Effect of simultaneous administration of cadmium, magnesium and alcohol on liver, kidney and biomarkers of oxidative stress in Wistar rats (2023)

Effect of simultaneous administration of cadmium, magnesium and alcohol on liver, kidney and biomarkers of oxidative stress in Wistar rats

Article information

Chidi John Ogham,^1,2*Jonathan Dabak¹, Left Jaryum¹

¹Department of Biochemistry, Faculty of Basic Medical Sciences, Faculty of Health Sciences, University of Jos, Jos, Nigeria

²Standards Organization of Nigeria, Company Headquarters, Abuja, Nigeria.

^*Corresponding author:Chidi John Ogham. 1 Department of Biochemistry, Faculty of Basic Medical Sciences, Faculty of Health Sciences, University of Jos, Jos, Nigeria

Received:April 29, 2023;Accepted:May 10, 2023;Published:xxxxx

Quote:Chidi John Ogham, Jonathan Dabak, Kiri Jaryum. Effect of simultaneous administration of cadmium, magnesium and alcohol on liver, kidney and oxidative stress biomarkers in Wistar rats. Archives of Nephrology and Urology. 6 (2023): 61-74.

Abstract

Keywords

cadmium; Magnesium; Alcohol; Oxidative stress; Biomarkers; Liver; Kidney.

Cadmium articles, magnesium articles, alcohol articles, oxidative stress articles, biomarker articles, liver articles, kidney articles.

Item details

1. Introduction

Bar is a landlocked village separated by the Bununu River to the north and surrounded by hills to the east and west with a population of about 1,300 people. The narration says that some mineral resources have already been exploited in the area in unattainable quantities for commercial purposes.

A few years ago, the village experienced a health crisis of epic proportions after the public water source was switched from surface water to underground water for drinking, cooking and irrigation. The move away from using surface water for drinking and cooking was initially hailed as a huge advance for public health because of a dramatic drop in deaths from waterborne diseases caused by microbes. However, the organization that dug these wells for the community did not know that the groundwater in the village was contaminated with some toxic metals of hydrogeological origin. An assessment of water quality carried out earlier in the area showed that it is contaminated with cadmium and has a high magnesium content [1]. This led to the outbreak of a strange disease characterized by migraine, fever, anemia, hypertension, degenerative dementia and liver enlargement which consequently led to the death of dozens of people in this village. The disease, according to residents, began in October 2003 and was attributed to drinking water from a hand-dug well in the area. This incident was reported by the local Daily Trust newspaper on Wednesday 31 May 2006, and the once lively villagers lived in fear of the disease [2].

An epidemiological study conducted by the public health department of the Bauchi State Ministry of Health revealed that those who died of these diseases were mostly those who consumed locally produced alcoholic beverages in the area. However, the study was very suggestive because it did not show a clear link between health effects, concurrent exposure to toxic metals, and alcohol consumption.

There are numerous literatures that point to the fact that environmental pollution is considered one of the biggest challenges of today's modern human society [3]. Heavy metal pollution and contamination in the environment is a threat to the environment and a serious health problem in this modern age, mainly due to the growth of industrialization and urbanization [4]. The rates at which these metals are mobilized and transported into the environment have also accelerated significantly since the 1940s [5]. These metals occur naturally in the environment through the weathering of metal-bearing rocks and volcanic eruptions, while industrial emissions, mining, smelting and agricultural activities such as the application of pesticides and phosphate fertilizers are the main anthropogenic sources [6]. Combustion of fossil fuels also contributes to the release of heavy metals such as cadmium (Cd) into the environment [7]. Heavy metals pollute food chains, remain in the environment and cause various health problems due to their toxicity. Chronic exposure to heavy metals in the environment is a great threat to living organisms [8], because the population living in polluted areas is not even aware of the lurking danger.

The microbiological balance of the soil is affected by metal concentrations above threshold levels and can reduce soil fertility [9]. Bioaccumulation of toxic heavy metals in the biota of river ecosystems can have negative effects on animals and humans [10]. Higher levels of heavy metals in biota can have negative effects on the ecological health of aquatic animal species and can contribute to the decline of their populations [11].

It must be emphasized that exposure to these toxic metals usually involves exposure to a combination of toxic metals, not just one metal; therefore, an accurate assessment of health risk from the consumption of metal-contaminated groundwater must take into account the potential interactions between metals at the chemical, biochemical and physiological levels. When individuals are exposed to metals in combination, each metal may have its own typical health effects; synergistic or antagonistic. When assessing the risk of contaminants in drinking water, it is important to consider not only the health risks due to individual contaminants, but also the risks due to combinations of contaminants. The intake of metals in combination can increase or decrease the absorption and distribution of certain metals in the digestive tract and circulatory system, as well as affect the excretion of other metals [12,13].

