Protective role of nigella sativa oil and vitamins C and E against aflatoxin M₁-induced hematology and hepatic toxicity in neonatal rats

Abstract

This study explores the protective efficacy of Nigella sativa oil (NSO), Vitamin C, and E in countering the toxic effects of Aflatoxin M1 (AFM1) in neonatal Wistar rats. A total of 117 neonatal rats were randomized into 13 groups (A–M) and observed over 12 weeks. Group A served as the control, while the remaining groups were exposed to varying concentrations of AFM1, either alone or in combination with the supplements. Hematological parameters, including Packed Cell Volume (PCV), Red Blood Cells (RBC), Hemoglobin (HB), White Blood Cells (WBC), and liver enzyme activities, were measured using Microhematocrit Reader, Hemocytometer, Hemoglobinometer, and Spectrophotometer respectively. Histopathological assessments of liver tissues were conducted using staining techniques to assess cellular damage, inflammation, and fibrosis. The results demonstrated that simultaneous supplementation with NSO, Vitamin C, and E significantly improved PCV, HB, and RBC levels while reducing WBC counts and liver enzyme activities in comparison to AFM1-exposed groups. Notably, NSO effectively restored hematological parameters and facilitated liver tissue regeneration, even at higher concentrations of AFM1. In contrast, Vitamins C and E displayed protective effects predominantly at lower toxin levels. These findings suggest that NSO, in conjunction with Vitamin C and E, plays a pivotal role in mitigating the detrimental effects of AFM1 toxicity. NSO shows superior potential in reversing blood dyscrasia and liver damage. The study highlights the promising therapeutic utility of Nigella sativa oil as a natural remedy for alleviating aflatoxin-induced oxidative stress and organ damage, reinforcing its potential to safeguard public health against aflatoxin exposure.

Introduction

Aflatoxin contamination in food might kill us all, especially those in developing countries. Aflatoxins are toxic metabolites produced mainly by Aspergillus flavus and Aspergillus parasiticus during pre-harvest, post-harvest and storage of cereal grains, particularly maize, rice, wheat, barley, and oats¹. Aflatoxin B1 (AFB₁), the most potent mycotoxin, is also the most toxic naturally occurring liver carcinogen². AFB₁ metabolism in the liver produces several metabolites, including a hydroxylated derivative, aflatoxin M1 (AFM₁).

Aflatoxin M1 is a metabolite of aflatoxin B1 (AFB₁): it is formed by enzymatic hydroxylation of the B1 carried over from contaminated feed, primarily cereal grains. AFM₁ was reported in the milk of nursing women who eat food containing AFM₁^3,4. ⁵ also reported the presence of AFM₁ in milk from free-grazing cows meant for human consumption in Nigeria with a value that ranged between 9 ng/l and 456 ng/l. Although the values were reported as critically not high, the mean levels exceeded the European Union maximum levels by a factor of two.

The presence of AFM₁ in milk is transient⁶. It usually reaches a peak within two days of ingestion of the contaminated commodity and disappears within 4–5 days of withdrawal from a contaminated food source³.

Although AFM₁ is ten times less carcinogenic and mutagenic than AFB₁, acute and chronic exposure to it exhibits a high level of genotoxic activity and certainly represents a health risk due to its possible accumulation and binding to DNA^7,8.

AFM₁ poses a worldwide concern, particularly for infants and children who consume large quantities of milk and therefore are more susceptible to the adverse effects of AFM₁⁹. Although acute aflatoxicosis caused by aflatoxins especially AFB₁ has been implicated in the pathogenesis of malnutrition diseases, AFM₁ has mostly been indicted in increased neonatal susceptibility to infections, and jaundice^10,11 and importantly also in oxidative stress. Oxidative stress happens when the production of Reactive Oxygen species (ROS) exceeds the body’s natural antioxidant defence mechanisms, causing damage to macromolecules such as DNA, proteins and lipids.

