Introduction

Mus musculus (laboratory mouse) is distributed worldwide and has developed into a cosmopolitan rodent species [1]. The laboratory mouse is descended from the house mouse, so its high reproductive rate and adaptability make it a rapidly growing population that can adapt to its environment [1, 2]. This laboratory mouse is often used as a model animal in toxicological and pharmacological research [2]. Herbicides are toxic agrochemicals. They are used to kill weeds in agricultural areas and gardens [3]. Atrazine is a well-known herbicide. It is used to control weeds in crops (asparagus, sorghum, pineapple, and sugar cane) on lawns and golf courses [4]. It is an odorless compound that comes in granular form. It is a synthetic, water-soluble, reactive, flammable, and non-volatile compound with low degradability. Different studies indicate that atrazine residues in surface waters and groundwater cause serious health problems for humans and animals [5-7] have linked serious health problems to atrazine exposure. [8] reported adverse effects of atrazine exposure on the hematology and biochemistry of snow trout. In humans, atrazine exposure can have adverse effects on the reproductive system, causing changes in blood hormones that impair reproductive function. It also causes the formation of tumors and can damage the liver, kidneys, and heart [9]. The results of various studies indicate that pesticides induce lipid peroxidation, which causes harmful biological effects in living organisms. It is known that the administration of atrazine is responsible for the formation of reactive oxygen species (ROS), which are excreted via the kidneys. This leads to atrophy of the glomerulus and vacuolization of the epithelial cells in the juxtamedullary renal tubules. This leads to a reduction in the excretion of creatinine and urea from the body, and the creatinine level in the blood rises [10-12, 13] reported that atrazine exposure at doses of 23, 90, and 360 mg/kg in female mice causes severe toxicity to hematologic parameters and liver and liver function tests with increasing atrazine dose increases. High doses of atrazine significantly reduce the number of lymphocytes, white blood cells, and monocytes. In female mice, it significantly increased the levels of blood urea nitrogen (BUN), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Plants are natural sources of therapeutic substances. They are used to promote health and fight different diseases. For centuries, herbal extracts have been used in several indigenous systems of medicine to treat diseases. In recent decades, people’s interest in the therapeutic use of natural products has increased as medicinal plants are considered safer compared to modern allopathic treatment [14, 15]. Herbal extracts are commonly used to cure various diseases, such as cancer, diabetes, asthma, and kidney diseases [16, 17].

N. sativa is a dicotyledonous herb and belongs to the family Ranunculaceae. It is native to North Africa, Southern Europe, and Southwest Asia [18, 19]. N. sativa is of great importance among Muslims, and it is considered an important remedy. The Prophet Muhammad (peace be upon him) referred to it as a panacea, meaning “universal healer”. It is considered a remedy for all diseases except death and the aging process [20]. In the past, several active compounds have been isolated, identified, and reported from different varieties of black cumin. Several studies have reported nutritional components of N. sativa seeds, such as vitamins, carbohydrates, minerals, fats, and proteins [21]. The most important active ingredient of N. sativa is thymoquinone [22]. Thymoquinone is the main constituent of N. sativa, which is thought to have a protective effect on hepatotoxicity and nephrotoxicity as well as reproductive toxicity. Thymoquinone has a strong antioxidant effect, which could protect organs from oxidative damage caused by free radicals. Some studies have shown that thymoquinone and carvacrol have remarkable free radical scavenging properties that prevent tissue damage in organs affected by oxidizing substances in males [23, 24]. [25] reported that in mice receiving oral Nigella sativa oil at a dose of 10 mL/kg for 5 days, hematologic parameters and liver function tests improved to normal values compared to X-ray-exposed mice. [26] revealed that mice receiving a hydroalcoholic extract of N. sativa seeds orally at doses of 50, 100, and 200 mg/kg for 2 weeks exhibited improved kidney function test parameters, including blood urea nitrogen and creatinine kinase, in male mice. Considering the efficient curative and remedial potential of N. sativa and the lack of knowledge on its curative and protective effects against atrazine (herbicide), this study aimed to determine the ameliorative potential of N. sativa against atrazine-induced liver, kidney, and hematological toxicity through biochemical analysis.

