Ameliorative Effects of Zinc and Vitamin E on Physiological Changes after Exposure to Heavy Metal

Abstract

Heavy metals have been known to have great deteriorative impacts on the physiology of the body, altering the normal functioning of the body. These impacts cut across the various systems of the body including cardiopulmonary, endocrine, neurological, gastrointestinal, hematological, etc. However, not every exposure will leave such effects in the aftermath. The level of exposure to one heavy metal that is considered harmful may not be with another metal. This chapter examines the various levels of exposure that may be considered unhealthy to the human body, and the mechanisms by which the metals exert their impacts, with the aim of educating readers on how to keep exposure below such threshold level. This chapter also explains that not all heavy metals are considered unhealthy as there are essential heavy metals that may have some beneficial effects to the physiology of the human system.

1. Introduction

Heavy metals are defined as metallic elements that have a relatively high density compared to water. These metals may be toxic or poisonous even at low concentrations. They are described as those elements having atomic number greater than 20 and atomic density above 5 g cm⁻³ and must exhibit the properties of metals [1, 2].

Examples include cadmium (Cd), mercury (Hg), zinc (Zn), copper (Cu), chromium (Cr), lead (Pb), arsenic (As), and nickel (Ni).

Natural phenomena like weathering and volcanic eruptions have also been implicated in heavy metal pollution [3, 4]. Some of them are exploited for various industrial and economic purposes. They are grouped into essential and non-essential heavy metals.

While essential heavy metals such as iron (Fe), magnesium (Mg), copper (Cu), and the like, are essential nutrients required for various biochemical and physiological functions such as growth, metabolism, and development of different organs, non-essential heavy metals such as cadmium (Cd), antimony (Sb), lead (Pb), vanadium (V) have no established biological functions, yet they still find their way into the body system, and have been reported to affect cellular organelles and components in biological systems [5].

There are numerous essential heavy metals required by plants as they form cofactors that are structurally and functionally vital for enzymes and other proteins. Essential elements are often required in trace amounts in the level of 10–15 ppm and are known as micronutrients.

Due to their high levels of toxicity, arsenic, cadmium, chromium, lead, and mercury are among the priority metals that are important for public health. Even at the modest exposure levels, these metallic elements are known to cause numerous organ damage and are regarded as systemic toxicants.

Despite the fact that some of these metals only affect human physiology at high amounts, others, including cadmium, mercury, lead, chromium, silver, and arsenic, have significant effects on the body even in minute quantities, leading to acute and chronic toxicities in humans [6].

Exposure to these heavy metals has been associated with certain physiological changes ranging from mental, hematological, and hormonal.

This chapter discusses major physiological changes that exposure to these metals can cause to the human body, as well as the risk factors that can lead to changes in human physiology. The impact of cadmium on the central nervous system (CNS) is used as a case study. It also discusses how these changes could be ameliorated.

2. Effects of cadmium on some physiological parameters in human

Cadmium chloride is a colorless heavy metal that can dissolve in ethanol, methanol, and water. It is considered a major environmental pollutant as a result of its widespread industrial use. It is present not only in soil and food, but also in water and air. So, it could be contaminated through food intake and could be released into water as a by-product. Combustion of coal and oil could also expose individuals to it [6]. It has a long half-life of between 15 and 30 years in humans due to its low rate of excretion from the body [7, 8].

International Agency for Research on Cancer (IARC) has identified cadmium as a known or probable human carcinogen. It has also been listed in the International Register of Potentially Toxic Chemicals (IRPTC) of the United Nations Environment Program (IRPTC), even as the World Health Organization (WHO) estimated 500 micrograms per week cadmium as the safe level for human ingestion [9].

Fish, liver, grains, and vegetables remain major sources of dietary cadmium [10].

Cadmium chloride has various lines of applications and is mostly used industrially. The major industrial applications of cadmium include the production of alloys, pigments, and batteries [11].

Invariably, people are exposed to cadmium on a daily basis, with common exposure in industrial work places, plants, soils, and from smoking. Due to its low permissible exposure to humans, over exposure may occur even in situations where trace quantities of cadmium are found [12]. Shortness of breath, pneumonitis, and pulmonary edema can all be signs of more serious respiratory system injuries [13, 14].