Expunging Aflatoxins from contaminated feed and foodstuffs remains a major problem, and the non-avoidance of aflatoxin-contaminated food has necessitated the development of numerous detoxification strategies to alleviate its impact¹². reported on the efficacy of an herbal mycotoxin binder (a unique combination of minerals, antioxidants and enzymes) for the control of aflatoxin in broiler/breeder diets. Little or no attention has been paid to the decontamination and toxic effects of AFM₁ that occur as a result of its intake and absorption into the body system. However, contemporary studies reported the likely potential of antioxidants such as vitamins in protecting living organisms against the toxic effects of environmental chemicals.

Vitamins C and E are known to be protective anti-oxidants^13,14. They cause the inhibition of peroxidation, mopping up of free oxygen radicals and disorganization and breakage of peroxidation chain reactions by inhibition of glutathione peroxide, Protein Kinase C (PKC) inhibition and calcium metabolism¹⁴. ¹⁵ also reported the protective effects of vitamins A, C and E on AFM1-induced oxidative stress in human lymphocytes.

Nigella sativa (NS) is a promising medicinal plant that possesses potent antioxidant properties in its seeds known as black cumin or “Habatul-Barakah”¹⁶. NS oil has long been used in folk medicine in the Middle and Far East as a traditional medicine for various illnesses. Several pharmacological properties of NS seeds, including anti-inflammatory, anticancer, anti-fertility, anti-diabetic, antimicrobial, anti-histaminic, hypotensive, and anti-gastrointestinal problems have been reported^16–19. The seeds of NS contain more than 30% fixed oil and 0.4–0.45% wt/wt volatile oil including 18.4–24% thymoquinone (TQ) and 46% of various monoterpenes, examples such as p-cymene and α-pinene²⁰. A more recent study by²¹, reported concentrations of the aroma compound in NS seed to be thymoquinone (38.23%), p-cymene (28.61%), 4-isopropyl-9-methoxy-1-methyl-1 cyclohexene (5.74%), longifolene (5.33%), -thujene (3.88%), and carvacrol (2.31%) compounds have known for antimicrobial and pharmacological properties. Advanced research has shown that antioxidative chemicals such as tocopherols and polyphenols are potentially beneficial to human health (Karrar et al.., 2019). Levels of α-tocopherol, β-tocopherol, and γ-tocopherol in NS seed oils 1according to²² were found to be 25.59, 14.21, and 242.83 mg/100 g, respectively, while the level of polyphenols was 315.68 mg GAE/kg oil, higher than previous reports. The protective ability of NS oil against toxicity belongs to its radical scavenging (anti-oxidative) activity^18,23, its inhibition of 5-lipoxygenase products during inflammation²⁴, as well as to its suppression of cell proliferation.

Aflatoxin M1 (AFM₁), a toxic metabolite of Aflatoxin B1 (AFB₁), poses significant health risks due to its presence in milk and dairy products. Despite being less carcinogenic than AFB1, its genotoxic potential and oxidative stress induction in humans, especially in infants, highlight the need for effective intervention. This study aims to demonstrate the protective role of Nigella sativa oil, vitamins E and C, known for their potent free-radical scavenging properties, in mitigating AFM₁ toxicity by reducing oxidative stress, enhancing antioxidant defence, and improving overall health. The use of these natural antioxidants may offer a promising solution for reducing the harmful effects of AFM₁ on human health.

Methodology

Standards and antioxidants

AFM₁ was purchased from Sigma Aldrich, USA and was diluted with phosphate-buffered saline (PBS) according to the manufacturer’s instructions to prepare a stock solution at working concentrations of 9 ng, 235 ng and 456 ng per 100µL and was stored at 4 °C till use. The working concentrations were deduced from the previous study of⁵. Commercially sold Nigella sativa oil manufactured by Hemani International Limited, Karachi, Pakistan, was purchased from Rukdol Islamic Ventures in Lagos State, Nigeria.

Ascorbic acid (Vitamin C), produced by Memphis Company, was purchased from Precious Pharmacy in Abeokuta and used at a concentration of 500 mg/ Ml. dl-(α-Tocopherol acetate (15%)) (Vitamin E), 500 mg/ mL, produced by Kunimed Pharmaceutical Company, was purchased from Precious Pharmacy in Abeokuta, Nigeria.

