Figure 1.
Structures of 4,4’-DMAR (4-Methyl-5-(4-methylphenyl)-4,5-dihydrooxazol-2-amine), 4-MAR (4-Methyl-5-phenyl-4,5-dihydrooxazol-2-amine) and Aminorex (5-phenyl-4,5-dihydro-1,3-oxazol-2-amine) copied from the Cayman Chemical website (
https://www.caymanchem.com, accessed date 17 May 2021).
Figure 1.
Structures of 4,4’-DMAR (4-Methyl-5-(4-methylphenyl)-4,5-dihydrooxazol-2-amine), 4-MAR (4-Methyl-5-phenyl-4,5-dihydrooxazol-2-amine) and Aminorex (5-phenyl-4,5-dihydro-1,3-oxazol-2-amine) copied from the Cayman Chemical website (
https://www.caymanchem.com, accessed date 17 May 2021).
Figure 2.
The frontal cortex of the treated mice ((±)cis 30; (b) showed mild edema (i.e., vacuolization areas are indicated by yellow arrows) compared to controls (a).
Figure 2.
The frontal cortex of the treated mice ((±)cis 30; (b) showed mild edema (i.e., vacuolization areas are indicated by yellow arrows) compared to controls (a).
Figure 3.
(A) Quantification and comparison of 8-OHdG positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). Representative image (light microscopy, 40×) of 8-OHdG immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 30, a’) and controls (a). (B) Quantification and comparison of iNOS positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). Comparison of (±)cis 60 versus (±)trans 30 + (±)cis 60. Representative image (light microscopy, 40×) of iNOS immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 60, b’) and controls (b). (C) Quantification and comparison of NT (nitrotyrosine) positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). Representative image (light microscopy, 40x) of NT immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 30, c’) and controls (c). (D) Quantification and comparison of NOX2 positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). Representative image (light microscopy, 40×) of NOX2 immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 30, d’) and controls (d). * p < 0.05, ** p < 0.01 and *** p < 0.001 different from control; ## p < 0.001 different from (±)cis 60.
Figure 3.
(A) Quantification and comparison of 8-OHdG positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). Representative image (light microscopy, 40×) of 8-OHdG immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 30, a’) and controls (a). (B) Quantification and comparison of iNOS positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). Comparison of (±)cis 60 versus (±)trans 30 + (±)cis 60. Representative image (light microscopy, 40×) of iNOS immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 60, b’) and controls (b). (C) Quantification and comparison of NT (nitrotyrosine) positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). Representative image (light microscopy, 40x) of NT immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 30, c’) and controls (c). (D) Quantification and comparison of NOX2 positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). Representative image (light microscopy, 40×) of NOX2 immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 30, d’) and controls (d). * p < 0.05, ** p < 0.01 and *** p < 0.001 different from control; ## p < 0.001 different from (±)cis 60.
Figure 4.
(A) Quantification and comparison of Smac/DIABLO positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60).). Comparison of (±)cis 60 versus (±)trans 30 + (±)cis 60. Representative image (light microscopy, 40×) of Smac/DIABLO immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)trans 30+(±)cis 60, a’) and controls (a). (B) Quantification and comparison of NF-κB positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). Representative image (light microscopy, 40x) of NF-κB immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 10, b’) and controls (b). *** p < 0.001 different from control; ### p < 0.001 different from (±)cis 60.
Figure 4.
(A) Quantification and comparison of Smac/DIABLO positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60).). Comparison of (±)cis 60 versus (±)trans 30 + (±)cis 60. Representative image (light microscopy, 40×) of Smac/DIABLO immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)trans 30+(±)cis 60, a’) and controls (a). (B) Quantification and comparison of NF-κB positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). Representative image (light microscopy, 40x) of NF-κB immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 10, b’) and controls (b). *** p < 0.001 different from control; ### p < 0.001 different from (±)cis 60.
