Abstract

Background: Astaxanthin, a marine-derived carotenoid, possesses potent antioxidant and anti-inflammatory properties with potential therapeutic effects in liver diseases. Specific Background: Diazinon, a widely used organophosphorus pesticide, induces hepatotoxicity through oxidative stress and inflammation. Knowledge Gap: Although astaxanthin’s antioxidant potential is recognized, its protective role against diazinon-induced liver injury remains underexplored. Aim: This study investigated the hepatoprotective effects of astaxanthin against diazinon-induced liver toxicity in male rats. Results: Thirty rats were divided into five groups and treated with diazinon alone or in combination with astaxanthin (50 mg/kg and 100 mg/kg), vitamin C, or distilled water for 30 days. Diazinon exposure significantly elevated serum ALT, AST, and IL-1β levels, reduced hepatic GPx activity, and caused histopathological liver damage. Astaxanthin pre-treatment, particularly at 100 mg/kg, significantly attenuated these effects by normalizing enzyme levels, reducing inflammatory markers, enhancing antioxidant activity, and preserving liver histology. Novelty: This study is among the first to demonstrate astaxanthin’s dose-dependent hepatoprotective effect against organophosphate-induced toxicity. Implications: Astaxanthin shows promise as a therapeutic agent for mitigating liver damage caused by environmental toxins, warranting further clinical and mechanistic investigations.

Highlights:

1. Astaxanthin lowers AST, ALT, IL-1β in treated rats.

2. Liver histology improved with astaxanthin pre-treatment.

3. Shows promise as hepatoprotective agent against diazinon toxicity.

Keywords: Diazinon, Astaxanthin, vitamin C, Antioxidants, hepatotoxicity

Introduction

Oxidative stress, which results from an imbalance between the concentration of reactive oxygen species and the antioxidant defense system, is thought to be the cause of several illnesses, including cardiovascular, inflammatory disorders, and Hepatotoxicity.[1] a primary concern in the field of toxicology, refers to liver damage often resulting from environmental pollutants such as Diazinon.

Diazinon (O, O-diethyl O-[2-isopropyl-6-methyl-4-pyrimidinyl] phosphonothioate) is an organophosphate pesticide extensively utilized in agriculture to control various pests. Its main toxicity mechanism is the inhibition of acetylcholinesterase, leading to the buildup of acetylcholine at nerve synapses, causing neurotoxicity in both insects and mammals[2]. However, Diazinon is also known to induce hepatotoxicity via many interrelated mechanisms. It generates oxidative stress by increasing reactive oxygen species (ROS) and lipid peroxidation, leading to membrane damage and hepatocyte dysfunction. It disrupts mitochondrial function, impairing membrane permeability and the electron transport chain, which results in ATP depletion and triggers apoptosis in liver cells. Diazinon interferes with cytochrome P450 enzymes, disturbing the liver's detoxification processes and leading to the bioactivation of toxic metabolites. Diazinon alters lipid metabolism by disturbing the phospholipid bilayers of hepatocyte membranes, compromising cell integrity and function. [3]

Antioxidants are molecules that prevent oxidative damage to cells and tissues by donating electrons to free radicals. Antioxidants are divided into two categories: enzymatic and non-enzymatic. Glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) are examples of enzymatic antioxidants that function by catalyzing reactions that neutralize free radicals.[4] Vitamins C and E, glutathione (GSH), flavonoids, and polyphenols are examples of non-enzymatic antioxidants that either directly scavenge free radicals or enhance cellular antioxidant responses. [5]Astaxanthin is a natural carotenoid that has strong antioxidant properties, surpassing more conventional antioxidants like vitamin E and β-carotene. It is primarily found in marine organisms like the microalga Haematococcus pluvialis and seafood such as salmon and shrimp[6]. Astaxanthin is widely used in medicine and nutrition due to its various health benefits, including cardiovascular and brain health support, immune system enhancement, liver protection against oxidative damage, prevents lipid peroxidation, and regulates autophagy and apoptosis[7]. This research aims to examine the hepatoprotective effects of Astaxanthin against Diazinon-induced hepatotoxicity by assessing liver function indicators, inflammatory cytokines, and oxidative stress markers.

Methods

Experimental Animals

Thirty mature male albino rats 2–3 months of age (weighted average 250–350 gm) participated in the experiment. Iraq's breeding research in Baghdad provided the animals. The rats were housed in plastic cages with wood shavings, and Pellet and vegetable food and an enough supply of tap water for drinking were provided for the rats. Appropriate conditions were maintained for them, with humidity and temperature regulated at 25 ± 3 degrees Celsius. Each of the boxes contained four rats. The rats were exposed to 12 hours of darkness and 12 hours of normal light. After two weeks and before starting the experiment and giving any treatments, the rats' weights were noted using a sensitive electronic balance. All procedures were authorized by the Pharmacy College of the University of Basra's Ethical Committee (EC 40) in October 2023.

