4(5)-methylimidazole (4(5)-MEI), used as an additive in the food industry. A neurotoxic effect is reported in experimental animals, with alterations in the cellular structure of the central nervous system. Evidence to demonstrate alterations in synaptic connectivity is lacking. The objective was to determine the neurotoxic effect of 4(5)-MEI, at the structural level of the synaptic connectivity of the rat dorsal hippocampus. Eighteen Sprague Dawley rats divided into 3 groups: control and 2 experimental with oral intake of 4(5)-MEI (625 and 1250 ppm), for 24 weeks. Morris water maze test was performed. Animals were sacrificed, serum malondialdehyde (MDA) levels were determined, and the dorsal hippocampus was removed for quantification of dendritic spines. The MDA determination did not show significant differences between the groups. The groups treated with 4(5)-MEI, showed a deterioration in spatial memory with respect to the control group and a decrease in the density of dendritic spines of the dorsal hippocampus. Chronic consumption of 4(5)-MEI does not increase serum MDA levels. However, exerts a neurotoxic effect by decreasing the performance of the rat on a spatial memory task, this can be correlated with the decrease in the density of dendritic spines.

4(5)-Methylimidazole (4(5)-MEI), is a nitrogen-containing heterocyclic compound, belongs to class III and IV of ammonia caramel dyes, and is widely used as an additive in the food industry, unfortunately Carcinogenic and neurotoxic properties have been attributed to it in laboratory animals, so efforts are being made to limit its use in food [1]. Because of this, various studies have focused on identifying the presence of 4(5)-MEI in food and beverages, using analytical techniques such as high-performance liquid chromatography coupled to mass spectrometry, to quantify small amounts of this compound [2]. On the other hand, toxicological studies continue to be carried out to determine the deleterious effects that 4(5)-Methylimidazole, when consumed in high concentrations and for long periods of time, since in 2007 The United States Institute of Public Health through d The National Toxicology Program carried out a long-term toxicity study, evaluating the effects of 4(5)-MEI on female and male rodents exposed for 2 years at concentrations of 625, 1250 and 2500 ppm and 312, 625 and 1250 ppm. Respectively. At the end of the study, the development of lung cancer in mice and leukemia in rats was observed with the highest concentration [3, 4].

In another study, it was reported that the administration of 4(5)-MEI at concentrations of 300, 800 and 2500 ppm in B6C3F1 mice and F344/N rats for 15 days, did not induce histopathological changes in these rodents. Instead, the administration for 14 weeks of the same compound in these laboratory specimens at concentrations of 625, 1,250, 2,500, 5,000 and 10,000 ppm; they did not modify the serum levels of T(3), T(4) and TSH and did not produce thyroid lesions. Tremors and ataxia were observed only in the high-dose groups.5 Anemia, hepatocytic vacuolation, testicular degeneration, and prostatic atrophy were also observed in both mice and rats.6 However, the study of the neurotoxic effect has not been investigated so far of 4(5)-MEI, most toxicological studies have focused on the carcinogenic effect [6, 7]. Furthermore, there is evidence that at high doses and long exposure times 4(5)-MEI affects the central nervous system,[8] so it is of particular interest to assess whether chronic exposure to oral administration of 4(5)-MEI in water ad libitum is capable of modifying the function of cognitive activities of learning and memory, and the spinal plasticity of the hippocampus to through the production of oxidative stress in this anatomical structure, which can thus affect the rat's spatial memory.

Animals and experimental groups

Eighteen rats of the Sprague-Dawley strain, weighing 200 ± 5 g at the start of the experiment, divided into three groups with n = 6 each were used. All the rats were placed in 40x60x30cm transparent acrylic boxes, in a room that had a controlled temperature of 22 to 25 ° C, and a light-dark cycle of 12:12 hours, beginning the light period at 7:00. Hours . They were divided into 3 groups: 1) Control Group (CG), with food and water ad libitum; 2) Group that was administered 4 (5) -methylimidazole orally (v.o.) dissolved in water ad libitum at 625ppm, (G625ppm); and 3) Group that was administered 4(5)-methylimidazole by v.o. dissolved in water ad libitum at 1250ppm, (G1250pmm), for 24 continuous weeks[5].