Experimental animals

Fifty Albino rats (40 female and 10 male) in the mating stage weighing 70 to 120 g were obtained from the Animal house of the University of Ibadan Nigeria. They were kept in cages with wooden shavings at room temperature in the Department of Pure and Applied Zoology, Animal House of the Federal University of Agriculture Abeokuta, Ogun State. The animals were maintained on Chunkum rats growers mash and water ad-libitium and were allowed to mate freely for two weeks. The pregnant female rats were then separated into another cage to get the litters for the experiment.

Experimental rat design

Immediately after birth, rat litters were separated from their mothers to initiate the treatments. A total of 117 neonatal rats, with body weights ranging from 10.15 to 12.25 g at birth, were included in the study. The rats were randomly divided into 13 groups of 9 rats. A 3 × 3 × 3 × 1 factorial design was employed, with daily toxins and/or antioxidants administration. Sacrifices occurred at the end of the 4th, 8th, and 12th weeks. The study included three doses of aflatoxin (9, 235, and 456 ng/L) and three antioxidants (Nigella sativa oil, vitamin C, and vitamin E), each at a concentration of 2%.

Ethical considerations for the use of rats adhered to the guidelines set forth by the Nuffield Council on Bioethics. Weight changes in the rats administered aflatoxin M1 (AFM₁) and other treatments were recorded weekly. Stock solutions of 50 ml of milk were prepared in various extraction bottles, each spiked with the three levels of AFM₁ and stored in the refrigerator until administration. Additionally, rats in certain groups received 2 ml of antioxidants immediately following the AFM₁ treatment. The animals were fed every two hours from dawn until sunset, according to the experimental protocol detailed below.

Experimental protocol

Group 1: Milk (control rats).

Group 2: Milk + 9ng/L AFM₁.

Group 3: Milk + 9 ng/L AFM₁ + Vit.C.

Group 4: Milk + 9 ng/L AFM₁ + Vit.E.

Group 5: Milk + 9 ng/L AFM₁ + NSO.

Group 6: Milk + 235 ng/L AFM₁.

Group 7: Milk + 235 ng/L AFM₁ + Vit.C.

Group 8: Milk + 235 ng/L AFM₁ + Vit.E.

Group 9: Milk + 235 ng/L AFM₁ + NSO.

Group 10: Milk + 456 ng/L AFM₁.

Group 11: Milk + 456 ng/L AFM₁ + Vit.C.

Group 12: Milk + 456 ng/L AFM₁ + Vit. E.

Group 13: Milk + 456 ng/L AFM₁ + NSO.

A report of a survey of AFM₁ in cows’ milk from free-grazing cows in Nigeria indicated that toxin levels in positive milk samples ranged from 9.0 to 456.0 ng/L. Therefore, milk samples were spiked with AFM₁ at three concentrations of 9, 235 and 456 ng/L AFM₁ and were administered to the rats at 2 ml/dose via oral intubation.

Ethical approval statement

The ethical considerations of the use of the rats were in line with the specifications of the Nuffield Council on Bioethics, an international body. Also, local institutional standards of the Federal University of Agriculture, Abeokuta, Nigeria, care and use of laboratory animals were followed and approval number FUNAAB/13/252 was obtained. All experiments were performed following relevant guidelines and regulations.

Sacrifice and sample collection

After receiving AFM ₁ and /or antioxidant treatments, the rats were fasted overnight at the end of the 4th, 8th, and 12th week, and three rats from each group were sacrificed. Body weights of the rats were recorded prior to the procedure. Anaesthesia was induced via intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Euthanasia was subsequently performed through intraperitoneal administration of sodium pentobarbital. Blood sample (4 ml) was collected with a disposable syringe and needle and divided into two parts, 2 ml was immediately transferred into sterile ethylene Diamine Tertra-acetic Acid (EDTA) embedded vials for haematological study, while the remaining 2 mL was dispensed into plain tubes, the sera separated from the clotted blood by routine procedures was stored at − 20 °C until it was used for biochemical analysis. The rats were observed daily for feeding patterns, clinical signs and mortality for 12 weeks after administration of the varying concentrations of the treatments: AFM₁, AFM₁ with 2% Nigella sativa oil, vitamins C or E.