Figure 5.
(A) Quantification and comparison of HSP27 positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). ). Comparison of (±)cis 60 versus (±)trans 30 + (±)cis 60). Comparison of (±)cis 10 versus (±)trans 30 + (±)cis 10 [°]. Representative image (light microscopy, 40×) of HSP27 immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 60, a’) and controls (a). (B) Quantification and comparison of HSP70 positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). Comparison of (±)cis 10 versus (±)trans 30 + (±)cis 10 [°]. Representative image (light microscopy, 40x) of HSP70 immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 60, b’) and controls (b). (C) Quantification and comparison of HSP90 positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). Representative image (light microscopy, 40×) of HSP90 immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 30, c’) and controls (c). * p < 0.05, ** p < 0.01 and *** p < 0.001 different from control; ° p < 0.05 and °°° p< 0.001 different from (±)cis 30; ## p < 0.001 different from (±)cis 60.
Figure 5.
(A) Quantification and comparison of HSP27 positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). ). Comparison of (±)cis 60 versus (±)trans 30 + (±)cis 60). Comparison of (±)cis 10 versus (±)trans 30 + (±)cis 10 [°]. Representative image (light microscopy, 40×) of HSP27 immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 60, a’) and controls (a). (B) Quantification and comparison of HSP70 positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). Comparison of (±)cis 10 versus (±)trans 30 + (±)cis 10 [°]. Representative image (light microscopy, 40x) of HSP70 immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 60, b’) and controls (b). (C) Quantification and comparison of HSP90 positive areas in controls versus 4-4′-DMAR-mice ((±)cis 10, (±)cis 30, (±)cis 60, (±)trans 30, (±)trans 30 + (±)cis 10, (±)trans 30 + (±)cis 60). Representative image (light microscopy, 40×) of HSP90 immunostaining (brown areas) in the cortex of mice receiving 4-4′-DMAR ((±)cis 30, c’) and controls (c). * p < 0.05, ** p < 0.01 and *** p < 0.001 different from control; ° p < 0.05 and °°° p< 0.001 different from (±)cis 30; ## p < 0.001 different from (±)cis 60.
Figure 6.
Structures of cis-4,4’-DMAR and its principal markers: hydroxylated (M1, M3) and carboxylated (M2).
Figure 6.
Structures of cis-4,4’-DMAR and its principal markers: hydroxylated (M1, M3) and carboxylated (M2).
Figure 7.
(A) Percentage ratio of excretion of M1, M2 and M3, the principal metabolites of cis-4,4′-DMAR for the administration of a dose of 10 mg/kg of 4,4′-DMAR (cis) and the same dose of both isomers (cis + trans). The box plots report maximum and minimum value, median and average (+). (B) Excretion data of 4,4′-cis-DMAR and its principal metabolites normalised to the sum of excretion of 4,4′-DMAR and its metabolites. Data normalised and reported, respectively, for the administration of cis or cis + trans isomers at a dose of 10 mg/kg.
Figure 7.
(A) Percentage ratio of excretion of M1, M2 and M3, the principal metabolites of cis-4,4′-DMAR for the administration of a dose of 10 mg/kg of 4,4′-DMAR (cis) and the same dose of both isomers (cis + trans). The box plots report maximum and minimum value, median and average (+). (B) Excretion data of 4,4′-cis-DMAR and its principal metabolites normalised to the sum of excretion of 4,4′-DMAR and its metabolites. Data normalised and reported, respectively, for the administration of cis or cis + trans isomers at a dose of 10 mg/kg.
Table 1.