Long-term accumulation of cadmium in a number of tissues, including the kidneys, liver, CNS, and peripheral neuronal systems, may have hazardous effects at the peripheral level. It could cross the blood-brain barrier at the CNS and enter the CNS through the nasal mucosa or olfactory pathways. Exposure to cadmium is implicated in hyperactivity, increased aggression, impaired social memory processes, and altered drinking behavior [15, 16].

2.1 Mechanism of action

Cadmium acts as catalysts for biochemical reactions, regulators of gene expression, second messengers in signaling pathways, and co-factors for vital enzymes notorious for regulating physiological, pathological, and behavioral functions [7, 17].

In comparison with other brain regions, the hippocampus collects the divalent metals to a larger amount. The hippocampus impairment that results from heavy metal exposure has been linked to behavioral changes. Animal studies using Cd exposure also show behavioral changes in this approach. Reduced memory and altered anxiety and fear responses have been seen in rats exposed to Cd [18].

2.2 Zinc and Vitamin E

2.2.1 Zinc

Zinc (Zn), a trace element necessary for live cells and an important heavy metal for many enzymes, is involved in DNA replication, transcription, and protein synthesis, which all have an impact on cell division and differentiation [19]. It performs the task of attaching particular genes to tetrahedral bonds, causing transcription, and is thus directly implicated in the translation stage of DNA element gene expression.

Zinc deficiency may prevent the production of new proteins, which would reduce the amount of protein and cause a buildup of amino acids. This is due to zinc, a ribosome structural element that maintains the structural integrity of the ribosomes. In the absence of it, ribosomes break down [19].

By the antioxidant system’s action, it stops cell damage. It performs many different roles and is a crucial part of the antioxidant defense system [20, 21].

2.2.2 Vitamin E

The collection of eight fat-soluble compounds includes vitamin E. It can be discovered in many foods and oils. Alpha-tocopherol is mostly found in nuts, seeds, vegetable oils, fortified cereal, and green vegetables. Significant levels are also present in green leafy vegetables and fortified cereals. Food-based vitamin E is not known to be harmful. However, there is proof that extremely high doses of vitamin E supplementation might cause pro-oxidant damage [22, 23].

Vitamin E plays a role in the prevention of diseases like cancer, Alzheimer’s disease, HIV/AIDS, and others by preventing oxidative stress, protecting cell membranes, controlling platelet aggregation, and activating protein kinase C. According to other theories, vitamin E regulates gene expression and cell signal transmission [24, 25, 26].

2.2.3 Anxiety

Many conditions that produce trepidation, fear, concern, and worrying are together referred to as anxiety. It is described as a feeling that is accompanied by tense sensations, anxious thoughts, and physical changes like raised blood pressure. Fear is a reaction to an immediate threat, actual or perceived; anxiety is the anticipation of an impending threat [27, 28].

Muscle tension, agitation, exhaustion, and attention issues are frequently present in conjunction with it. Although experiencing anxiety occasionally is normal, a person may develop an anxiety disorder. Drug addiction, drug withdrawal, and genetic factors are all possible causes of anxiety disorders [27].

Therapy, medication, and lifestyle modifications are all potential treatment options. Worry, which is considered to be a result of metacognitive beliefs, is something that metacognitive treatment aims to eliminate [29].

This chapter addresses how cadmium chloride impacts the CNS to create anxiety as well as the role of zinc and vitamin E in reducing its effects on anxiety levels. Exposure to cadmium chloride may change the physiology of biological organs and systems.

3. Materials and methods

3.1 Animal preparation

For the investigation, 25 healthy CD1 mice, 8–10 weeks old, and weighing 18–30 g were employed. The animals were given unlimited access to food and water. For the course of the trial, the food and water troughs were replaced daily, and the beddings were also changed every three to five days. They were kept in a room with standard temperature and humidity levels (between 18 and 23°C and 40 and 60%, respectively), as well as a 12/12-hour light/dark cycle.