The absorption of metals from drinking water can be affected by certain diets, behaviors and addictions. Interactions with dietary components and total nutrient levels also affect outcomes of toxic metal exposures [14]. The health effects of co-exposure to toxic metals are further influenced by behavioral and cultural practices (eg smoking) and dietary habits (alcoholism). Of particular interest are interactions between xenobiotics to which exposure is uncommon. Examples of these substances are cadmium and ethanol. Interactions of cadmium and ethanol are an important problem in the field of modern toxicology, since both substances pose a risk to human and animal health [15]. Dabak et al. (2016) also reported that magnesium has some level of protective effect against cadmium toxicity [16].

Alcoholism is a serious problem in most societies. Excessive consumption of ethanol in the form of alcoholic beverages may be common among some industrial workers exposed to cadmium, including smokers [15]. Ethanol has been reported to increase the permeability of biological membranes to cadmium [17], which may make alcoholics more susceptible to the effects of cadmium toxicity. The findings suggest that, with typical exposure patterns, multiple mechanisms likely contribute to proximal tubule cadmium uptake in vivo [ 18 ]. Regardless of the uptake mechanisms involved, it is clear that cadmium can accumulate over time in the epithelial cells of the proximal tubules. The traditional view is that when tissue cadmium levels exceed a critical concentration of about 150 µg/g tissue, intracellular defenses such as metallothionein (MT) and glutathione (GSH) are overwhelmed, and cells suffer damage and begin to die [19,20] .

Based on the above-mentioned studies, the dose, duration, chemical form of Cd in kidney, liver and intestinal tissue and the way of exposure to Cd should be considered an important factor in the assessment of long-term chronic effects and application of Cd. short-lived cd. The liver and kidneys are important organs of metabolism, detoxification, storage and excretion of xenobiotics and their metabolites, and are particularly sensitive to damage. The liver is an important target organ for ethanol [21], and the kidney for Cd toxicity [22]. Exposure to particles results in the delivery of metals to various extrapulmonary sites, where they form reactive centers that continuously catalyze the formation of reactive oxygen species and cause oxidative stress.

This work was therefore designed to investigate the possible effects that co-administration of water containing cadmium and magnesium with graded concentrations of alcohol would have on the liver, kidney and redox status, in order to document a plausible explanation for the cause(s) of these health problems leading to deaths of mostly alcoholics in our research area. This in vivo research using mouse models used methods to assess interactions between parameters in a subject to support relevant in vitro data, findings and correlations.

2. Materials and methods

2.1 Experimental animals

Thirty-two (32) 10-week-old white albino rats with an average initial body weight of 230 g were obtained from the vivarium at the University of Jos, Nigeria and were fed commercial feed (Vital Feed) and received various treatments with water ad libitum. They were housed in ventilated cages and maintained on a regular daylight cycle (12:12 light:dark). Shredded corn cob served as bedding. They were also allowed to acclimatize for 7 days in standard environmental conditions before treatment.

2.2 Chemicals/reagents

The feed used was vital feed (grower mash) produced by Grand Cereal and Oil Mills Ltd, Jos Plateau State. Cadmium chloride used was a product of May and Baker (M&B) Ltd, Gagenhan, England, while ethanol was a product of British Drug House (BDH), England. The magnesium chloride used was a Sigma-Aldrich product. Other chemicals used were of analytical grade purchased by the Ministry of Education, Bauchi State for Misau State Science Secondary School and also from NanaRich Bauchi Medical Laboratories and Prestige Laboratories Jos. All chemical preparations were made with distilled water that was distilled from a Pyrex apparatus. Cadmium standards were obtained from the Water and Sanitation Agency (WATSAN) laboratory established by the United Nations Children's Fund (UNICEF) in Bauchi. Alanine aminotransferase (ALT; E-BC-K235-S), aspartate aminotransferase (AST; E-BC-K236-S), alkaline phosphatase (ALP; E-BC-K009-M), lactate dehydrogenase (LDH; E-BC -K766-M), Albumin (ALB; E-BC-K057-5), Protein Biuret (E-BC-K165-S), Total Birilubin (E-BC-K760-M), Urea (BUN; E- BC -K183-S) and creatinine activity assay kit (Cr; E-BC-K188-M) from Elabscience purchased from Hospilab, FCT. A rat glucose 6 phosphate dehydrogenase Elisa kit (G6PH; E-EL-R0428) from Elabscience was also used.

2.3 Grouping and treatment of experimental rats

Rats were randomly divided into eight groups of 4 rats per group in cages and all groups drank the solution assigned to each group ad libitum. Administration of ethanol in a dose of 5 g of 96% ethanol/kg body weight/24 h was performed by intragastric intubation at 12-hour intervals throughout the duration of the experiment. During the entire experiment, the animals were kept in the same conditions and had unlimited access to food. As a case study for the events that occurred in the village of Bar/Bula, we used exposure levels of cadmium and magnesium to match those that occurred in groundwater sampled in the area. Preparations of aqueous solutions containing 0.16 mg/L CdCl2 and 105 mg/L MgCl2 served as representative samples of water given to rats.