Effect of (±)cis-4,4′-DMAR (0.1–60 mg/kg i.p.) on neuro-behavioural changes (psychomotor agitation, aggressiveness, convulsions), physiological alterations (sweating, salivation, hyperthermia) and lethality in mice. The data here shown (see material and methods) refers to the mean ± SEM values relating to six animals for each treatment. The statistical analysis of the effects of the (±)cis-4,4′-DMAR in different concentrations were performed using a one-way ANOVA, followed by a Bonferroni test for multiple comparisons. A Student’s t-test was used to determine statistical significance (p < 0.05) between the two groups. a p < 0.05, versus cis 3 mg/kg; b p < 0.05, versus cis 10 mg/kg; c p < 0.05, versus vehicle; d p < 0.05, versus cis 30 mg/kg.
Table 1.
Effect of (±)cis-4,4′-DMAR (0.1–60 mg/kg i.p.) on neuro-behavioural changes (psychomotor agitation, aggressiveness, convulsions), physiological alterations (sweating, salivation, hyperthermia) and lethality in mice. The data here shown (see material and methods) refers to the mean ± SEM values relating to six animals for each treatment. The statistical analysis of the effects of the (±)cis-4,4′-DMAR in different concentrations were performed using a one-way ANOVA, followed by a Bonferroni test for multiple comparisons. A Student’s t-test was used to determine statistical significance (p < 0.05) between the two groups. a p < 0.05, versus cis 3 mg/kg; b p < 0.05, versus cis 10 mg/kg; c p < 0.05, versus vehicle; d p < 0.05, versus cis 30 mg/kg.
COMPOUND | | cis-4,4’-DMAR |
---|
Doses (mg/kg) | vehicle | 0.1 | 1 | 3 | 10 | 30 | 60 |
---|
Psychomotor agitation | Frequency % (n° of mice) | - | - | - | 100% (6) | 100% (6) | 100% (6) | 100% (6) |
Duration (min) | - | - | - | 54.2 ± 10.30 | 121.0 ± 12.35 a | 235.1 ± 15.25 ab | 280.1 ± 12.35 ab |
Latency (min) | - | - | - | 61.2 ± 6.30 | 28.2 ± 7.20 a | 3.2 ± 0.25 ab | 2.2 ± 0.15 ab |
Sweating | Frequency % (n° of mice) | - | - | - | - | - | 100% (6) | 100% (6) |
Salivation | Frequency % (n° of mice) | - | - | - | - | - | 100% (6) | 50% (3) |
Hyperthermia | Variation (Δ°C) | −0.28 ± 0.12 | −0.18 ± 0.14 | −0.22 ± 0.17 | −0.34 ± 0.22 | −0.41 ± 0.13 | 1.52 ± 0.11 c | 2.22 ± 0.12 c |
Latency (min) | - | - | - | - | - | 25 ± 0.25 | 7.12 ± 0.23 d |
Aggressiveness | Spontaneus | Frequency % (n° of mice) | - | - | - | - | - | - | nd |
Stimulated | Frequency % (n° of mice) | - | - | - | - | 100% (6) | 100% (6) | nd |
Score (n° of bites) | - | - | - | - | 6 ± 0.23 | 10 ± 0.23 b | nd |
Convulsion | Frequency % (n° of mice) | - | - | - | - | - | 50% (3) | 100% (6) |
Episodes (n°) | - | - | - | - | - | 3.5 ± 0.5 | 2.0 ± 0.41 d |
Latency of first episode (sec) | - | - | - | - | - | 10.5 ± 1.5 | 8.25 ± 1.1 |
Duration of each episode (sec) | - | - | - | - | - | 4.86 ± 1.26 | 16.14 ± 3.2 d |
Lethality | Frequency % (n° of mice) | - | - | - | - | - | 50% (3) | 100% (6) |
Time of death (min) | - | - | - | - | - | 59.3 ± 2.3 | 31.9 ± 5.6 d |
Surviving mice % (n° of mice) | - | - | - | - | - | 50% (3) | 0% (0) |
Table 2.