Morris Aquatic Maze Test (MAM)

The MAM used is a black circular plastic tank with a diameter of 150 cm and a height of 60 cm placed on a metal base that supports it at a height of 58 cm. It was filled with water to a height of 30 cm maintaining a temperature of 25 ° C ± 1 ° C. The four starting positions were marked on the external face of the tank with the four cardinal axes North (N), South (S), East (E), West(W), thus dividing into 4 quadrants. A black plastic platform with dimensions of 10 x 10 cm, submerged at 1 cm, was placed under the surface of the water.

The hidden platform aquatic maze paradigm was used, the training and test schedules ranged between 10:00 and 13:00. The training procedure was as follows: Animals were tested in one session of 8 trials. In each, the animal was placed inside the tank facing the wall at one of the randomly designated starting points (North, South, East,West); the animal could escape the aversive stimulus of the water by finding and climbing on the escape platform. Different starting points were used in each trial, and each of these points was used 2 times within the trials. The escape platform was in the lower left quadrant in all trials and the first exit point was west of the tank; in the second the exit point was placed to the North of the tank, the third point was placed to the South and the fourth point to the East; at the fifth point she moved back to the north, the sixth exit point to the south, the seventh point to the east and the eighth exit point to the west.

If any animal did not enter the escape platform, during the 60 second test time, it was manually guided to the escape platform, only during the first test. After climbing on the platform, the rats remained there for 20 seconds and were then removed from the maze, dried with a cotton towel and placed in a resting box for an interval of 30 seconds, to then start the next test. The latency of arrival at the escape platform, measured in seconds (s), was used to determine the acquisition.

In the analysis of the data obtained by the repeated measures ANOVA test during the three learning sessions with four trials per day, no statistically significant differences were observed between the control group and the experimental groups (F(2,18) = 0.414, p = 0.6672), on the other hand regarding the training day if statistically significant differences were observed (F(2,15) = 7.989, p = 0.0002), as well as in the interaction between the variables (F(2,6) = 9.521, p <0>

Retention was assessed 24 hours after the end of training. The retention consisted of a 2 min session where the escape platform was removed from the tank. The rat was positioned facing the labyrinth wall at the west exit point; and she was allowed to swim freely during this time;At the end of this, the rat withdrew from the maze, to dry it and place it in a resting box for 30 seconds, after which it was returned to its room box to finish the task. For its evaluation, the latency in seconds and the number of entrances to the place where the platform was located were considered.

Obtaining the brain and blood plasma

To obtain the brain and blood plasma, the animals were decapitated after anesthesia with xylazine / ketamine (40 mg/kg). At the time of decapitation, the blood was collected in test tubes of 1 x 10 cm, they were placed inside a clinical centrifuge at a speed of 3000 rpm for 30 min separating the serum into eppendorf tubes. On the other hand, the head was dissected to the cranial cavity and the brain was separated from it and immersed in a mixture with mercury chloride, potassium dichromate and distilled water for its preparation by means of Golgi Cox staining.

Once the brains were obtained by decapitation, they were immersed in 50 milliliters of a mixture with mercury chloride, potassium dichromate and distilled water, for a period of 30 days. Subsequently, they were rinsed and dehydrated in alcohol graduated from 50 to 95% for 30 min in each change. They were transferred to a mixture of alcohol/acetone 50ml/50ml, for 24 hours. They were included in a low nitrocellulose solution at consecutive concentrations of 5, 10, 15 and 30%. They were placed in molds with 30% nitrocellulose in chloroform vapors for subsequent cutting in a sliding microtome at 120 micrometers thick, collecting the sections in a container with 70% alcohol. They were hydrated in distilled water and differentiated in a photographic developer solution for 2 min. They were washed with distilled water. They were dehydrated in alcohol graduated from 70 to 95% and placed in isopropyl alcohol for 10 min, for subsequent mounting in resin and observation under a microscope.