Material and Methods

Results

Comparison of Hematological Parameters Among

Discussion

Chemicals like pesticides, industrial chemicals, plastics, plasticizers, and pharmaceuticals are well-known endocrine disruptors and toxicants. These chemicals are present in our environment and have an adverse effect on human health [28]. The increased use of pesticides has introduced serious, novel hazards to humans and their livestock [29]. Atrazine is an herbicide. It is used to control or prevent broad-leaf weeds in crops such as corn, sugarcane, etc. In the present study, the atrazine-administered group showed a highly significant reduction in Hb, MCH, MCV, PCV, and RBC counts in the ATZ-treated group as compared with the control. Similarly, [30] reported that atrazine-treated rats at different doses (150 mg/kg, 200 mg/kg, and 300 mg/kg) for 28 days showed a significant (p˂ 0.05) reduction in PCV, RBC, and Hb levels. While [8] reported that atrazine-treated snow trout (at doses of 2-4 ppm) showed a significant reduction in RBC count, MCH, and MCHC, Nigella sativa alone treatment induced a significant increase in RBC, Hb, MCV, MCH, and PCV as compared with the control. These results are in line with [31], who reported that the oral treatment of rats with the fixed oil of N. sativa (1 mL/kg/day) for 14 days maintained normal levels of RBC, Hb, and PCV. While co-administration of N. sativa with atrazine administration led to an improvement in hematological parameters as compared with only atrazine-treated mice. These results are in line with [32], who investigated that oral administration of N. sativa (400 mg/kg) extract for 5 weeks showed protection against carbendazim-induced (150 mg/kg) hematologic, hepatic, and renal toxicities in rats. Similarly, [33] reported that pre-treatment of N. sativa oil (2 mL/kg) for five weeks can stimulate the production of erythropoietin and restore the mechanism of erythropoiesis in AlCl₃-treated rats. These beneficial effects of N. sativa oil are due to its antioxidant activity. In the present study, a significant increase in total leucocyte count was observed in both ATZ-treated groups. White blood cells play an important role in body defense mechanisms. Leukocytosis is a clear indication that the body is taking atrazine as a foreign chemical agent [30] has significantly higher (p < 0.001) concentrations of granulocytes (leukocytes, eosinophils, and neutrophils) and agranulocytes (monocytes) than in ATZ-treated mice. The present results are in agreement with [34], who reported that intraperitoneal injection of ATZ (72 mg/kg for 14 days) leads to a significant (p <0.05) increase in granulocyte and agranulocyte counts. In the present study, a high lymphocyte count was observed in atrazine-treated animals, which reflects the immunogenic abilities of atrazine. An increase in WBC, granulocytes, and agranulocytes was also found in the present study in the N.s treated group. Nigella sativa oil treatment may enhance the immune response. Similarly, [33] reported that rats administered with 2 mL/kg N. sativa for 5 weeks significantly (p <0.01) increased their WBC count as compared with the control group. The increase in WBC count treated with N. sativa oil alone is related to the increase in lymphocyte count, and that is confirmed by [35]. Lymphocyte count was significantly increased in mice treated with N. sativa oil alone as compared with the control and other groups of the study, and these results are consistent with [35]. Lymphocytes represent an adaptive immune response. The co-administrated (ATZ+N.s) group showed a significant (p < 0.05) increase in TLC, lymphocytes, and monocytes as compared with the ATZ exposed group, which showed that the effects of atrazine were reversed by N. sativa co-administration at the cellular immunity level. In several studies, it has been reported that atrazine induces hepatotoxicity, nephrotoxicity, and abnormal changes in hematological parameters [7, 36, 37]. A significantly (p ˂ 0.001) higher level of AST, ALT, and ALP was observed in the current study in ATZ groups as compared with the control group. These results are similar to the findings of [38], who reported that 50 mg/kg atrazine administration for 30 days caused a significant elevation in serum concentrations of AST and ALT in rats. These liver enzymes are important in assessing liver injury [39]. Atrazine causes the production of reactive oxygen species that are responsible for the oxidative stress of various organs. Oxidative stress causes lipid peroxidation, which results in hepatic injury [38]. Cellular damage may release these enzymes into circulation. Alanine aminotransferase is considered a more specific enzyme for hepatic injury. It is mainly found in the cytosol of the liver, with low concentrations elsewhere [40]. Hyperbilirubinemia was observed in the present study due to atrazine exposure in both atrazine-administered groups, which supports the above results of hepatic injury. In the present study, N. sativa oil alone treatment did not show any notable effect on serum concentrations of AST, ALT, ALP, and bilirubin in N.s and ATZ+N.s groups as compared to the control. Therapy with N. sativa oil causes a significant reduction in serum concentrations of liver enzymes (AST, ALT, ALP, and bilirubin) in the co-administered N. sativa group (ATZ+N.s) as compared with the ATZ group. These results were in agreement with [41], who reported that N. sativa oil therapy (1 mg/kg/day for 30 days) reduced the serum liver enzyme concentration of tramadol-treated rats. [42] reported that thymoquinone (an important constituent of N. sativa oil) can antagonize lipid peroxidation and counterpoise the integrity of the cellular membranes of hepatic cells, which causes a reduction in the leakage of liver enzymes. Proteins are the most essential and copious macromolecules in living organisms and play a chief role in the structure and function of the cell and cellular metabolism [43]. In the present study, administration of atrazine caused hypoalbuminemia in the atrazine group as compared with the untreated group. Serum total protein was also significantly reduced in the ATZ group. A decline in total protein content can be considered an index of cellular dysfunction. [44] reported a reduction in serum total proteins in atrazine at doses of 0, 60, 150, and 300 mg administered to male albino rats for 30 days. A reduction in serum total proteins may be due to changes in enzyme activities and impaired protein synthesis by the liver through the production of ROS during the metabolism of cypermethrin. In the present study, N. sativa oil alone treatment (N.s group) did not show any notable effect on serum total protein or albumin concentration as compared with the control. Results of the current study revealed that N. sativa oil treatment in the co-administered group may induce restoration of the serum total protein and albumin levels, which may cause a significant rise in albumin and serum protein concentration in the ATZ+N.s group as compared with the ATZ group. These results indicate an improvement in the repair mechanisms of the liver and kidney. The present results were consistent with the findings of [45]. They reported the beneficial effect of N. sativa on the serum albumin level after infection with Schistosoma mansoni in mice. While [46] reported an increase in serum protein concentration by the use of N. sativa against paracetamol toxicity in male mice. A high concentration of urea and creatinine was observed in ATZ groups as compared with untreated animals. These results are in agreement with the previous findings of [47]. Their findings suggested that atrazine administration in rats causes an increase in urea and creatinine concentrations. Atrazine administration causes the generation of reactive oxygen species, which are eliminated through the kidney. It caused atrophy of the glomerulus and the induction of vacuolation in the epithelial cells of the juxtamedullary renal tubules. It leads to a decrease in creatinine and urea clearance from the body, due to which creatinine and urea levels rise in the blood [10, 48]. The serum concentrations of urea and creatinine remain unchanged in the N.s treated group as compared with the control. Administration of N. sativa oil in the co-administrated group induced a significant reduction in the concentration of urea and creatinine in the serum of the ATZ+N.s group as compared with the ATZ group. Several studies on N. sativa in various models of oxidative stress revealed that most of its pharmacological effects are due to its antioxidant potential. It can scavenge free radicals and ROS. It also inhibits oxidative enzymes, such as cytochrome P450, and prevents lipid peroxidation [49-51] also reported that N. sativa oil prevented lipid peroxidation in liposomes and acted as a scavenger of free radicals. In the current study, presumably, the antioxidant action of the N. sativa oil prevents the oxidative damage of renal tissues.