Haematology and serum chemistry

The rats’ haematological parameters: Packed Cell Volume (PCV), Haemoglobin (Hb), Red Blood Cells (RBC), and Leukocyte (WBC) count were determined as described by the International Committee for Standardization in Haematology Serum biochemical parameters, electrolytes (sodium, potassium and chloride), urea, creatinine, total protein, albumin, globulin, and total bilirubin ) as well as the activities of Alanine Aminotransamine (ALT), Aspartate amino transaminase (AST) were determined using the method of²⁵.

Histology

The livers were excised for routine histopathology. Succinctly, the livers were fixed in 10% formalin and dehydrated with ethanol of different grades (70, 80, 90, 95 and 100%). Dehydration was then followed by cleaning the samples in 2 changes of xylene. Samples were impregnated with 2 changes of molten paraffin wax, then embedded and blocked out. Paraffin sections of 5–6 μm thick transverse sections of the liver were cut using a rotary microtome and mounted on glass slides previously stained with routine haematoxylin and eosin (H&E). Stained sections of control and treated rats were examined under the light microscope (Olympus CH Japan) for possible alterations. Photomicrographs were obtained with an Amscope camera fitted on an Accu-scope microscope. Images were analyzed with the aid of ToupView software.

Statistical analysis

Data obtained for the body weights were analysed using the Statistical Package for Social Sciences (SPSS) version 20.0 (IBM Corp., 2011). Mean values were compared using Analysis of Variance (ANOVA), Results were presented as Mean ± Standard deviation. P < 0.05 was taken as significant. A post hoc test was done using the Student-Newman-Keuls (SNK) to compare mean values between the treatment groups. All other results were analysed using R (version 4.3.1). The figures were made using a faceted line plot in R studio version 2023.09.1.

Results

Effects of aflatoxin M1 on neonatal rats

Effects on haematological parameters

The significance of the toxic effects of the low (9 ng/ L), medium (235 ng/L) and high (456ng/ L) doses of Aflatoxin M1 administered to the rats is demonstrated in Table 1. Interactions between the aflatoxin dosages and the Packed Cell Volume (PCV), Haemoglobin (Hb), Red Blood Cells (RBC), and Leukocyte (WBC) showed significance (p < 0.05) for toxic effects on the parameters investigated at weeks 4,8 and 12 Duration and exposure to AFM₁ played a crucial role in changes that occurred in the haematology of the neonatal rats. 70% mortality was recorded in rats that received 456ng/ L AFM₁. Rats were anaemic, pale and exhibited loss of appetite (Fig. 1a, b,c, d).

Table 1.

Summary table indicating Anova F values and significances for the main effect of aflatoxin dosage across weeks after administration on haematological parameters of neonatal Wistar rats.

Factors	PCV	HB	WBC	RBC
Aflatoxin dosage (A)	478.8 ± 203.76***	63.32 ± 251.9***	38.07 ± 93.25***	6.016 ± 166.34***
WAA	253.8 ± 107.98***	28 ± 111.4***	18.08 ± 44.29***	2.101 ± 58.09***
A*W	39.2 ± 16.66***	4.58 ± 18.2***	6.00 ± 14.70***	0.422 ± 11.66***

***F-values are presented as mean ± standard deviation.

WAA = Weeks After Administration.

p ≤ 0.001, indicating highly significant main effects and interactions for all parameters .

Fig. 1.

Effects of AFMI dosage on Packed cell volume (PCV), Haemoglobin (Hb), Red Blood Cells (RBC), Leukocyte (WBC) Haematological Parameters of Neonatal rats at 4 ,8 and 12 weeks.