The chemicals and materials used in these studies were Diazinon 10% EC liquid, purchased from Endimaj for the specialized chemical and pharmaceutical industry, CO., Jordan. The astaxanthin soft gelatin capsule 12mg was acquired from Ventura, Turkey. Vitamin C tablet 1000 mg was from Nutritional L.L.C., USA. Formaldehyde came from Loba Chemie PVT. LTD. India. Chloroform was from Thomas Baker, India.

Experiment design and animal treatment protocol

Five groups were formed, each consisting of six rats. The modified dosage of the medication was given first to animals. Followed by the appropriate dose of toxin for each group by intraoral gavage for 30 days.

Group 1: Administer diazinon 20 mg/kg with an equal amount of distilled water as a vehicle to induce toxicity.

Group 2: Treatment with astaxanthin (50 mg/kg) before giving diazinon (20 mg/kg).

Group 3: Treatment with astaxanthin (100 mg/kg) before giving diazinon (20 mg/kg).

Group 4: Treatment with Vitamin C (100 mg/kg) before giving diazinon (20 mg/kg).

Group 5: Administration of distilled water (D.W.) as a vehicle in a quantity equivalent to that which was used to dilute the agents given to the remaining groups, as the control group.

After the trial ended, the weights of rats in all groups were noted the next day. Rats were put in a tight container with a piece of cotton soaked in chloroform, an anesthetic, to put them to sleep and end their lives. After the rats' anesthesia was verified, they were removed from the container, their chests were opened, their livers were removed for histological examination, and blood was drawn for biochemical analysis.

Estimation of body weight and organs

While the experiment was underway, the weights of animals in all groups involved were logged weekly, before and after giving any agents. The relative weight of livers was determined by utilizing the equation (organ weight/animal weight) * 100.

Blood Sampling collection

After the rats' chests were opened, blood was drawn from the vena cava by syringe (G23) and carefully into a tube for biochemical examination after centrifugation at 3000 rpm for 10 min. Then, the serum was divided into a small amount in Eppendorf using a micropipette stored in the refrigerator.

Tissue sampling collection (homogenate)

In the operative extract way, the 4 livers were taken, washed with phosphate-buffered saline (PBS PH 7.4), and dried on qualitative filter paper, then weighed in a sensitive electronic balance. Then, they were crushed by adding 1:9 BPS (each 1 gm of tissue added 9 ml of BPS). By using a glass tissue grinder. Then, it was centrifuged at 5000 rpm for 5 minutes to assemble the supernatant in Eppendorf tubes and stored at -20 C for IL1B and GPX tests.

Histopathological preparation

For histopathological examination, liver tissue was collected from two rats per experimental group. Following euthanasia, the livers were excised, rinsed in physiological saline to remove blood, blotted dry, and weighed. Subsequently, tissue samples were fixed in 10% neutral buffered formalin solution and stored at room temperature for 3 to 5 days for histopathological analysis.

Biochemical examination

Estimation of Hepatic enzymes

Estimation of serum aspartate aminotransferase (AST)

A spectrophotometric method (Abbott Architect i1000SR Immunoassay System) was used to measure (AST) levels for all groups of animals by using a previously prepared sample. [8]

Estimation of serum alanine aminotransferase (ALT)

A spectrophotometric method (Abbott Architect i1000SR Immunoassay System) was used to measure (ALT) levels for all groups of animals by using a previously prepared sample. [8]

Estimation of tissue glutathione peroxidase levels (GPX)

Estimation of GPX level in liver tissue homogenizer sample of rats by enzyme-linked immunosorbent assay ELISA kit for all groups of animals. [9]

Estimation of tissue interleukin -1B levels (IL1B)

Estimation of IL-1B level in liver tissue homogenizer sample of rats by enzyme-linked immunosorbent assay ELISA kit for all groups of animals. [10]

Histopathological assessment

The experiment involved preparing microscope glass slides for histopathological study. Livers were preserved in 10% formalin, then alcohol was used to remove tissue water. Paraffin was embedded in molds, hardened, and microtome to 3-5mm thickness. Staining was used to color specimens, and Photomicrography at different objectives (10,40 X) for our study.[11]

Statistical analysis

The results were presented by using the mean ±standard error of the mean, comparing the result between treated and control groups by using one-way ANOVA analysis

and Tukey post hoc. The difference in value is considered significant at a P value less than 0.05. using Graph Pad Prism software version 10.2.3 to represent all data analysis. [12]

Result and Discussion

Results

1- Effect of toxin and treatment on relative liver weight

In our experiment, the relative liver weight had a statistically insignificant difference (p=0.74) between all groups.