3.2 Experimental design

The 25 mice were randomly assigned into four (5) groups of five (5) animals;

Group A: Control

Group B: CdCl₂ (14 days)

Group C: CdCl₂ (28 days)

Group D: CdCl₂ + Zinc

Group E: CdCl₂ + Vitamin E

3.3 Drug administration

The test drugs were reconstituted into appropriate concentrations as follows: 500 mg of CdCl₂ was dissolved in 10,000 ml of distilled water; (50 ppm). 400 mg of vitamin E was dissolved in 5 ml of castor oil. 100 mg of zinc was dissolved in 69 ml of normal saline.

The CdCl₂ was administered orally with the aid of an orogastric cannula to the mice in group B for a period of 14 days (short-term exposure), while to the mice in group C, it was administered for 28 days (long-term exposure). Mice in groups D and E received same dose of CdCl₂ for 14 days but in addition they were given zinc and vitamin E, respectively. Following the Light and Dark Transition Box (LDTB) and Elevated Plus Maze (EPM) paradigms, anxiety-like behaviors were assessed at the conclusion of the treatment session.

3.4 Determination of anxiety

The test for anxiety was done using:

1. Elevated Plus Maze (EPM)

2. Light and Dark Transition Box (LDTB)

3.4.1 Elevated plus maze (EPM)

Rodents are used in the Elevated Plus Maze (EPM), a test for detecting anxiety in lab animals, as a general research tool in the study of neurobiological anxiety as well as a screening test for potential anxiolytic or anxiogenic substances. The animal in the EPM displays this concern by spending greater time in the enclosed arms [30, 31].

A raised, plus-shaped (+) apparatus with two open and two enclosed arms is used for the test. The behavioral model is based on rodents’ typical dislike of open areas. Due to this aversion, a behavior known as thigmotaxis develops, which is the desire for staying in enclosed areas or close to the boundaries of a confined region. This results in the animals restricting their movement to the confined arms of the EPM. An increase in the proportion of time spent in the open arms (time in open arms/total time in open or closed arms) and an increase in the proportion of entries into the open arms (entries into open arms/total entries into open or closed arms) are indicators of reduced anxiety in the plus maze. Occasionally, the total number of closed-arm entries is used as a gauge of overall activity [31, 32].

The EPM was created in accordance with Lister’s specifications (1987). From a center square (5 x 5 cm), the maze contains two open arms (45 5 cm²) with 0.25 cm high borders and two closed arms (40 5 cm²) with 15 cm high walls. There is a small ledge in the open arms (4 mm high) to stop the mice from losing their footing and going off the edge. Because they are contained, like most anxiety tests, the closed arms give a feeling of security. This job takes advantage of mice’s natural desire to investigate novel surroundings and their aversion to wide open spaces. Anxiety is also quantified by the open arm avoidance score [33].

To remove olfactory cues as well as feces and urine, the surfaces and closed sides of the plus maze arms were washed with methylated spirit prior to the test. The mouse was positioned in the plus maze’s middle square so that it initially faced an open arm away from the experimenter. Mouse was given five minutes to examine the device after placement before a silent stopwatch was started. The testing procedure was documented. Open arm movements and head dipping were deemed exploratory behaviors, and a higher frequency of these actions indicates a higher level of investigation [34].

Following are the behaviors scored:

1. The animal’s total distance traveled while participating in the test.

2. The animal’s open arm entries: the frequency with which it did so. For a mouse to be eligible for entrance, all four of its paws have to be inside the arm.

3. Closed arm entries: the animal’s frequency of entry into the closed arms. For a mouse to be eligible for entrance, all four of its paws have to be inside the arm.

4. The animal’s time in the open arms was measured in open arm duration.

5. Time spent in closed arms: how long the animal was held there.

6. Center square entries: how frequently the animal used all four paws to enter the center square.

7. Time spent in the core square: the amount of time the animal was there.

8. Head dipping: the animal frequently dropped its head over the sides of the open arm and down toward the ground.

9. Stretch attend postures: the animal frequently extends its head and shoulders forward before retracting back to its starting position.