Effects on the weekly bodyweight of rats

Rats treated with only doses of AFM₁ had significant (p > 0.05) increases in their body weight from the initial 10.15 g to 13.18 g during the first week of the low dose of AFM₁ (9 ng/ L) administration, however, the tenth and eleventh week recorded a stationary weight as compared with the control, while there was a non-significant loss in weight from 45.65 g of control to 43.45 g in the twelfth week of the 9 ng/ L treatment. Treatment with medium dose (235 ng/ L) of AFM₁ showed a significant increase p < 0.05 in the weight of the rats from the first to the ninth week, at the ninth week there was no increase nor decrease in the weight, while at the eleventh and twelfth week, a significant (p < 0.05) reduction was observed in their weight. With the high dose of AFM1, significant (p < 0.05) increase in weight was observed till the eighth week, while very significant weight loss from 49.95 g to dead animals were observed at twelfth week (Fig. 2).

Fig. 2.

Comparison of efficacy of antioxidants used in treating weight (g) of aflatoxicosed neonatal rats.

Protective effects of nigella sativa oil, vitamin C and E

Packed cell volume ( PCV)

The antioxidative effects of the 2% Nigella sativa (NSO), Vitamins C and E on the packed cell volume (PCV) of neonatal rats at 4, 8 and 12th week is shown in Fig. 3.

Fig. 3.

Effects of Nigella Sativa oil (Blackseed oil), Vitamins C and E on the Packed Cell Volume of Neonatal rats.

Haemoglobin concentration ( Hb)

Concentrations of haemoglobin increased after significant reduction as a result of AFM₁ contamination. The most significant increase was observed in rats administered NSO as shown in Fig. 4.

Fig. 4.

Effects of Nigella Sativa oil ( Blackseed oil), Vitamins C and E on the Haemoglobin of Neonatal rats.

Red blood cell count ( RBC)

The results of Red blood cell count as shown in (Fig. 5) indicate a significant (P ≤ 0.05) increase in the values of the RBC of rats that received NSO, Vitamins C and E .

Fig. 5.

Effects of Nigella Sativa oil (Blackseed oil), Vitamins C and E on the Red blood cell of Neonatal rats.

White blood cell count. (WBC)

The difference in the counts of the white blood cells is shown in Fig. 6. After the twelfth week of AFM₁ administration, there was a significant increase in the WBC of rats compared to the control rats. For rats that received 9ng/ L AFM₁, the WBC was increased from 8450.0 ± 327.1^b of the control to 10000.0 ± 569.2^c. However, it was observed that the higher the AFM₁ dosage the lower the counts of WBC. The WBC of rats in the 235ng/ L and 456ng /l groups dropped from 8450.0 ± 327.1^a and 8454.0 ± 320.6^a to 3966.7 ± 186.2^b and 3616.7 ± 325.1^b respectively. The intervention with NSO after the twelfth week showed a significant increase in the WBC of the rats and with Vitamins C and E.

Fig. 6.

Effects of Nigella Sativa oil ( Blackseed oil), Vitamins C and E on the White blood cell of Neonatal rats.

Serum biochemistry and liver function enzymes

No significant changes were observed in the values of serum proteins, albumin and globulin, urea, creatinine and electrolytes between control rats and those given either varying concentrations of AFM₁ alone or in combination with Vitamins C, E and Nigella sativa oil (NSO) (data not shown). Also, 9ng and 235ng concentrations of AFM₁ administered did not cause any significant changes (p > 0.05) to ALT, AST and ALP levels in the rats (Figs. 7, 8 and 9). However, significant increases (p < 0.05) were observed in the values of serum ALT, AST and ALP levels of rats that received 456ng AFM₁, while the levels of serum ALT, AST and ALP increased (p < 0.05) progressively in the test rats. Vitamin E was able to restore partial changes in the enzyme activity, while Vitamin C, was not effective, however, Nigella Sativa oil was able to restore the levels of ALT, AST and ALT comparable with the control rats.

Fig. 7.

Effects of Nigella Sativa oil ( Blackseed oil), Vitamins C and E on liver function enzyme ( ALP) of Neonatal rats.

Fig. 8.

Effects of Nigella Sativa oil ( Blackseed oil), Vitamins C and E on the liver function enzyme ( ALT) of Neonatal rats.

Plate 1.

Photomicrograph of liver tissue section from control Wistar rats stained with hematoxylin and eosin (H&E), viewed at ×400 total magnification. Scale bar: 50 μm.