Fig. 1.

(A) Mean value of AST and ALT in serum of control, N.s, ATZ and ATZ+N.s groups. (B) Concentration of ALP in control and other treated groups. (C) Mean serum concentration of Albumin in control and other treated groups. (D) Mean serum concentration of total protein in control and other treated groups. a=Control vs. treated groups, b= N.s vs. treated groups, c= ATZ vs. ATZ+N.s treated group. *p ˂ 0.05, **p ˂ 0.01, ***p ˂ 0.001. Values are expressed as Mean ± SEM

Fig. 2.

(A) Mean concentration of serum bilirubin in control and treated male mice. (B) Effect of atrazine alone or in combination with N. sativa on serum urea concentration of male mice dose. (C) Effect of atrazine alone or in combination with N. sativa on serum creatinine. a=Control vs. treated groups, b= N.s vs. treated groups, c= ATZ vs. ATZ+N.s treated group. *p ˂ 0.05, **p ˂ 0.01, ***p ˂ 0.001. Values are expressed as Mean ± SEM

Conclusions

It is concluded from the present study that hematological parameters showed that atrazine administration has a profound influence on the blood profiles of the mice. Atrazine exposure may cause anemia, probably due to a decrease in the rate of RBC production or an impairment of erythropoiesis. Atrazine administration induced a toxic effect on the function of the liver and kidney in an animal model. Moreover, the protective effect of Nigella sativa crude oil was discerned in almost all parameters of this research. The findings of the present research support the use of crude N. sativa oil as a protective and therapeutic agent against atrazine toxicity, to which the human population is intentionally or unintentionally exposed. It is suggested that Nigella sativa oil be used and added to the daily routine. More research work is required on its phytochemical analysis and its pharmaceutical components. Thus, it can be used to treat several toxicities caused by several chemicals or diseases. Further, it is suggested to use it for more toxicants or several chemicals that are harmful to one’s health. Also, more research is required against atrazine-induced toxicity in male-based models.