10. Rearing: the number of times an animal stands on its hind legs or leans its front paws against a maze wall.

11. Grooming: the amount of time an animal spends stationarily licking or scratching itself.

12. Urination: the quantity of urine pools or streaks.

13. Number of fecal boli formed during defecation.

3.4.2 Light/Dark transition box (LDTB)

Two compartments make up the LDTB device. The light compartment occupies two-thirds of the box and is both open and well-lit. A covered and dark compartment makes up one-third of the entire box. The two chambers are connected by a 7-cm door. Rodents choose shadowy environments than bright ones. Rodents, on the other hand, show a propensity to explore when placed in an unfamiliar habitat. There are visible indications of anxiety as a result of these two opposing feelings. The dark compartment is often where rodents spend more time than the bright one. The percentage of time spent in the light compartment will rise in animals given anxiolytic injections. Rearing, or when a rodent raises up on its hind legs, is an indication of motion and nighttime exploration increase in compartment as well. The amount of time spent in the dark compartment increases after receiving anxiogenic injections. There is no prerequisite training for the LDTB. No food or water is restricted and only natural stressors like as light are utilized [35, 36].

The wooden light/dark box (45 x 27 x 27 cm) has two compartments that are of different sizes. Two-fifths of the box is painted white for the bigger compartment (27 x 27 cm), while two-fifths of the box is painted black for the smaller compartment (18 x 27 cm). A door (7.5 x 7.5 cm) that is situated in the middle of the wall between the two compartments at floor level connects them. The Plexiglas-covered floor is separated into 9 x 9 cm squares. The covers of both sections are made of transparent Plexiglas.

The apparatus’s light box is filled with a mouse, which is given free rein to move about. The mouse will typically explore the compartment’s edges before discovering the door. The mouse is given five minutes to investigate the device, and the rodent’s actions inside the box recorded. To be deemed an entry, all four paws must be inserted into the opposing chamber. The mouse is then taken out, and the box is cleaned with cotton wool and 70% ethyl alcohol. The box is then allowed to dry in between experiments [37].

3.4.3 Behaviors score

1. Number of transitions: how often the animal enters the opposing compartment (The mouse’s four paws must enter the new compartment for it to be scored and to be regarded as having been entered.) [38].

2. Number of times the animal stepped over a line marked on the box’s floor.

3. Rearing: the regularity with which the animal stands up straight or leans its front paws against the box wall.

4. Stretch attend postures: the animal frequently exhibits forward extension of the head and shoulders, followed by retraction to the starting position.

5. The length of time the animal spent stationarily licking the body.

6. The animal’s time in the box’s dark side is measured in terms of how long it was there.

7. The animal’s time in the light side of the cage, measured in minutes.

8. Defecation: the quantity of fecal boli that are formed (light vs. dark).

9. The quantity of pee pools or streaks (light vs. dark).

4. Statistical analysis

Data from the tests were analyzed, and the outcomes were displayed as graphs of means and standard error of means (SEM). To determine whether there was any significant variation between the test and control groups, analysis of variance (ANOVA) and a post-hoc Student’s t-test were utilized. P < 0.05 was adopted as the threshold for significance.

5. Results

5.1 Comparison of frequency of rearing among the different experimental groups in the light/dark transition box

The mean ± SEM rearing frequency for the control, CdCl₂ (14 days), CdCl₂ (28 days), CdCl₂ + Zinc, and CdCl₂ + vitamin E groups were 59.60 ± 5.12, 35.40 ± 3.41, 26.50 ± 5.11, 55.40 ± 3.04, and 53.60 ± 3.66, respectively.

The results revealed that as compared to the control group, the rearing frequency in the CdCl₂ group was significantly lower (p < 0.05). However, compared to the CdCl₂ group, the rearing frequency of the CdCl₂ + Zinc and CdCl₂ + vitamin E groups was significantly greater (p < 0.05) (see Table 1).

Groups	Rearing	Stretch Attend Posture	Transition between Light and Dark	Dark Duration in Light and Dark	Light Duration in Light and Dark
Control	59.60 ± 5.12	2.00 ± 0.71	21.60 ± 2.01	85.00 ± 12.98	215.00 ± 12.98
CdCl2 (short-term exposure)	35.40 ± 3.41	3.60 ± 0.51	7.40 + 1.03	213.50 ± 17.06	86.55 + 17.09
CdCl2 (long-term exposure)	26.50 ± 5.11	4.70 ± 0.41	5.30 + 1.13	227.66 ± 15.87	72.48 ± 13.87
CdCl2 + Zn	55.40 ± 3.04	1.60 ± 0.51	17.40 ± 1.63	81.63 ± 15.87	218.37 ± 15.87
CdCl2 + Vitamin E	53.60 ± 3.66	1.60 ± 0.51	21.40 ± 2.01	63.76 ± 18.35	236.24 ± 18.35

Table 1.