Fig. 9.

Effects of Nigella Sativa oil ( Blackseed oil), Vitamins C and E on the liver function enzyme ( AST) of Neonatal rats.

Histology of liver samples

Histopathological profile of the liver of aflatoxin M1 treated rats showed vacuole degeneration of hepatocytes, congestion of hepatic sinusoids and hepatic necrosis with inflammatory cell infiltration, Kupffer cells hyperplasia in 235ng AFM₁-treated rats at 12 weeks while rats that received 456ng AFM₁ showed all the defects right from the 4th week when compared with the finding of control rats. Vitamins C and E partly restored the changes caused by AFM₁, However, Nigella sativa oil was able to restore the liver to its original state (Plates. 1, 2, 3, 4, 5, 6, 7 and 8).

Plate 2.

Photomicrograph of liver tissue section from rats exposed to 235 ng/L Aflatoxin M1 (AFM₁) for 12 weeks, stained with hematoxylin and eosin (H&E), viewed at ×400 total magnification. Red arrow: Kupffer cell hyperplasia; black arrow: hepatocellular necrosis; blue arrow: bile duct hyperplasia; green arrow: hepatic sinusoids; star: congested blood vessels. Scale bar: 50 μm.

Plate 3.

Photomicrograph of liver tissue section from rats exposed to 456 ng/L Aflatoxin M1(AFM₁) for 4 weeks, stained with hematoxylin and eosin (H&E), viewed at ×400 total magnification. Blue arrow: inflammatory cells; black arrow: hepatocellular necrosis; red arrow: Kupffer cell hyperplasia (KCH) laden with black pigments. Scale bar: 50 μm.

Plate 4.

Photomicrograph of liver tissue section from rats exposed to 456 ng/L Aflatoxin M1 (AFM₁) for 8 weeks, stained with hematoxylin and eosin (H&E), viewed at ×400 total magnification. Blue arrow: aggregate mononuclear cells in portal tracts; black arrow: hepatocellular necrosis; red arrow: moderate Kupffer cell hyperplasia. Scale bar: 50 μm.

Plate 5.

Photomicrograph of liver tissue section from rats exposed to 456 ng/L Aflatoxin M1 (AFM₁)for 12 weeks, stained with hematoxylin and eosin (H&E), viewed at ×400 total magnification. Blue arrow: hepatic sinusoids; black arrow: hepatocellular necrosis; red arrow: marked Kupffer cell hyperplasia; green arrow: vacuolar changes in hepatocytes. Scale bar: 50 μm.

Plate 6.

Photomicrograph of liver tissue section from rats exposed to 456 ng/L Aflatoxin M1 (AFM₁) and treated with black seed oil for 12 weeks, stained with hematoxylin and eosin (H&E), viewed at ×400 total magnification. Blue arrow: closely packed hepatocytes; green arrow: very mild megalocytosis. Scale bar: 50 μm.

Plate 7.

Photomicrograph of liver tissue section from rats exposed to 456 ng/L Aflatoxin M1 (AFM₁)and treated with vitamin C for 12 weeks, stained with hematoxylin and eosin (H&E), viewed at ×400 total magnification. Blue arrow: fairly closely packed hepatic cords; black arrow: few hepatocellular necrotic cells. Scale bar: 50 μm.

Plate 8.

Photomicrograph of liver tissue section from rats exposed to 456 ng/L Aflatoxin M1 (AFM₁)and treated with vitamin E for 12 weeks, stained with hematoxylin and eosin (H&E), viewed at ×400 total magnification. Black arrow: closely packed hepatic cords with single-cell hepatocellular necrosis. Scale bar: 50 μm.

