME + COC (n = 3). CTRL, control; AEME, anhydroecgonine methyl ester; COC, cocaine; and AEME + COC, AEME-cocaine combination. p < 0.05, p < 0.01 and *p < 0.001 for all groups when compared with control group. After 6 h of exposure (B), p < 0.05 for the 1 mM AEME when compared with either 2 mM COC or AEME+COC (ANOVA and Newman-Keuls multiple comparisons test). Results are presented as mean ± SEM.
	Fig. 4. Glutathione S-transferase (GST) activity, expressed in percentage of control group, after 3 (A) and 6 h (B) of exposure to 1 mM AEME, 2 mM COC and AEME + COC (n = 3). CTRL, control; AEME, anhydroecgonine methyl ester; COC, cocaine; and AEME + COC, AEME-cocaine combination. p < 0.05 and *p < 0.01 for all groups when compared with control group (ANOVA and Newman-Keuls multiple comparisons test). Results are presented as mean ± SEM.
	Fig. 5. Malondialdehyde (MDA) levels, expressed in percentage of control group, after 3 (A), 6 (B), 24 (C) and 48 h (D) of exposure to 1 mM AEME, 2 mM COC and AEME + COC (n = 3). CTRL, control; AEME, anhydroecgonine methyl ester; COC, cocaine; and AEME + COC, AEME-cocaine combination. p < 0.01 and p < 0.0001 for all groups when compared with control group. After 48 h of exposure (C), *p < 0.001 for the AEME+COC when compared with either 1 mM AEME or 2 mM COC (ANOVA and Newman-Keuls multiple comparisons test). Results are presented as mean ± SEM.