Group 1 (Normal Control) – This group was placed in redistilled drinking water throughout the experiment. Group 2 (test control) was treated only with 6% (v/v) aqueous ethanol solution. Group 3 was treated with a 1% (v/v) aqueous ethanol solution and a drinking aqueous solution of a combination of 0.16 mg/L CdCl2 and 105 mg/L MgCl. Group 4 treated with 2% (v/v) aqueous ethanol solution and drinking water solution of 0.16 mg/L CdCl2 and 105 mg/L MgCl. Group 5 was treated with a 3% (v/v) aqueous ethanol solution and a drinking water solution of 0.16 mg/L CdCl2 and 105 mg/L MgCl. Group 6 was treated with a 4% (v/v) aqueous ethanol solution and a drinking water solution of 0.16 mg/L CdCl2 and 105 mg/L MgCl. Group 7 was treated with a 5% (v/v) aqueous ethanol solution and a drinking water solution of 0.16 mg/L CdCl2 and 105 mg/L MgCl. Group 8 was treated with a 6% (v/v) aqueous ethanol solution and an aqueous drinking solution of 0.16 mg/L CdCl2 and 105 mg/L MgCl. Animals were included in the study if they underwent successful intragastric intubation throughout the study period. Animals were excluded if they died prematurely before collection of analytical samples.

Four different teams of researchers were involved during the study; the first investigator was responsible for treatment preparation. Another researcher was responsible for conducting the cluster-based treatment. The third examiner was responsible for the anesthesia procedure and the performance of the surgical procedure, while the fourth group of researchers (without knowledge of the treatment) evaluated the biochemical and histopathological tests.

2.4 Sample collection and preparation

After 21 days of treatment, rats were sacrificed by cervical dislocation. Serum was obtained after centrifugation and kept frozen (-20oC) until required for biochemical analyses. The kidneys and liver were harvested and separated into two parts. A part was immediately frozen in liquid nitrogen and stored at -80oC for the examination of antioxidant status, and the other part was preserved in 10% formalin for histopathological examinations.

2.5 Biochemical tests

Serum alanine aminotransferase (ALT; E-BC-K235-S) and aspartate aminotransferase (AST; E-BC-K236-S) activities were determined according to the method of Reitman and Frankel (1957) [23], alkaline phosphatase ( ALP ; E -BC-K009-M, PNPP method), lactate dehydrogenase (LDH; E-BC-K766-M, WST-8 method), albumin (ALB; E-BC-K057-5, Bromocresol Green method), biuret Protein ( E-BC-K165-S), total bilirubin (E-BC-K760-M), urea (BUN; E-BC-K183-S, urease method) and creatinine (Cr; E-BC -K188-M , method sarcosine oxidase) using Elabscience activity assay kits purchased from Hospilab, FCT. Serum electrolytes; sodium ions (Na), potassium ions (K), bicarbonate ions (H3O-) and chloride ions (Cl) were measured using kits (Teco Diagnostics, California, USA)

The activity of ALT, AST and ALP in serum was determined by the method of Reitman and Frankel.23 To analyze the activity of ALT, ALT reacted with alanine and a-ketoglutarate and produced pyruvic acid.

Glutamate + pyruvate ⇌ α-ketoglutarate + Alanine

Pyruvic acid was then reacted with 2,4-dinitrophenylhydrazine (DNPH) to form phenylhydrazone, which could be dissolved in sodium hydroxide (10 ml 0.4 M NaOH) and then detected at 505 nm. To analyze the activity of AST, AST catalyzed aspartate and α-ketoglutarate to form oxaloacetate and glutamate. Oxaloacetate can be automatically decarboxylated to pyruvic acid, which can react with DNPH and then be detected at 510 nm. To assay ALP activity, ALP was catalyzed by disodium phenyl phosphate to form phenol, which can react with 4-aminoantipyrine and potassium ferricyanide and can be detected at 520 nm

Determination of nitrogen from urea in the blood was carried out using the urease method. 5 µl of serum is added to the microplate well with the urease reagent mixture. The reaction mixture was incubated for 15 minutes, after which 150 µl of alkaline hypochlorite was added and incubated for another 10 minutes. The absorbance was read at 620 nm. Serum creatinine was determined by the sarcosine oxidase method. 10 µl of serum is added to a microplate well with 180 µl of reagent mixture. The reaction mixture was incubated at 37°C for 5 minutes, after which 60 µl of reagent 2 (peroxidase) was added and incubated for 2 minutes. The absorbance was read at 515 nm.

The bromocresol green method for albumin determination involved mixing 10 µl of sample with 2500 µl of reagent at pH 4 – 4.2 for 10 min to form a yellow-green complex that is measured at 628 nm. The protein biuret method for protein determination involved mixing 50 µl of sample with 2500 µl of working biuret solution for 10 min to form a purple complex that is measured at 540 nm.