Comparing the occurrence of Rearing, Stretch Attend Posture (SAP), Transition between Light and Dark (TLD), Dark Duration in Light and Dark (DDLD) Transition Box Test, and Light Duration in Light and Dark (LDLD) Transition Box Test among various groups.

5.2 Comparison of stretch attend posture frequency (SAPLDT) in the light/dark transition box among the experimental groups

The mean ± SEM SAP frequency for the control, CdCl₂ (14 days), CdCl₂ (28 days), CdCl₂ + Zinc, and CdCl₂ + vitamin E groups were 2.00 ± 0.71, 3.60 ± 0.51, 4.70 ± 0.41, 1.60 ± 0.51, and 1.60 ± 0.51 respectively.

The results showed that there was no discernible change in the SAP frequency between the CdCl₂ group and the control group. Yet when compared to the CdCl₂ group, the SAP frequency in the CdCl₂ + Zinc and CdCl₂ + vitamin E groups was considerably lower (p < 0.05) (see Table 1).

5.3 Comparison of frequency of transition between the light/dark (TLD) compartments among the different experimental groups in the light/dark transition box

The mean ± SEM frequency of transition for the control, CdCl₂ (14 days)_, CdCl₂ (28 days)_, CdCl₂ + Zinc, and CdCl₂ + vitamin E groups were 21.60 ± 2.01, 7.40 + 1.03, 5.30 + 1.13, 17.40 ± 1.63, and 21.40 ± 2.01 respectively.

The findings demonstrated that the frequency of transition was substantially lower (p< 0.05) in the CdCl₂ group than in the control group. When compared to the CdCl₂ group, the frequency of transition was significantly higher (p < 0.05) in the CdCl₂ + Zinc and CdCl₂ + vitamin E groups (see Table 1).

5.4 Comparison of dark duration in light/dark (DDLD) transition box test among the different experimental groups

The mean ± SEM dark duration for the control, CdCl₂ (14 days), CdCl₂ (28 days), CdCl₂ + Zinc, and CdCl₂ + vitamin E groups were 85.00 ± 12.98, 213.50 ± 17.06, 227.66 ± 15.87, 81.63 ± 15.87, and 63.76 ± 18.35 respectively.

The results showed that the CdCl₂ group’s time spent in the dark chamber was substantially longer (p < 0.05) than that of the control group. When compared to the CdCl₂ group, the dark chamber duration was considerably shorter (p < 0.05) in the CdCl₂ + Zinc and CdCl₂ + vitamin E groups (see Table 1).

5.5 Comparison of light duration during light/dark (LDLD) transition box test among the different experimental groups

The mean ± SEM light duration for the control, CdCl₂ (14 days), CdCl₂ (28 days), CdCl₂ + Zinc, and CdCl₂ + vitamin E groups were 215.00 ± 12.98, 86.55 + 17.09, 72.48 ± 13.87, 218.37 ± 15.87, and 236.24 ± 18.35, respectively.

Results showed that the CdCl₂ group’s time spent in the light chamber was substantially shorter (p < 0.05) than that of the control group. The light chamber duration was substantially longer (p < 0.05) for the CdCl₂ + Zinc and CdCl₂ + vitamin E groups than for the CdCl₂ group (see Table 1).

5.6 Comparison of grooming frequency during light/dark transition box test among the different experimental groups

The mean ± SEM grooming frequency for the control, CdCl₂ (14 days), CdCl₂ (28 days), CdCl₂ + Zinc, and CdCl₂ + Vitamin E groups were 1.80 ± 0.37, 7.20 ± 0.86, 9.33 ± 0.38, 2.60 ± 1.08, and 4.20 ± 0.58 respectively.

The findings revealed that the CdCl₂ group’s grooming frequency was substantially higher (p < 0.05) than that of the control group. However, when compared to the CdCl₂ group, the grooming frequency in the CdCl₂ + Zinc group was considerably lower (p < 0.05) (see Table 2).