Effect of (±)trans-4,4′-DMAR (30 and 60 mg/kg i.p.) on neuro-behavioural changes (psychomotor agitation, aggressiveness, convulsion), physiological alterations (sweating, salivation, hyperthermia) and lethality in mice. Data expressed (see material and methods) represents the mean ± SEM of six animals for each treatment. The statistical analysis of the effects of the (±)trans-4,4′-DMAR in different concentrations were performed using a one-way ANOVA followed by a Bonferroni test for multiple comparisons. A Student’s t-test was used to determine statistical significance (p < 0.05) between the two groups.
Table 2.
Effect of (±)trans-4,4′-DMAR (30 and 60 mg/kg i.p.) on neuro-behavioural changes (psychomotor agitation, aggressiveness, convulsion), physiological alterations (sweating, salivation, hyperthermia) and lethality in mice. Data expressed (see material and methods) represents the mean ± SEM of six animals for each treatment. The statistical analysis of the effects of the (±)trans-4,4′-DMAR in different concentrations were performed using a one-way ANOVA followed by a Bonferroni test for multiple comparisons. A Student’s t-test was used to determine statistical significance (p < 0.05) between the two groups.
COMPOUND | | trans-4,4’-DMAR |
---|
Doses (mg/kg) | vehicle | 30 | 60 |
---|
Psychomotor agitation | Frequency % (n° of mice) | - | - | - |
Duration (min) | - | - | - |
Latency (min) | - | - | - |
Sweating | Frequency % (n° of mice) | - | - | - |
Salivation | Frequency % (n° of mice) | - | - | - |
Hyperthermia | Variation (Δ°C) | −0.28 ± 0.12 | −0.37 ± 0.13 | −0.35 ± 0.16 |
Latency (min) | - | - | - |
Aggressiveness | Spontaneus | Frequency % (n° of mice) | - | - | - |
Stimulated | Frequency % (n° of mice) | - | - | - |
Score (n° of bites) | - | - | - |
Convulsion | Frequency % (n° of mice) | - | - | - |
Episodes (n°) | - | - | - |
Latency of first episode (sec) | - | - | - |
Duration of each episode (sec) | - | - | - |
Lethality | Frequency % (n° of mice) | - | - | - |
Time of death (min) | - | - | - |
Table 3.
Effect of co-administration of (±)cis-4,4′-DMAR (1, 10 and 60 mg/kg i.p.) and (±)trans-4,4′-DMAR (30 mg/kg i.p.) on neuro-behavioural changes (psychomotor agitation, aggressiveness, convulsion), physiological alterations (sweating, salivation, hyperthermia) and death in mice. Data expressed (see material and methods) represents the mean ± SEM of six animals for each treatment. The statistical analysis of the effects of the interactions between (±)cis-4,4′-DMAR and (±)trans-4,4′-DMAR were performed using a one-way ANOVA followed by a Bonferroni test for multiple comparisons. A Student’s t-test was used to determine statistical significance (p < 0.05) between the two groups. e p < 0.05, versus cis 10 mg/kg; f p < 0.05, versus cis 60 mg/kg.
Table 3.
Effect of co-administration of (±)cis-4,4′-DMAR (1, 10 and 60 mg/kg i.p.) and (±)trans-4,4′-DMAR (30 mg/kg i.p.) on neuro-behavioural changes (psychomotor agitation, aggressiveness, convulsion), physiological alterations (sweating, salivation, hyperthermia) and death in mice. Data expressed (see material and methods) represents the mean ± SEM of six animals for each treatment. The statistical analysis of the effects of the interactions between (±)cis-4,4′-DMAR and (±)trans-4,4′-DMAR were performed using a one-way ANOVA followed by a Bonferroni test for multiple comparisons. A Student’s t-test was used to determine statistical significance (p < 0.05) between the two groups. e p < 0.05, versus cis 10 mg/kg; f p < 0.05, versus cis 60 mg/kg.