Lipid peroxidation technique (LP)

To determine the plasma PL, the measurement of malondialdehyde levels (MDA) was used by the fluorometric method, using the lipid peroxidation test kit (colorimetric/fluorometric), (abcam 118970), according to the manufacturer's specifications.

Ethical and legal considerations

All the animals were housed, handled, and slaughtered based on the provisions of the Official Mexican Standard NOM-062-ZOO-1999, Technical Specifications for the production, care and use of laboratory animals [9].This project was authorized by the research committee of the University of Guanajuato Campus Celaya-Salvaterra and registered with No. CIDSC2700923.

The IBM SPSS Statistics version 23 program was used. For the statistical analysis of weight gain and latency time during acquisition, the repeated measures ANOVA test was applied. For the analysis of retention latency time, entrances to the platform site and quantification of dendritic spines, the one-way ANOVA test was applied. Followed by Tukey's post hoc test. The level of significance was established with p<0.05.

In the analysis of the weight gain of the animals using the repeated measures ANOVA test, statistically significant differences were observed in the category by group (F(2.90) = 7.566, p = 0.0053), in the category by weight it was observed a statistically significant difference (F(2, 6) = 114.052, p <0 xss=removed xss=removed>

Figure1

Acquisition Test

In the analysis of the data obtained by the repeated measures ANOVA test during the three learning sessions with four trials per day, no statistically significant differences were observed between the control group and the experimental groups (F(2,18) = 0.414, p = 0.6672), on the other hand regarding the training day if statistically significant differences were observed (F(2,15) = 7.989, p = 0.0002), as well as in the interaction between the variables (F(2,6) = 9.521, p <0>

Figure:3

The analysis of the levels obtained from MDA, by means of the fluorometric test by means of the ELISA technique, was carried out by applying the one-way ANOVA test. The data obtained from plasma MDA levels do not show statistically significant differences between the control group and the experimental groups (F(2.15) = 0.165, p = 0.8493). (Figure 2).

Figure:2

The analysis of the results obtained by the one-way ANOVA, regarding the arrival latency during the memory retention test, statistically significant differences were observed between the control group and the experimental groups (F(2.15) = 13.947, p = 0.0004), Tukey's post hoc test determined significant differences between the control group and the group that received 625 ppm of 4(5)-MEI (p = 0.0003) and between the control group and the group that received 1250 ppm of 4(5)-MEI (p = 0.0006), not so, between the groups that received 625 ppm and 1250 ppm of 4(5)-MEI (p = 7007). (Figure 4).

Figure:4

Regarding the number of entries to the place where the platform was located in the memory retention test, the analysis of the data using the one-way ANOVA showed statistically significant differences (F(2.15) = 6,743, p = 0.0081) , between the CG and the experimental groups G625ppm and G1250ppm, not so between both experimental groups G625ppm and G1250ppm (p = 0.6075) (Figure 5).

Figure:5

The one-way ANOVA test on the density of dendritic spines shows statistically significant differences between the CG and the experimental groups (F(2.78) = 5.526, p = 0.0057). This shows that the density of spines along the apical dendrite is modified by the consumption of 4(5)-MEI. Tukey's post hoc test reveals that the GC maintains a higher density of dendritic spines with respect to G625 (p = 0.007), and G1250 (p = 0.0036). However, no differences are shown when comparing both experimental groups. (Figure 6).