Lactate and NAD+ are converted into pyruvate and NADH by the action of lactate dehydrogenase. NADH strongly absorbs light at 340 nm, while NAD+ does not. The rate of increase in absorbance at 340 nm is directly proportional to the LD activity in the sample. This involved mixing 1000 µl and 10 µl and incubating at 37oC for 1 minute, measuring the change in absorbance per minute (340 nm) for 3 minutes

2.6 Redox status analysis

Liver and kidney tissues for assessment of redox status were rapidly excised, washed in ice-cold 0.9% NaCl and homogenized in nine volumes of buffer (0.1 mol/L phosphate buffer, pH 7.4) [24]. Homogenization was performed with a basic homogenizer T10 Ultra-Turrax (IKA, Staufen, Germany). Part of the homogenate was used for the determination of malondialdehyde (MDA), while the rest was centrifuged for 10 min at 800 g and then for 20 min at 9500 g to obtain the post-mitochondrial supernatant (PMS). Evaluation performance of the following parameters: MDA, catalase level, total thiol group (SH) level, glucose 6 phosphate dehydrogenase (G6PH) and superoxide dismutase (SOD) activity on the ILAB 300 plus analyzer (Instrumentation Laboratory, Milan, Italy) and Cary 60 UV -VIS spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). The concentration of MDA was determined as a thiobarbituric acid reactive substance (TBARS) by a spectrophotometric test based on the maximum absorption of the complex of malondialdehyde and other TBARS with thiobarbituric acid at 535 nm [25]. The principle of the method for determining AOPP is a two-stage measurement of sample absorption in the wavelength range 200-400 nm, with a maximum at 340 nm. The measured difference in the measured absorbance values ​​at 340 nm indicates the AOPP value for a given sample [26]. The principle of the method for determining total SH groups is as follows: aliphatic thiol compounds in a basic environment react with DTNB (2,2'-dinitro-5,5'-dithio-benzoic acid), in which one mole of thiol produces one mole of p-nitrophenol. The resulting dye has an absorption peak at 412 nm [27]. Determination of superoxide dismutase (SOD, EC 1.15.1.1) enzyme activity was established in 1971 by Misra and Fridovich (1972) [28]. The method uses the ability of SOD to inhibit the spontaneous autoxidation of adrenaline at pH 10.2.

A whole blood G-6-PD screening assay was performed using mouse Elisa Sandwich G-6-PD kits. Screening was performed on the day of blood collection. Wells for diluted standard, blank and sample were designated. A 100 μL sample was added to the appropriate wells and the plate was covered with the sealing agent provided in the kit followed by incubation for 90 minutes at 37°C. The liquid from each well was then decanted with the immediate addition of 100 μL of Biotinylated Detection Ab working solution to each well, and the plate was covered with a new coverslip and incubated for 1 hour at 37°C. 350 µL of wash buffer was added to each well and soaked for 1 minute with subsequent aspiration and patting on clean absorbent paper until dry. The washing was then repeated 3 times. 100 µL of HRP conjugate working solution was added to each well, covered with a new cap and incubated for 30 minutes at 37°C. The solution was decanted from each well and washed repeatedly 5 times. 90 μL of substrate reagent was added to each well, covered with a new seal and incubated for about 15 minutes at 37°C. The plate is carefully protected from light. Then 50 µL of stop solution was added to each well. Absorbance was determined at 450 nm.

2.7 Histopathological studies

Slices (thickness 0.5 cm) of the left lobe of the liver and the left kidney (from two animals in each group) were fixed in 10% formalin solution for 24 hours, after which they were transferred to 70% alcohol for dehydration. The tissues were soaked in 90% alcohol and chloroform for different times, then transferred to two melted paraffin embeds for 20 minutes each in an oven at 57ºC. Serial sections with a thickness of 5-6 µm were obtained from a solid block of tissue and stained with hematoxylin and eosin, after which they were passed through a mixture of equal concentration of xylene and alcohol. After clearing in xylene, the tissues were dried in an oven. Photomicrographs were taken with a JVC digital color video camera (JVC, China) mounted on an Olympus light microscope (Olympus UK Ltd, Essex, UK) to demonstrate the cytoarchitecture of the liver. All changes compared to the normal structure were recorded.

2.8 Statistical analysis

The mean values ​​obtained for the tested metals in the samples of alcoholic beverages were compared by one-way ANOVA at the significance level of 95% using statistical software IBM SPSS version 20; assuming that there are significant differences between them when the statistical comparison gives p < 0.05.

3. Results

3.1 Effect of treatment on total proteins, albumin. Bilirubin and direct bilirubin

Figure 1 shows the effect of treatment on total protein, albumin, bilirubin and direct bilirubin. The results show that total protein and albumin concentrations were not significantly different (p>0.05) between the normal control and groups 3, 4, 5 and 6, but there was a significant decrease (P<0.05) in groups 7 and 8. when compared to a normal control. With increasing alcohol concentration in the tested groups, there was a significant decrease (p<0.05) in total protein and albumin concentrations compared to the test control. On the other hand, with the increase in alcohol concentration in the tested groups, there was a significant increase (p < 0.05) in the concentration of bilirubin and direct bilirubin compared to the test control.