Figure 1.Comparison of mean ± SEM relative liver weight among the different experimental groups shows the effect of Astaxanthin and vitamin C following diazinon administration.

D.W: Distill water group (negative control group) /Diazinon: inducer group (positive control group)/Vit. C: Vitamin C group (comparison group)/Astax. 50 mg: Astaxanthin 50 mg group (treatment group 1)/Astax. 100mg: Astaxanthin 100 mg group (treatment group 2)

Hepatic effect

1. Effect of Treatments and toxin on serum AST levels

The important liver enzyme is Aspartate aminotransferase; if AST is elevated in the bloodstream, this means liver damage. In this experiment, there was a statistically significant difference in the level of AST between all groups. According to the figure (2) below, the diazinon group has a highly elevated level of AST compared to all the other groups. Also, there was a statistically significant difference between diazinon and the control groups. The level of AST was decreased in treatment groups, and the best result was from the astaxanthin 50mg group. The statistical difference between the astaxanthin and the diazinon groups was significant, while the difference between the Vit C and the diazinon groups was considered insignificant.

2. Effect of Treatments and toxins on serum ALT levels

Alanine aminotransferase (ALT), a crucial liver enzyme, if it is increased in the blood. This is indicative of liver injury. Between all groups, there was a statistically significant difference in the level of ALT. Compared to the control group, a statistically significant difference relationship with the diazinon group, which had a highly elevated level of ALT. Treatment groups had a drop in ALT levels, with astaxanthin 50 mg producing the highest results. There was a statistically significant difference between the diazinon group and 2 groups of astaxanthin, while the difference between the Vit c and diazinon groups was deemed insignificant. Figure (3)

3- Effect of Treatments and toxin on tissue GPX level

Glutathione peroxidase is an essential antioxidant enzyme that protects cells from oxidative damage. Figure 4 showed no significant difference in GPX levels among all groups. The diazinon group showed a statistically insignificant decrease in GPX levels compared to the control group. All treatment groups showed a statistically insignificant increase in GPX levels compared to the diazinon group. The best result was from the 100 mg astaxanthin group.

4- Effect of Treatments and toxins on hepatic tissue IL-1B level

Interleukin-1 beta is a pro-inflammatory cytokine that is produced from monocytes and macrophages in several inflammatory diseases or stressors. Statistically significant relationship between all groups in the experiment. Diazinon significantly increased IL1B levels compared to the control group. Vitamin C and astaxanthin 100 mg groups had statistically significant reduced IL1B levels compared to the Diazinon group. Figure (5)

Figure 2.The level of each parameter in different experimental groups shows how astaxanthin and vitamin C affect the following diazinon administration.

* Represent significant difference between groups p < 0.05

1. Effect of Treatments and toxins on hepatic histopathology

In the negative control group (D.W) liver was within normal limits, and the central areas showed normal central vein and normal architecture and orientation of hepatocytes around the central vein, the portal area consists of normal components of the portal triad which are the portal vein, hepatic artery, and bile duct, figure (6). In the induction diazinon group, hepatocytes show massive vacuolar degeneration in both the centrilobular and periportal area figure (7). Comparison of the treated groups with the induction group shows that there were variable degrees of hepatocellular protection; the best results were obtained in the Astaxanthin (100 mg), in which the hepatocytes were normal in both centrilobular and periportal area, figures (10). Less improvement was seen in the Vit c group figure (8); a large number of hepatocytes in the field at 40X magnification showed vacuolation while the rest were normal. Then the astaxanthin (50mg) figure (9) group shows moderate vacoulation of hepatocytes, and the remainder was typical.

Figure 3.Liver section of negative control (D.W) group shows normal hepatocytes in both periportal area (black arrow) and centrilobular area (green arrow), normal central vein (blue arrow), normal portal triad (yellow circle) that include portal vein, hepatic artery and bile duct in the porta area. H&E A) 10X B) 40X

Figure 4.Liver section of induction diazinon group shows massive vacuolation of hepatocytes in the centrilobular area (black arrow) with swelling. H&E A) 10X B) 40X

Figure 5.Liver section of Vit C treated group shows massive vacoulation of some hepatocytes in the centrilobular area (black arrow) with swelling, other normal hepatocytes (blue arrows). H&E A) 10X B) 40X

Figure 6.Liver section of Astaxanthin (50 mg) treated group shows moderate vacoulation of hepatocytes in the centrilobular area (black arrow) normal hepatocytes in the same site (blue arrow). H&E A) 10X, B) 40X

Figure 7.Liver section of Astaxanthin (100 mg) treated group shows normal hepatocytes in centrilobular area (black arrow), normal central vein (blue arrow). H&E A) 10X B) 40X