COMPOUND | | cis-4,4’-DMAR | trans | (cis + trans)-4-4’DMAR |
---|
Doses (mg/kg) | vehicle | 1 | 10 | 60 | 30 | 1 + 30 | 10 + 30 | 60 + 30 |
---|
Psychomotor agitation | Frequency % (n° of mice) | - | - | 100% (6) | 100% (6) | - | 50% (3) | 100% (6) | 100% (6) |
Duration (min) | - | - | 121.0 ± 12.35 | 220.1 ± 12.35 | - | 55.0 ± 5.0 | 185.5 ± 11.0 e | nd |
Latency (min) | - | - | 28.2 ± 7.20 | 2.2 ± 0.15 | - | 45.2 ± 5.20 | 6.2 ± 2.20 e | 0.45 ± 0.10 f |
Sweating | Frequency % (n° of mice) | - | - | - | 100% (6) | - | - | 33% (2) | 100% (6) |
Salivation | Frequency % (n° of mice) | - | - | - | 50% (3) | - | - | 33% (2) | 50% (3) |
Hyperthermia | Variation (Δ°C) | −0.28 ± 0.12 | −0.22 ± 0.17 | −0.41 ± 0.13 | 2.22 ± 0.12 | −0.37 ± 0.13 | −0.70 ± 0.15 | 1.6 ± 0.11 e | 2.25 ± 0.09 |
Latency (min) | - | - | - | 7.12 ± 0.23 | - | - | 28.0 ± 0.21 | 5.0 ± 0.23 f |
Aggressiveness | Spontaneus | Frequency % (n° of mice) | - | - | - | nd | - | - | - | nd |
Stimulated | Frequency % (n° of mice) | - | - | 100% (6) | nd | - | - | 100% (6) | nd |
Score (n° of bites) | - | - | 6 ± 0.23 | nd | - | - | 10 ± 0.05 e | nd |
Convulsion | Frequency % (n° of mice) | - | - | - | 100% (6) | - | - | - | 100% (6) |
Episodes (n°) | - | - | - | 2.0 ± 0.41 | - | - | - | 1.0 ± 0.0 f |
Latency of first episode (sec) | - | - | - | 8.25 ± 1.1 | - | - | - | 6.67 ± 2.73 |
Duration of each episode (sec) | - | - | - | 16.14 ± 6.2 | - | - | - | 40.0 ± 5.0 f |
Lethality | Frequency % (n° of mice) | - | - | - | 100% (6) | - | - | - | 100% (6) |
Time of death (min) | - | - | - | 31.9 ± 5.6 | - | - | - | 11.0 ± 3.9 f |
Surviving mice % (n° of mice) | - | - | - | 0% (0) | - | - | - | 0% (0) |
Table 4.
Effect of co-administration of (±)cis-4,4′-DMAR (10 mg/kg i.p.) and (±)trans-4,4′-DMAR (10 mg/kg i.p.) on neuro-behavioural changes (psychomotor agitation, aggressiveness, convulsion), physiological alterations (sweating, salivation, hyperthermia) and death in mice. Data expressed (see material and methods) represents the mean ± SEM of four animals for each treatment. The statistical analysis of the effects of the interactions between (±)cis-4,4′-DMAR and (±)trans-4,4′-DMAR were performed using a one-way ANOVA followed by a Bonferroni test for multiple comparisons. A Student’s t-test was used to determine statistical significance (p < 0.05) between the two groups. e p < 0.05, versus cis 10 mg/kg.
Table 4.
Effect of co-administration of (±)cis-4,4′-DMAR (10 mg/kg i.p.) and (±)trans-4,4′-DMAR (10 mg/kg i.p.) on neuro-behavioural changes (psychomotor agitation, aggressiveness, convulsion), physiological alterations (sweating, salivation, hyperthermia) and death in mice. Data expressed (see material and methods) represents the mean ± SEM of four animals for each treatment. The statistical analysis of the effects of the interactions between (±)cis-4,4′-DMAR and (±)trans-4,4′-DMAR were performed using a one-way ANOVA followed by a Bonferroni test for multiple comparisons. A Student’s t-test was used to determine statistical significance (p < 0.05) between the two groups. e p < 0.05, versus cis 10 mg/kg.