3.2 Effect of treatment on ALP, AST and ALT

Figure 2 shows the effect of treatment on ALP, AST and ALT. The results show that the ALP concentration in group 3, which had 1% alcohol supplemented with Cd and Mg, was not significantly different (p>0.05) from the normal control, but was significantly different (P<0.05) from the test control . As the alcohol concentration was increased in the tested groups, there was a significant (p<0.05) increase in ALP compared to the test control. On the other hand, as the concentration of alcohol was increased in the tested groups, there was a significant increase (p < 0.05) in the concentrations of AST and ALT, which were significantly different from the test and normal control.

3.3 Effect of treatment on LDH activity after 1 and 3 minutes

Figure 3 shows the effect of treatment on lactic acid dehydrogenase (LDH) activity after 1 and 3 minutes. The results show that there was no significant difference (p > 0.05) between the normal controls and the LDH activity test after 1 and 3 minutes; but there was a significant difference (P<0.05) in the enzyme activity between the normal control and the test control after 3 minutes. As the alcohol concentration was increased in the tested groups, the enzyme activity increased significantly (p<0.05) after 1 minute and also after 3 minutes. The increase in enzyme activity in one minute was greater than the increase in 3 minutes.

3.4 Effect of treatment on the concentration of Na+, K+, Cl-, HCO3^-

Figure 4 shows the effect of the treatment on the concentrations of Na+, K+, Cl- and HCO3-. The results show that there was no significant difference (p > 0.05) between the normal control and the test in the concentrations of Na+, K+, Cl- and HCO3-. However, when the same concentration of Cd and Mg was applied with 1%, 2%, 3%, 4%, 5% and 6%, there was a significant (P<0.05) increase in Na+ concentration, which was expressed in groups 7 up to 8 compared to normal and test control. On the other hand, there was a significantly lower concentration (P<0.05) of K+ in all tested groups compared to the normal and test control. The results also show that Cl ion concentration was significantly (P<0.05) increased especially in groups 6, 7 and 8 compared to normal and test control. There was no significant difference (p > 0.05) between HCO3 concentration in groups 3, 4, 5, 6 and test controls, but there was a significant difference (P<0.05) between HCO3 concentration in control groups and groups 7 and 8 .

3.5 Effect of treatment on urea and creatinine concentration

Figure 5 shows the effect of treatment on BUN and creatinine concentrations. The results show that there was no significant difference (p > 0.05) between normal and test controls in urea concentrations. However, when the same concentration of Cd and Mg was applied with 1%, 2%, 3%, 4%, 5% and 6%, there was a significant increase (P<0.05) in urea concentration. The increase depended on the increase in alcohol concentration. The same pattern was observed with creatinine concentration in the same treatments.

3.6 Effect of treatment on the concentration of glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), catalase and glucose-6-phosphate dehydrogenase (G-6-PDH)

Figure 6 shows the effect of treatment on GSH, MDA, SOD, catalase and G-6-PDH concentrations. The results show that there was a significant decrease (p < 0.05) in the concentration of GSH in the test control compared to the normal control. However, when the same concentration of Cd and Mg of 1%, 2%, 3%, 4%, 5% and 6% was applied, there was a significant decrease (P<0.05) which was progressive with increasing Cd concentration. . The same pattern was observed with the concentration of SOD, catalase and G-6-PDH. On the other hand, there was a significant increase (p < 0.05) in the concentration of MDA in the test control compared to the normal control. As the same concentration of Cd and Mg was applied with 1%, 2%, 3%, 4%, 5% and 6%, there was a significant increase (P<0.05) which was progressive with increasing alcohol concentration

3.7 Effect of simultaneous application of cadmium, magnesium and graded concentrations of alcohol on the integrity of liver and kidney cells

Panels 1-10 show the results of histopathological examinations of the liver and kidney of rats treated with Cd and Mg with added graded concentrations of alcohol. Panel 1 is a section of the liver of control rats showing the normal arrangement of hepatocytes radiating from the central vein and separated by sinusoids containing Kupffer cells. They are regular and contain a large spheroidal nucleus with a sharply defined nucleolus and a peripheral distribution of chromatin. Some cells have two nuclei each. Panel 2 is a section of kidneys from normal control mice showing normal kidney morphology. The tubules are intact with podocytes and mesangial cells in the cortex.

The results of the histopathological analysis of the liver of the studied groups show that there are several changes, such as hepatocyte hypertrophy, disorganization of the liver cords, chronic portal inflammation with mild periportal necrosis, chronic lobular inflammation with some focal necrosis, noticeable lymphocytic infiltrates, portal inflammation with periportal necrosis, severe cellular infiltration, chronic lobular inflammation with punctate necrosis and sinusoidal enlargement as seen in panels 3, 4, 5 and 6.