Figure:6

4(5)-Methylimidazole (4(5)-MEI), is a neurotoxic agent, which exerts a convulsive effect at high doses and changes in the cellular structure (necrosis) of various regions of the central nervous system.8 Few are the studies sponsored in the different databases that show a direct relationship between the toxic effect on the effective brain region and its function. This study is a first attempt to deepen this relationship. The data obtained from the plasma MDA levels of the groups exposed to both experimental treatments showed no differences with respect to the plasma MDA levels of the control animals, the treatment time was probably not sufficient to alter the activity of the antioxidants. endogenous. This is probably due to the fact that the lipid unsaturation that promotes the autoxidation rate increases the complexity of the product distribution; and it can change depending on the experimental conditions (or different in vivo environments) [10]. The results obtained by subjecting the animals to a test that values ​​learning and spatial memory showed that under this scheme of chronic exposure with 625 and 1250 ppm of 4(5)-MEI during 24 weeks, the control group and the experimental ones behaved similarly in the performance of the training stage (learning). However, when performing the 24 hour retention test, after the training sessions, both experimental groups showed a low performance of the task, increasing the arrival latency time and decreasing the number of entries to the quadrant where the platform, with respect to the control group. However, the behavior of both experimental groups was similar when compared to each other. This behavior exhibited by both experimental groups could represent that despite the fact that the animals exposed for 24 weeks with the 4(5)-MEI, had an acquisition similar to the controls, this could not be remembered, so the test where memory recall was required was overcome only by the control group.

In the same way, we have not found studies that directly correlate the exposure by 4(5)-MEI, with alterations of cellular metabolism at the cerebral level, proposing in this case that these results are the first report in the field of neurodegeneration. Our results, therefore, support a possible relationship between the ability of 4(5)-MEI to produce oxidative stress, through the formation of lipid oxidation products, that is, the formation of MDA adducts, mainly at the hippocampus level. the rat. This product is a mutagenic α, β-unsaturated aldehyde that is commonly used as a biomarker for LPO through testing of the substance that reacts with thiobarbituric acid (TBARS) [11]. Probably the oxidative state as a result of exposure to 4(5) -MEI, explain how every neurodegeneration process, where in a balanced redox state, neurons and their supporting cells coexist in a healthy environment [12]. After induction of nitrative oxidative stress in an unbalanced redox state, a Neuroinflammatory condition due to microglial activation that undergoes morphological changes as factories producing EROS / RNS through positive regulation and release of cytokines such as interleukin-1, interleukin-6, tumor necrosis factor α, interferon-γ; increased inducible nitric oxide synthase (iNOS), which increases NO levels; increased production of EROS; and the stimulation of the formation of dopamine quinone in which dopamine-quinones regulate the expression of the neuroprotective gene [13]. In addition to activating the chain function of the microglia, keeping it activated and generating changes in neighboring neurons, which can eventually present alterations functional or structural [14].Several articles support the neurotoxic effect of 4(5)-MEI, characterized by episodes of confusion, motor agitation or hyperexcitability, tremors and seizures.15 The findings obtained in experimental studies where very low concentrations of 4(5)-MEI in the diet and determined in the plasma of experimentally intoxicated animals, [15,16] has given way to being considered a seizure agent, [17] not being possible to differentiate between the clinical signs of experimental exposure with 4(5)-MEI and food poisoning [18]. Furthermore, this observation has been verified in animals under concentrations and controlled application. Laboratory tests such as those carried out in long-term fed and intoxicated rats [4]. Despite lethal doses, these experimental animals show signs of excitation of the central nervous system (tremors, squeaks and jumps, among others), which later become seizures and death [19, 20]. Which implies that perhaps 4(5)-MEI is involved in the activity of various neurotransmitters such as gamma-aminobutyric acid (GABA) [21, 22].

Chronic consumption of 4(5)-MEI does not increase serum MDA levels in experimental rats, which may suggest a time-dependent efficiency of antioxidant systems. However, it exerts a neurotoxic effect by decreasing the rat's performance on a spatial memory task. A correlation study is required between the function and anatomy of the brain areas involved in the performance of memory tasks. It is suggested that more studies be carried out that allow the analysis of the mechanisms that underlie the toxic effect caused by 4(5)-MEI, as well as the possible protection mechanisms that the organism develops when chronically exposed to this substance.

All the authors declare that they have no conflict of interest.