Groups	Grooming; (Light/Day Transition)	Regaining consciousness during Elevated Plus Maze	Stretch Attend Posture during the Elevated Plus Maze	Head Dips during the Elevated Plus Maze	Grooming during the Elevated Plus Maze Freq.
Control	1.80 ± 0.37	40.60 ± 3.89	7.60 ± 1.54	11.00 ± 2.70	2.60 ± 0.40
CdCl2 (short-term exposure)	7.20 ± 0.86	23.20 ± 3.73	11.00 ± 1.26	14.40 ± 1.72	7.20 ± 0.58
CdC2 (long-term exposure)	9.33 ± 0.38	19.42 ± 3.86	13.00 ± 1.65	17.00 ± 1.12	10.40 ± 0.28
CdCl2 + Zn	2.60 ± 1.08	35.00 ± 3.54	7.00 ± 1.64	8.00 ± 1.22	2.20 ± 0.58
CdCl2 + Vitamin E	4.20 ± 0.58	40.40 ± 1.86	4.00 ± 0.71	9.80 ± 1.16	1.80 ± 0.37

Table 2.

Comparing the frequency of grooming during the Light/Dark Transition Box Test, the occurrence of regaining consciousness during the Elevated Plus Maze, the occurrence of the Stretch Attend Posture, the occurrence of Head Dips, and the occurrence of grooming during the Elevated Plus Maze among the various experimental groups.

5.7 Comparison of the elevated plus maze (REPM) test’s frequency of rearing among the several experimental groups

The mean ± SEM rearing frequency for the control, CdCl₂ (14 days), CdCl₂ (28 days), CdCl₂ + Zinc, and CdCl₂ + Vitamin E groups were 40.60 ± 3.89, 23.20 ± 3.73, 19.42 ± 3.86, 35.00 ± 3.54, and 40.40 ± 1.86, respectively.

The results revealed that as compared to the control group, the rearing frequency in the CdCl₂ group was considerably lower (p < 0.05). However, when compared to the CdCl₂ group, the rearing frequency in the CdCl₂ + Vitamin E group was considerably greater (p < 0.05) (see Table 2).

5.8 Comparison of the elevated plus maze test’s stretch attend posture (SAPEPM) frequency in the various experimental groups

The mean ± SEM SAP frequency for the control, CdCl₂ (14 days), CdCl₂ (28 days), CdCl₂ + Zinc, and CdCl₂ + Vitamin E groups were 7.60 ± 1.54, 11.00 ± 1.26, 13.00 ± 1.65, 7.00 ± 1.64, and 4.00 ± 0.71, respectively.

The findings showed that there was no discernible change in the SAP frequency between the CdCl₂ group and the control group. In contrast to the CdCl₂ group, the SAP frequency in the CdCl₂ + Vitamin E group was considerably lower (p < 0.05) (see Table 2).

5.9 Comparison of the variable experimental groups’ head dip frequency in the elevated plus maze test

The mean ± SEM head dip frequency for the control, CdCl₂ (14 days), CdCl₂ (28 days), CdCl₂ + Zinc, and CdCl₂ + Vitamin E were 11.00 ± 2.70, 14.40 ± 1.72, 17.00 ± 1.12, 8.00 ± 1.22, and 9.80 ± 1.16, respectively.

The results showed that there was no discernible difference in the frequency of head dips between groups (see Table 2).

5.10 Comparison of the various experimental groups’ grooming frequency throughout the elevated plus maze test

The mean ± SEM grooming frequency for the control, CdCl₂ (14 days), CdCl₂ (28 days), CdCl₂ + Zn, and CdCl₂ + Vitamin E groups were 2.60 ± 0.40, 7.20 ± 0.58, 10.40 ± 0.28, 2.20 ± 0.58, and 1.80 ± 0.37, respectively.

Results showed that the CdCl₂ group’s grooming frequency was substantially higher (p < 0.05) than that of the control group. When compared to the CdCl₂ group, grooming frequency was considerably reduced (p < 0.05) in the CdCl₂ + Zinc and CdCl₂ + Vitamin E groups (see Table 2).