The results of histopathological analysis of the kidneys of the examined groups show that there are several changes, such as normal macroscopic morphology with focal tubular necrosis, severe degeneration of glomerular contents and severe epithelial necrosis, focal tuberitis with necrosis, tubular epithelial cell degeneration and necrosis with infiltration of inflammatory cells, glomerular inflammation with focal hyalinization, rapidly progressive proliferation of mesangium, neutrophilic infiltrates, degeneration of glomerular content and glomerular enlargement due to proliferation and swelling of mesangial cells are evident as in plaques 7, 8, 9 and 10.

4. Discussion

4.1 Effect on the liver

Our results showed that there were significantly higher concentrations (P<0.05) of total protein and albumin in the test control compared to the normal control. On the other hand, there were significantly lower concentrations (P<0.05) for the tested groups compared to the normal control. The decrease was progressive as the alcohol concentration increased. A low serum protein concentration in the whole group may indicate liver damage, since the liver is known to be the main site of plasma protein synthesis, mainly albumin [29], or an altered nutritional status of the animal [30]. Studies have shown that liver injury can qualitatively and quantitatively affect the synthesis of hepatic plasma proteins [31]. Plasma albumin and total plasma protein loss may be due to hepatocellular dysfunction or liver disease [32].

The results also show a significantly higher concentration (P<0.05) of bilirubin and direct bilirubin in all tested groups compared to the control group. In this study, high concentrations of total bilirubin and unconjugated bilirubin indicate damage to the heme molecule. This is indicative of liver dysfunction which may be due to oxidative damage to the liver [33].

The activity of the enzymes liver transaminases (AST and ALT) and alkaline phosphatase (ALP) in the serum are most often measured to diagnose liver diseases, especially hepatitis infections, alcoholic cirrhosis, biliary obstruction and hepatocellular carcinoma. The liver enzymes, AST and ALT, are considered important biomarkers of liver damage and are therefore used in the detection of cadmium hepatotoxicity. The results show that there were significantly higher concentrations (P<0.05) of ALP, AST and ALT compared to the normal control. ALP, AST and ALT concentrations increased with increasing alcohol concentration. Leakage of AST and ALT from the liver cytosol into the bloodstream increases their plasma concentration in the studied groups, which could mean liver damage. Aminotransferases (AST and ALT) are enzymes that are mainly found in the liver. ALT is stored only in the cytoplasm, while AST is found both in the cytosol and in the mitochondria of hepatocytes [34]. A similar cause that led to a slight increase in ALP concentration could lead to its leakage into the circulation, which could be a consequence of the destruction of the structural integrity of the liver [35].

The results also show that there was a significant increase (P<0.05) in LDH levels in the test groups compared to normal controls. There was no significant difference between the control and test groups. Increasing alcohol concentration in the tested groups caused a progressive increase in LDH levels in rats. LDH is an intracellular enzyme. When serum LDH levels are elevated, it indicates hepatocellular damage [36]. From this study, this parameter appears to be the best tool to explain the dose-dependent effect of alcohol on liver damage. Analysis of LDH levels from liver homogenates can be considered a good biomarker for the diagnosis of liver diseases and cancer. The dose-dependent effect of alcohol on liver damage was clearly demonstrated by LDH levels in the studied groups. Increasing alcohol concentration caused a progressive increase in LDH levels in rats.

Examination of the liver by histopathological analysis reveals several changes, including enlargement of hepatocytes and rupture of hepatic cords, mainly in the higher alcohol concentration groups. Swelling of intracellular organelles, especially mitochondria and endoplasmic reticulum, leads to the observed enlargement of liver cells. cellular vacuolization is a reaction of cellular defense mechanisms against toxic substances harmful to the liver. These harmful substances were aggregated and prevented the vacuoles from interfering with cell metabolism. Disturbance of lipid infiltrates and fat metabolism is also a cause of cytoplasmic vacuolization. The infiltration of lymphocyte cells observed in this study in the investigated groups shows evidence of cellular irritability, inflammation and hypersensitivity to the toxin used. The presence of lymphocytic infiltrate and sinusoidal blood congestion in groups that received higher doses of alcohol indicates significant liver damage after treatment.

4.2 Effect on the kidneys

In this study, treatments were observed to significantly (p<0.05) increase creatinine and chloride ion levels in a dose-dependent manner compared to control. There was a significantly higher (P<0.05) increase in urea levels in the tested groups compared to the normal control. The increase in alcohol concentration caused a progressive and highly significant (P<0.05) increase in urea levels in the tested groups. These changes indicate that, even at lower alcohol concentrations, renal excretory function can be impaired in the presence of Cd. Ingestion of cadmium through drinking water or through the food chain leads to its accumulation in the kidneys and liver of individuals [37,38]. BUN concentration increased significantly in all tested groups compared to controls, as well as creatinine. Increasing alcohol concentration caused a progressive and highly significant (P<0.05) increase in creatinine levels in rats.

Changes in urea and creatinine levels can be an indicator of impairment of renal excretory function by cadmium, even at the lowest alcohol concentration. The kidneys transport and eliminate creatinine as a chemical waste through the bloodstream. Its inability to filter creatinine leads to its elevation in the blood. This always means that damage to the kidney organ leads to an increase in the level of creatinine in the blood [39].