Discussion

The first 1000 days of life, from conception to 24 months, is a critical period for healthy growth and development; hence, dietary intake during and after pregnancy plays a fundamental role in a child’s future health status²⁶. Aflatoxin M1 (AFM₁) has been classified by²⁷ as a human carcinogen a change from the previous classification of being probably carcinogenic to human status⁵. reported varying concentrations of AFM₁ in raw milk of free-ranging cows and in the breast milk of lactating mothers in Nigeria⁴, exposures which have been indicted in the cause of stunted growth in children, especially in the developing nations of the world. The present study aligns with and extends the findings of recent studies on the protective effects of natural antioxidants against aflatoxin-induced toxicity. While previous studies have largely focused on aflatoxin B1 (AFB1), emerging evidence suggests that AFM₁, a hydroxylated metabolite of AFB1, also poses significant health risks, particularly in neonates and infants. For instance²⁸, emphasised the growing concern of AFM₁ contamination in dairy products and its impact on child health, yet few studies have investigated its sub-chronic effects on neonatal development.

Various epidemiological studies have reported that aflatoxin B1 exposure is linked with low birth weight and low weight for age score^10,29,30. However, no study has investigated the relationship between AFM₁ exposure and weight development in children. The results of the present study revealed weight gains in rats that received 456 ng/ L till the 9th week of treatment. After the ninth week, a sharp drop in weight was observed in the rats till the 12th week. Administration of low dose and medium AFM₁ showed a consistent increase in weight was observed for the first 9 weeks, no weight gain or loss was observed in the 10th and 11th week, and a slight loss in the 12th week was observed. This indicates clearly that AFM1 starting at birth is also responsible for changes in body weight and is dosage dependent. Chronic aflatoxicoses may be determined through changes that may be observed in either or both of haematology and serum biochemistry parameters before the manifestation of clinical symptoms³¹. Blood haematological parameters such as the Parked Cell Volume (PCV), White Blood Cell Count (WBC), Red Blood Cell (RBC) and Heamoglobulin (HB) levels significantly ( p < 0.05) decreased in rats intoxicated with low (9ng/l), medium (235ng/l) and high (456ng/l) doses of AFM₁ when compared with the control. Although no study has reported the direct effects of AFM₁ on haematology of infant mammals, this would be the first, but several studies have reported the effects of total aflatoxin on the haematology adult animals such as in broiler chicken³¹, Merino rams³² and male Buffaloes³³, all studies reported significant decrease in the parameters.

The toxicity of aflatoxin differs with concentration and duration of exposure³⁴. This was evident in this study where decreased PCV, HB, RBC and WBC were more pronounced in rats that were intoxicated with 456ng/l/l AFM₁ for 12 weeks.

Co-administration of NSO in the study showed a significant increase in the percentage of PCV, and values of HB, RBC and WBC, when compared with the control rats, which strongly indicate that NSO can prevent cell damage and reverse a loss of blood cells. However, co-administration of AFM₁ with vitamins C and E did not result in a significant increase as the concentration of AFM₁ in the neonatal rats increased. Therefore, the ameliorating ability of NSO as it relates to haematology in this study agrees with³⁵ who reported that N. sativa oil reversed most of the haematological and biochemical changes and markedly improved the antioxidant capacity of treated infected mice.

Dysfunction of the liver is a major indication of systemic toxicity. The data presented in this study revealed that AFM₁ was associated with the steady-state activities of biomarkers of liver function. It has been established that aflatoxin has a harmful and stressful effect on liver tissue and only in exceptional cases Kidney function. Evidence from animal and epidemiological studies has supported the correlation between hepatocellular carcinoma and aflatoxin exposure in populations with high aflatoxin exposure^36,37. AST, ALT, and ALP are cytosolic serum enzymes and are established biomarkers of liver damage; therefore, elevated serum enzyme levels in intoxicated rats are an indication of damaged structural integrity of the liver³⁸. In this study, the activities of the AST, ALT, and ALP were increased in the intoxicated rats. Co-administration of Nigella sativa oil prevented the elevation of ALT and AST activities which are associated with hepatic parenchymal injury. The protective effect of Nigella sativa oil against CC1₄ and D-galactosamine-induced hepatic toxicity in rats was reported by^24,39 who observed a reduction in the activities of serum AST, ALT, alkaline phosphatase, lactate and malate dehydrogenases. In addition, other previous studies demonstrated that Nigella sativa may be successful in the protection of liver fibrosis in rabbits⁴⁰ and that its oil may play a role against liver damage induced by Schistosoma mansoni infection in mice⁴¹. In this study, rats that received AFM₁ with NSO supplementation showed no increase in AST, ALT, and ALP activities when compared to the rats in the control group. This suggests a possible protective effect of Nigella sativa treatment in hepatic ischemia. Although there was a decrease in the serum activities with the co-administration of vitamins C and E, they failed to return to normal levels.