5.11 Comparison of grooming duration in the elevated plus maze test among the different experimental groups

The mean ± SEM grooming duration for the control, CdCl₂ (14 days), CdCl₂ (28 days), CdCl₂ + Zinc, and CdCl₂ + Vitamin E groups were 10.71 ± 2.79, 38.08 ± 5.61, 45.02 ± 2.68, 11.49 ± 3.33, and 7.39 ± 1.99, respectively.

The results revealed that the CdCl₂ group’s grooming time was substantially longer (p < 0.05) than that of the control group. In contrast to the CdCl₂ group, the grooming time was considerably shorter in the CdCl₂ + Zinc and CdCl₂ + Vitamin E groups (p < 0.05) (see Table 3).

Groups	Grooming(EPM) duration	Freq. CAEEPM	CAEEPM Duration	Freq. OAEEPM	OAEEPM Dur.
Control	10.71 ± 2.79	3.80 ± 0.80	112.27 ± 10.92	9.40 ± 0.75	185.73 ± 10.66
CdCl2 (short-term exposure)	38.08 ± 5.61	5.80 ± 0.66	238.29 ± 18.21	2.80 ± 0.37	61.71 ± 18.21
CdCl2 (long-term exposure)	45.02 ± 2.68	7.01 ± 0.58	310.25 ± 12.81	1.48 ± 0.51	39.46 ± 18.66
CdCl2 + Zinc	11.49 ± 3.33	3.00 ± 0.45	144.29 ± 7.96	8.20 ± 0.86	155.71 ± 7.96
CdCl2 + Vitamin E	7.39 ± 1.99	3.20 ± 0.58	106.34 ± 26.04	8.60 ± 0.51	193.66 ± 26.04

Table 3.

Compares the length of grooming in the Elevated Plus Maze Test, frequency of closed arm entry during the test, duration of closed arm entry during the test (CAE Dur.), frequency of open arm entry during the test (Freq. OAE), and duration of open arm entry during the test among the various experimental groups.

5.12 Comparison of the frequency of closed arm entry during elevated plus maze test among the different experimental groups

The mean ± SEM frequency of closed arm entry for control, CdCl₂ (14 days), CdCl₂ (28 days), CdCl₂ + Zinc, and CdCl₂ + Vitamin E groups were 3.80 ± 0.80, 5.80 ± 0.66, 7.01 ± 0.58, 3.00 ± 0.45, and 3.20 ± 0.58, respectively.

The results showed that the frequency of closed arm entry in CdCl₂ group had no significant difference when compared with the control group. However, the CdCl₂ + Zinc and CdCl₂ + Vitamin E groups had significantly lower (p < 0.05) frequency of closed arm entry when compared with the CdCl₂ group (see Table 3).

5.13 Comparison of the duration in the closed arm entry during (CAE Dur.) elevated plus maze test among the different experimental groups

The mean ± SEM closed arm duration for the control, CdCl₂ (14 days), CdCl₂ (28 days), CdCl₂ + Zinc, and CdCl₂ + Vitamin E were 112.27 ± 10.92, 238.29 ± 18.21, 310.25 ± 12.81, 144.29 ± 7.96, and 106.34 ± 26.04, respectively.

The results showed that the CdCl₂ group’s duration in the closed arm was substantially longer (p < 0.05) than that of the control group. When compared to the CdCl₂ group, the duration in the closed arm was considerably shorter for the CdCl₂ + Zinc and CdCl₂ + Vitamin E groups (p < 0.05) (see Table 3).

5.14 Comparison of the frequency of open arm entry (Freq. OAE) during elevated plus maze test among the different experimental groups

The mean ± SEM frequency of open arm entry for the control, CdCl₂ (14 days), CdCl₂ (28 days), CdCl₂ + Zinc, and CdCl₂ + Vitamin E groups were 9.40 ± 0.75, 2.80 ± 0.37, 1.48 ± 0.51, 8.20 ± 0.86, and 8.60 ± 0.51, respectively.

In comparison with the control group, the CdCl₂ group’s open arm entry frequency was significantly lower, according to the data (p < 0.05). The open arm entry frequency was significantly higher (p < 0.05) in the CdCl₂ + Zinc and CdCl₂ + Vitamin E groups as compared to the CdCl₂ group (see Table 3).