The study also shows that there is a significantly high increase (P<0.05) in Na+ concentration in the test groups compared to the control, with emphasis on the last two highest alcohol concentrations. In the tested groups, significantly lower concentrations (P<0.05) of HCO3 were recorded in the lowest concentrations of alcohol, but there was a significantly greater increase in the concentration of HCO3 in the groups treated with the highest concentrations of alcohol compared to the control. The impaired excretory function is also supported by the fact that the levels of some serum ions (Na, K, HCO3- and Cl) in the blood and kidneys have changed. The finding of serum electrolytes indicated a possible impairment of renal function. The pronounced effect on the concentration of HCO3- in the test groups that had the highest concentration of alcohol points to the degree of kidney damage. A decrease in potassium ions, which indicates renal failure, is important in assessing kidney integrity [40]. The decrease in potassium ion concentration, along with the increase in chloride ion concentration, supports the idea that chloride ion acts as a counterion to potassium (or sodium ion), which is consistent with previous studies. [40]. These observations may also be due to the fact that changes in membrane structure can affect water balance and ion flow and all processes within the cell [42]. Abnormalities or changes in specific membrane constituents can result in various diseases, indicating that the proper functioning of cells depends on the integrity of their membranes.

A significant (P < 0.05) increase in ALP activity in the test groups in this study may also indicate kidney damage. Increased ALP activity can lead to random removal of membrane phosphate esters, leading to depletion of energy-rich phosphate compounds, which can compromise organ or cell integrity, leading to cell death. The integrity of the cell plasma membrane can be determined by assessing ALP. [43].

The above phenomenon is additionally supported by pathological changes in the kidney ultrastructure (damaged brush-edge microvilli and swollen mitochondria in proximal convoluted tubular cells) and necrosis observed in the examined groups with higher alcohol concentrations. Kidney damage became more pronounced with increasing alcohol concentration. Histopathology revealed signs of tubular necrosis, interstitial fibrosis and hypertrophy of glomerular epithelial cells in small areas of the renal cortex. The observed effect of alcohol on Cd nephrotoxicity can be stated as follows; as the concentration of alcohol increases, the nephrotoxicity of Cd increases.

4.3 Redox state

Cadmium treatment had a negative effect on all investigated parameters of oxidative stress. These observed changes agree with evidence from previous studies that cadmium toxicity is mediated by reduction of antioxidant enzymes, production of reactive oxygen species, and lipid peroxidation [44]. The occurrence of lipid peroxidation in the liver and kidneys increased in treated rats as evidenced by significantly elevated values ​​(P<0.05) in MDA levels for all groups compared to control. Lipid peroxidation is the main cause of cadmium-induced hepatotoxicity, and it is a consequence of the depletion of compounds containing non-protein sulfhydryl compounds. Lipids interact with reactive oxygen species leading to peroxidative changes resulting in increased lipid peroxidation. A significant increase (p < 0.05) in lipid peroxidation with increasing alcohol concentration in treatments may be an indicator of a decrease in non-enzymatic antioxidants of defense mechanisms.

Cellular and organic damage caused by cadmium and alcohol is strongly associated with oxidative stress [45]. Disorientation of the liver is caused by these substances by changing the activity of the enzymes superoxide dismutase, catalase and glutathione (GSH) in the liver tissue. The activities of antioxidant enzymes CAT, SOD, GSH and G6PDH in groups of rats were reduced in the study revealing liver and kidney damage. SOD activity decreased with progressive increase in alcohol concentration in rat organs. Reduced SOD activity due to increased alcohol concentrations in the treatments may be responsible for the accumulation of O-, H2O2 or their degradation products [45].

GSH activity decreased significantly (p < 0.05) between the GSH concentration of the normal control and the test control group. After the addition of Cd, Mg and graded concentrations of alcohol in the tested groups, the level of GSH significantly decreased with increasing alcohol concentration. The decrease in the level of antioxidant enzymes in the studied groups could be the result of an increase in lipid peroxidation with an increase in alcohol concentration. Dinu et al. [46] reported that the enzymes responsible for the recycling and utilization of glutathione in the kidneys become more active when exposed to a higher amount of alcohol, resulting in reduced levels of GSH in the tissues of the experimental groups [47]. The use of alcohol and cadmium causes lipid peroxidation and depletes GSH stores. The synthesis of reactive oxygen intermediates during alcohol metabolism leads to the oxidation of GSH and, as a result, reduces its levels [48]. He reports that when tissue Cd levels exceed a concentration of about 150 μg/g, the intracellular defense mechanisms of cells, metallothionein (MT) and glutathione (GSH), are overwhelmed, leading to cell damage and death [19,20] .

The observed decrease in GSH levels in response to increasing alcohol concentration correlates with increased glutathione transferase activity. An increased involvement of GSH in the conjugation process, which is required as a result of increased GSH activity, appears to be a plausible explanation for decreased GSH levels as a result of alcohol consumption. Oxidative stress leads to the activation of increased GSH activity [49]. Then there is a depletion of catalase in the blood, which is consistent with the accumulation of cadmium in the blood and tissues due to increased absorption of cadmium by alcohol and increased production of reactive oxygen species (ROS).