The histological profile of the liver of AFM₁-treated rats in this study showed vacuolar degeneration of hepatocytes, congestion of hepatic sinusoids and hepatic necrosis with inflammatory cell infiltration, bile duct hyperplasia, Kupffer cell hyperplasia when compared with findings of control rats.

The treatment of rats receiving AFM₁ with Vitamin E or C showed an extended portal tract infiltrated with few mononuclear inflammatory cells, less fibrosis, and degeneration of some hepatocytes. Furthermore, NSO-treated rats showed no vacuolation of hepatocytes and fibrosis when compared to the aflatoxin-treated group. These observations were well correlated with the biochemical findings and gave clear evidence that there was not only improvement in the liver functions with the treatment of antioxidant agents, especially NSO, but also in the hepatic architecture.

The protective effects observed with Nigella sativa oil, Vitamin C, and Vitamin E against aflatoxin M1-induced toxicity are attributed to their well-established antioxidant and anti-inflammatory mechanisms. Nigella sativa oil contains thymoquinone, which has been shown to scavenge free radicals, inhibit lipid peroxidation, and downregulate pro-inflammatory cytokines, thereby reducing oxidative damage in tissues⁴². Vitamin C (ascorbic acid), a potent water-soluble antioxidant, neutralises reactive oxygen species (ROS), enhances the regeneration of other antioxidants like Vitamin E, and supports immune defence²². Vitamin E (α-tocopherol), a lipid-soluble antioxidant, protects cellular membranes from oxidative damage by preventing lipid peroxidation and stabilising membrane integrity⁴³. The combined or individual administration of these agents may synergistically mitigate aflatoxin-induced oxidative stress, preserving tissue architecture and improving physiological outcomes observed in the treated groups.

The hepatotoxicity induced by aflatoxin M1 in neonates can be effectively modulated using Nigella sativa oil (NSO) and partially by antioxidants: vitamin E or C, when the doses are not high. Moreover, NSO are more efficient in modulating the biochemical alteration, liver antioxidant enzymatic system and pathological changes. Notably, the present study is among the first to document AFM₁-induced haematological alterations in neonatal mammals, underscoring the early-life susceptibility to environmental toxins and the potential of Nigella sativa as a preventive nutritional intervention.

Conclusion

This study demonstrates that hepatotoxicity induced by aflatoxin M1 in neonates can be significantly mitigated through the use of Nigella sativa oil (NSO), as well as antioxidants such as vitamins E and C, provided that their dosages are judiciously managed. Notably, NSO exhibits superior efficacy in ameliorating biochemical disturbances, enhancing liver antioxidant enzyme activities, and alleviating histopathological alterations when compared to the other antioxidants studied. Given these findings, it is strongly recommended to incorporate NSO into dietary regimens as a proactive measure to combat the harmful effects of environmental hepatotoxins, including aflatoxin M1.

Incorporating NSO not only enhances blood cell and liver health but also aids in minimizing further liver damage, thus promoting a faster recovery from hepatotoxic insults. As environmental contaminants remain a significant concern, dietary interventions with NSO could serve as a viable strategy for improving neonatal health outcomes and safeguarding against the risks associated with aflatoxin exposure. Future research should explore the underlying mechanisms of NSO’s protective effects and its potential applications in clinical settings.

Author contributions

(A) O. (B) carried out the research and wrote the manuscript draft; E.O. O. gave methodology protocols and wrote the methodology drafts; A.A.A. supervised the research, carried out some of the analysis and corrected and revised the manuscript before submission. F.O. conceptualised the study, supervised and monitored the final output.

Data availability

The data that support the findings of this study will be made available upon request from the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.

ARRIVE guidelines

The study is reported following the ARRIVE guidelines⁴⁴.