5.15 Comparison of the elevated plus maze test time in the open arm among the several experimental groups

The mean ± SEM open arm duration for the control, CdCl₂ (14 days), CdCl₂ (28 days), CdCl₂ + Zinc, and CdCl₂ + Vitamin E were 185.73 ± 10.66, 61.71 ± 18.21, 39.46 ± 18.66, 155.71 ± 7.96, and 193.66 ± 26.04, respectively.

Results showed that the CdCl₂ group’s open arm duration was considerably shorter (p < 0.05) than that of the control group. However, compared to the CdCl₂ group, the open arm time was considerably longer (p < 0.05) in the CdCl₂ + Zinc and CdCl₂ + Vitamin E groups (see Table 3).

6. Discussion

The elevated plus maze (EPM) and light and dark transition box (LDTB) tests were employed to measure anxiety. In order to assess anxiety, signs such as grooming frequency and duration, rearing, and stretch attend posture were also used [27].

Longer duration in the light box compartment shows decreased anxiety and greater length in the dark box compartment reveals increased anxiety [36].

The results of this experiment’s light/dark transition box test revealed that the control group’s dark chamber duration was shorter and its light chamber duration was longer than that of the experimental group. This suggests that the mice who were not exposed to CdCl₂ experienced less anxiety.

However, there was a longer time in the dark room and a shorter time in the light chamber in the group exposed to CdCl₂, indicating that exposure to CdCl₂ enhanced anxiety. This is demonstrated in Table 1 as a longer time of exposure had a more significant effect.

Also, the zinc and vitamin E treated groups that had previously been exposed to CdCl₂ had a shorter time in the dark and a longer time in the light. This means that even after exposure to situations that had previously elevated anxiety, zinc and vitamin E may be able to reduce it.

Increased grooming frequency indicates higher levels of anxiety [39].

According to the experiment’s findings, the CdCl₂ exposed group groomed more frequently than the control group. Even more frequent grooming was observed in the group exposed for a longer period of time, indicating a proportionate rise in anxiety with CdCl₂ exposure, compared to less frequent grooming in the zinc and vitamin E treated group. This supports Table 2’s finding that zinc and vitamin E lessen anxiety.

The animal spends more time in the enclosed arm when it is anxious, indicating that an increase in duration in the closed arm represents an increase in anxiety level and an increase in duration in the open arm suggests a drop in anxiety levels [31].

When compared to the control group, the CdCl₂ group had a longer duration in the closed arm of the labyrinth and a shorter length in the open arm, according to the results of this experiment’s elevated plus maze test. While the zinc and vitamin E treated group had a reduced duration in the closed arm and a higher duration in the open arm of the labyrinth, these effects are much more significant in the group with longer period of exposure. Also, it revealed that the CdCl₂ group groomed more frequently and for longer than the control group, whereas the zinc and vitamin E treated groups groomed less frequently and for shorter periods of time.

This supports the finding that anxiety is caused by cadmium chloride (CdCl₂) exposure in mice, and that the longer the exposure, the more anxiety is caused. As shown in Table 3 [20, 24], it also suggests that zinc and vitamin E, two important antioxidants, aid in reducing anxiety and stress.

The primary mechanisms of heavy metal toxicity include free radical production, which leads to oxidative stress, damage to biological molecules such as enzymes, proteins, lipids, and nucleic acids, as well as damage to DNA, which is crucial for both neurotoxicity and carcinogenesis. See Figure 1.

Figure 1.

Showing mechanisms of heavy metal toxicity.

7. Conclusion

The results of this study suggest that exposure to cadmium chloride (CdCl₂) causes anxiety in CD1 mice. This anxiety is even more pronounced with longer period of exposure. Zinc and vitamin E, through their antioxidant property, show ameliorative effect by lowering anxiety levels in CD1 mice after exposure to cadmium chloride (CdCl₂).

Conflict of interest

Authors declare that there is no conflict of interest over the manuscript.

Funding

This work did not receive any form of funding or financial support whatsoever from anyone or any group. It is solely the personal efforts of the authors. It also did not receive any form of non-financial support from anyone.

Ethical clearance

Ethical approval was obtained: UCFMSECAE1047.