Superoxide radical, hydroxyl radical and nitric oxide radicals are generated indirectly through the Fenton reaction of non-radical hydrogen peroxide during cadmium toxicity, unlike other heavy metals [50]. Iron and copper in various cytoplasmic and membrane proteins, such as ferritin, are replaced by cadmium, which leads to the release and increased concentration of unbound iron or copper ions. These unbound ions are involved in oxidative stress through Fenton reactions [51]. Superoxide dismutase (SOD) is an important enzymatic antioxidant, which breaks down O2.− and catalase, and the GSH redox system, which alleviates the formation of H2O2. Manganese SOD (found in mitochondria), Cu-Zn SOD (found in cytoplasm), and extracellular SOD (which lines blood vessels) are three important forms of SOD. Also important is GSH, a low molecular weight water-soluble tripeptide (L-γ-glutamyl-L-cysteinyl glycine) present in high concentrations in every cell. It is also present extracellularly, and is especially abundant in the fluids of the lung epithelium [52]. The antioxidant, GSH, generates oxidized glutathione (GSSG) as an intermolecular disulfide non-radical end product. GSH is also a cofactor for several enzymes that reduce oxidative stress [53]. Always NADPH donates electrons to the reductase reaction for GSSG export from the cell or conversion to GSH. The abundant presence of GSH in the liver makes it the first line of defense against Cd hepatotoxicity, since Cd binds tightly to thiol groups, and depletion of hepatic GSH by diethyl maleate significantly enhances cadmium-induced hepatotoxicity [54]. Depletion of hepatic GSH by diethyl maleate leads to increased signals from bile cadmium-generated radical adducts, suggesting that disruption of the cellular GSH system is a key element for cadmium-induced oxidative stress in the liver [55].

Glucose-6-phosphate dehydrogenase (G6PDH) is an important enzyme in the pentose phosphate pathway (PPP) and plays a key role in the response to oxidative stress, generating the major intracellular reductant, nicotinamide adenine dinucleotide phosphate (NADPH). From this study, G-6-PDH liver function profiles of mice show significantly lower values ​​(P<0.05) as alcohol concentration was increased in the test groups compared to controls. Increasing alcohol concentration caused a progressive and highly significant (P<0.05) decrease in G-6-PDH levels in rats. Our result also showed that progressively lower levels of G-6-PDH in the test groups increased MDA levels than the control status of G-6-PDH. Since MDA is a byproduct of lipid peroxidation, this could mean increased lipid peroxidation in the low G-6-PDH groups. Protection of the body from the harmful effects of free radicals using endogenous and exogenous antioxidants leads to maintenance of redox balance [56]. In the plasma of rats, the use of G-6-PDH to neutralize the cause of oxidative stress leads to a decrease in its levels. Prolonged exposure and influence of free radicals, even at low levels, can cause damage to biologically important molecules and potentially lead to tissue damage and disease [56].

5. Conclusions

The results of this research show that the higher the concentration of alcohol administered with a constant concentration of Cd and Mg, the higher the concentrations of biomarkers of liver and kidney damage in the serum of rats. The results also show that there was a decrease in the concentration of oxidative stress biomarkers and an increase in oxidative stress marker byproducts. This result suggests that populations living in cadmium-contaminated water and soil and alcoholics are at increased risk of cadmium-induced hepatotoxicity and nephrotoxicity, and a wide range of diseases induced by the byproducts of oxidative stress. This may have been the case for the residents of our study area, where outbreaks of strange diseases killed mostly alcoholics in the community..

declarations

Ethical approval and consent to participate

All experimental procedures were approved by the Ethics Committee of the Laboratory Animal Unit of the University of Jos, Faculty of Pharmaceutical Sciences, reference number: F17-00379, in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85- 23, revised 1996).

Consent to publish

Not applicable

Availability of data and materials

Ogham CJ, Dabak JD and Jaryum KH. 2023. Physicochemical and metal analysis of Bar Village well water and the effect of simultaneous application of cadmium, magnesium and alcohol on some organs. Unpublished PhD Thesis, University of Jos, Plateau State, Nigeria, 148-149. https://doi.org/10.21203/rs.3.rs-1738108/v1

Competing interests

The authors declare that they have no conflicting interests.

Financing

This research received no specific grants from any funding agency in the public, commercial or non-profit sector.

Author's contribution

CO designed, investigated and funded the study; CO also wrote the manuscript; JD designed, supervised and made substantial contributions to all sections of the paper to improve the quality of the manuscript; KJ Supervised and participated in the literature search. All authors read and approved the final manuscript.

Recognitions

The authors posthumously thank the deceased for their support. Engr. A. Marafi, former Director, Ministry of Water Resources and Rural Development, Bauchi State, for his administrative support. The participation of Mr. Istifanus Gurumtet in various stages of this study cannot be left out.

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