Discussion

Diazinon induced hepatotoxicity via its ability to cause inflammation and oxidative stress. In our study, we tested the effect of astaxanthin as a hepatoprotective against diazinon-induced hepatotoxicity in thirty laboratory rats; six rats were divided into each of the five groups. the first group took diazinon by oral route, the second and third groups took varying doses of astaxanthin with diazinon, the fourth group received vitamin C with diazinon, and the last group received only distilled water for 30 days. The body weights and vital signs were monitored throughout the experimental period for all rats. The laboratory results show diazinon induces hepatotoxicity by increasing liver enzymes that are alanine aminotransferase (ALT) and aspartate aminotransferase (AST), interleukin-1B(IL-1B) is an inflammatory marker that also increased by the effect of diazinon toxicity[13], while the antioxidant enzyme glutathione peroxidase (GPX) was decreased[14]. Hepatotoxicity was clear in the pathological study through histological alteration that occurred in the liver[15]. administration of astaxanthin and vitamin C effectively decreases hepatotoxicity by improving histological and biochemical parameters. Astaxanthin is a naturally occurring carotenoid pigment. It is a potent antioxidant substance that is found in several marine organisms like shrimp, salmon, and microalgae.[16] It works as an antioxidant that helps to reduce inflammation and oxidative damage that results is due to the formation of free radicals.[17] Astaxanthin shows a more hepatoprotective effect than vitamin C, as was clear through the results of liver weight and other laboratory parameters.

Liver enzymes AST and ALT are normally found in liver cells involved in amino acid metabolism. When liver injury happens, these enzymes are released into blood bloodstream. The results we obtained show an elevated level of AST and ALT in the diazinon group due to suppression of acetylcholinesterase (AChE), raising the sympathetic nervous system activity and inducing oxidative stress, causing the formation of reactive oxygen species (ROS) that lead to nitric oxide reaction, lipid peroxidation, and mitochondrial damage, leakage enzymes to blood circulation due to membrane permeability damage[18][19]. Astaxanthin decreases levels of AST and ALT in treatment groups by its action as a hepatoprotective, which decreases ROS by scavenging free radicals. astaxanthin also stimulates superoxide dismutase (SOD) which have important protective role against oxidative stress. [14]Reducing lipid peroxidation is considered the effective role of astaxanthin in protecting mitochondria and increasing cellular membrane stability. [20] Interleukin 1B (IL-1B) is a pro-inflammatory cytokine that is increased in the diazinon group and contributes to the inflammatory response, causing liver injury. nuclear factor kappa (NF-KB) is a transcription factor involved in inflammatory cytokines, which is activated by ROS, that also stimulates NLRP3 inflammasome to stimulate mitogen-activated protein kinase (MAPKs) and Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways. This stimulation elicits an immune response and liver tissue damage[21]. Astaxanthin modulates immune response, decreases IL-1B production by antioxidant effects through inhibition of NF-KB pathway and NLRP3 inflammasome. Together, these actions will protect liver tissue and reduce cellular damage[17]. Glutathione peroxidase (GPX) is an important antioxidant enzyme that has a crucial role in inhibiting oxidative damage and decreasing the level of ROS by converting it to water[9]. Results of our experiment show a decreased level of GPX in the diazinon group, which indicates DZN drained the GPX level to neutralize the high level of ROS. astaxanthin upholds the level of the GPX by regeneration and recycling of glutathione GSH, decreasing oxidative stress by scavenging ROS, positive effect on the rate-limiting enzyme glutamate cysteine ligase (GCL) in the synthesis pathway of GSH, causing an increase in production of antioxidant enzymes[18]. Astaxanthin also has an important role in inflammatory cytokine inhibition, such as tumor necrosis factor (TNF-), to preserve a normal level of GSH enzyme. Finally, it inhibited lipid peroxidation to prevent the use of GSH in this process[22]. A histopathological study of the DZN group shows massive hepatic tissue damage in both centrilobular and periportal areas, increasing in liver weight due to inflammatory cells infiltration and edema. While in the astaxanthin groups, hepatic cellular features are normal in both centrilobular and periportal areas, and also normal liver weight due to the activity of astaxanthin as an inflammatory inhibitor and hepatoprotective effect[23]. Conforming to previous studies[19].

Conclusion

The result of this experiment shows the hepatoprotective effect of astaxanthin against diazinon toxicity by effect on decreasing liver enzymes such as AST, ALT, and improving antioxidant defense glutathione peroxidase (GPX); on the other hand, decreasing the inflammatory response IL-1B. these results insinuate that astaxanthin activity as an anti-inflammatory and antioxidant may be a promising drug for organophosphorus insecticide poisoning that causes liver damage. Further experimental studies are justified to explore the exact action of astaxanthin and to assess its potential treatment applications in clinical environments. More research can also investigate the optimal dose of the drug to increase the beneficial protective effect on the liver.

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