COMPOUND | cis-4,4’-DMAR | trans-4,4’-DMAR | (cis + trans)-4-4’DMAR |
---|
Doses (mg/kg) | 10 | 10 | 10+10 |
---|
Psychomotoragitation | Frequency % (n° of mice) | 100% (4) | - | 100% (4) |
Duration (min) | 125.0 ± 10.12 | - | 171.0 ± 9.25 e |
Latency (min) | 26.4 ± 6.25 | - | 8.0 ± 4.25 e |
Sweating | Frequency % (n° of mice) | - | - | - |
Salivation | Frequency % (n° of mice) | - | - | - |
Hyperthermia | Variation (Δ°C) | −0.21 ± 0.15 | −0.32 ± 0.1 | 1.58 ± 0.23 e |
Latency (min) | - | - | 32.0 ± 2.45 |
Aggressiveness | Spontaneus | Frequency % (n° of mice) | - | - | - |
Stimulated | Frequency % (n° of mice) | 100% (4) | - | 100% (4) |
Score (n° of bites) | 5 ± 0.23 | - | 8 ± 0.13 e |
Convulsion | Frequency % (n° of mice) | - | - | - |
Episodes (n°) | - | - | - |
Latency of first episode (sec) | - | - | - |
Duration of each episode (sec) | - | - | - |
Lethality | Frequency % (n° of mice) | - | - | - |
Time of death (min) | - | - | - |
Table 5.
Correlation between mouse doses (mg/kg) and human equivalent doses (HED, mg/kg). The table also reported the correlation between doses and effects in human.
Table 5.
Correlation between mouse doses (mg/kg) and human equivalent doses (HED, mg/kg). The table also reported the correlation between doses and effects in human.
Mouse Dose (mg/kg) | HED | Human Dose | Human Dosage | Effects |
---|
(mg/kg) | (mg) |
---|
0.1 | 0.0081 | 0.486 | Low | high state of vigilance, euphoria, decreased appetite, increased frequency of heartbeat and motor activity |
1 | 0.081 | 4.86 |
3 | 0.243 | 14.58 |
10 | 0.81 | 48.6 | Intermediate | restlessness, agitation and insomnia |
30 | 2.43 | 145.8 | high | involve severe anorexia, mild paranoia (sometimes hallucinations), hyperthermia, bruxism, facial spasms, an increase in aggression and desire for violence, seizures, an increased heart rate that will be involved in a cardiac arrest |
60 | 4.86 | 291.6 |
Table 6.
Antibodies used for immunohistochemical analyses with the relative dilutions and antigenic retrieval methods.
Table 6.
Antibodies used for immunohistochemical analyses with the relative dilutions and antigenic retrieval methods.
Marker | | Dilution | Retrieval |
---|
HSP27 | Santa Cruz Biotechnology, Inc. | 1:50 | HIER (0.25 mM EDTA buffer ) |
HSP70 | Santa Cruz Biotechnology, Inc. | 1:50 | HIER (0.25 mM EDTA buffer ) |
HSP90 | Santa Cruz Biotechnology, Inc. | 1:50 | HIER (0.25 mM EDTA buffer ) |
SMAC | Santa Cruz Biotechnology, Inc. | 1:100 | HIER (0.01 M citrate buffer) |
NF-kB | Santa Cruz Biotechnology, Inc. | 1:50 | HIER (0.25 mM EDTA buffer ) |
iNOS | Santa Cruz Biotechnology, Inc. | 1:100 | HIER (0.01 M citrate buffer) |
NOX-2 | Proteintech | 1:100 | HIER (0.01 M citrate buffer) |
NT | Santa Cruz Biotechnology, Inc. | 1:600 | HIER (0.01 M citrate buffer) |
8OHDG | Santa Cruz Biotechnology, Inc. | 1:500 | HIER (0.01 M citrate buffer) |