Biosynthesized Iron Oxide Nanoparticles from Petroselinum crispum Leaf Extract Mitigate Lead-Acetate-Induced Anemia in Male Albino Rats: Hematological, Biochemical and Histopathological Features.

This study aimed to investigate the ameliorative effects of iron oxide nanoparticles (IONPs) prepared from leaf extract of Petroselinum crispum compared to those prepared using a chemical method in lead-acetate-induced anemic rats. Twenty rats were divided into four groups (five rats each). Throughout the experimental period (8 weeks), the rats in group 1 were not given any therapy. The rats in groups 2, 3 and 4 were given 400 ppm lead acetate orally for 2 weeks to make them anemic. Following that, these rats were either left untreated, given 27 ppm of chemical IONPs orally or given 27 ppm of natural IONPs orally for the remaining 6 weeks of the experiment. TEM analysis indicated that the chemically and naturally prepared IONPs had sizes of 6.22-9.7  and 64-68 nm, respectively. Serum ferritin and iron concentrations were reduced, whereas the total iron-binding capacity (TIBC), ALT, AST, urea and creatinine were significantly increased in the non-treated lead-acetate-induced anemic rats compared to those of the control. In addition, congestion, hemorrhage, necrosis, vacuolation and leukocytic infiltration in the kidneys, liver and spleen were observed in non-treated lead-acetate-induced anemic rats compared to the control. The effects of lead acetate were mitigated by IONPs, particularly the natural one. In conclusion, IONPs produced from Petroselinum crispum leaf extract can be used as an efficient and safe therapy in lead-acetate-induced anemic rats.

1. Introduction

Anemia, a contributor to hidden hunger, is the most common disorder of the blood. It is defined as a diminishing of either total circulating red blood cell mass or hemoglobin (Hb) concentration below the normal level for people of the specified age and sex. Anemia exists in both developing and developed countries. Iron deficiency anemia is one type of microcytic hypochromic anemia [1]. Due to the increased demand for growth and development, it is one of the most frequent nutritional problems, especially in children and women in developing countries. The main causes of this anemia are dietary factors, iron absorption competitors, parasites and intestinal diseases.

Lead is an injurious heavy metal, which is widely spread in the environment. Its concentration in the air, food and water has elevated several folds in recent years as a result of its widespread use in industries such as gasoline, paint and battery manufacturing [2]. Lead can enter human and animal bodies by inhalation and/or ingestion [3,4]. Lead poisoning primarily affects the hematopoietic system, central nervous system and hepatic and renal systems, causing serious health problems [5,6]. Lead exposure is classified as the most common cause of anemia [7] and it is one of the main inducers of microcytic hypochromic anemia [8,9]. Lead interferes with iron metabolism [10,11], inhibits heme-synthesizing enzymes with concurrent elevation of protoporphyrin and shortens the life spans of circulating erythrocytes [8,9]. In addition, lead reduced the absorption of iron in the gastrointestinal tract [12] and suppressed hematopoiesis of the bone marrow, probably through its interaction with enteric iron absorption [13].

The treatment regimen for microcytic hypochromic anemia is based on preventing further iron loss and replenishing iron stores in the body using iron supplements [14]. Iron supplements are damaging to the gastrointestinal tract and microflora and generate free radicals. The encapsulation of iron in nanoparticles guarantees that it is absorbed securely and without causing harm to the digestive system [15]. The primary applications of nanomaterials in drug delivery offer a number of advantages. Nano applications of drugs make them safer and more biocompatible and increase drug stability, solubility, absorption and bioavailability. In addition, nano application of a drug decreases its toxicity without reducing its therapeutic potential [16,17,18].

Synthesis of nanoparticles by chemical and physical methods offers pure nanoparticles with good features. These nanoparticles are prohibitively expensive and may not be environmentally friendly [19,20]. Plant-mediated NP synthesis (green synthesis) has emerged lately as the most effective way of producing large quantities of NPs in a short amount of time [21]. In the synthesis process, the bioactive components in the plant extract function as reducing and coating agents, converting metal ions into nanoparticles [21]. Green synthesis can produce stable nanoparticles of the desired size and morphology, and the process is simple, cost-effective, reproducible and sustainable [22,23,24].

For nanoparticle synthesis, utilizing plant sources as reducing agents will replace the use of toxic chemicals. Recently, several studies were carried out for green synthesis of iron-based nanoparticles from various plants parts, such as fruit extract of Cynometra ramiflora, rind of Persea americana, seed extract of Punica granatum and flower extract of Avicennia marina [25,26,27,28]. Petroselinum crispum is a popular herbal crop valued for its nutritional and medicinal properties [29]. Its leaves contain phenolic acids (caffeic acid), flavonoids and volatile oil [30], ascorbic acid [31] and carotenoids [32].

Petroselinum crispum can be used for the synthesis of zinc oxide nanoparticles. When compared to chemically generated ZnO NPs, those made from Petroselinum crispum extracts had higher antibacterial activity (2–16 times) [33]. However, the use of this plant for the preparation of IONPs has not been investigated so far. Therefore, the present study is the first and foremost report on the synthesis of magnetic iron oxide nanoparticles (Fe3O4NPs) using Petroselinum crispum leaf extract. The goal of this study was to examine the effects of plant-derived iron oxide nanoparticles vs. chemically derived iron oxide nanoparticles in lead-acetate-induced anemic rats. The comparative criteria included hematological, biochemical and histopathological characteristics.

2. Materials and Methods

2.1. Chemicals and Plant Extraction

Ferric chloride hexahydrate (FeCl3 6H2O; 98%), ferrous sulphate heptahydrate (FeSO4 7H2O; 98%) and ammonia solution 25% were supplied by Sigma Aldrich, USA. Other reagents and buffers were of the highest purity and analytical grade. Double-distilled water was used for extraction and solution preparation purposes. Fresh Petroselinum crispum leaves were collected from the premises of Benha University, Egypt. The fresh leaves were washed multiple times with tap water followed by double-distilled water. The leaves were then dried in an oven for an hour and ground to form a fine powder. Five grams of the fine powder was boiled with 100 mL of double-distilled water while being continuously stirred for 15 min. The extract was then filtered using Whatman no. 42 filter paper. The filtrate was stored at −4 °C for further use.

2.2. Preparation of Chemical and Natural Magnetic Iron Oxide Nanoparticles

The Institutional Animal Care and Use Committee Research Ethics of Benha University, Faculty of Veterinary Medicine, Egypt, reviewed and approved all experimental protocols (BUFVTM, 01012021; 1 January 2021) conducted in this study. IONPs were prepared in the department of Corn Diseases and Sugar Crops, Agricultural Research Center, Egypt. Chemical iron oxide nanoparticles’ synthesis was conducted as described earlier [34,35]. Chemical-based synthesis methods are mostly adopted due to the low production cost and high yield. Briefly, magnetite was synthesized by adding a base to an aqueous mixture of Fe2+ and Fe3+ chloride in a 1:2 molar ratio, resulting in a black color [35]. The chemical reaction of Fe3O4 precipitation is given in Equations (1) and (2). The overall reaction [36] is written as [Fe2+ + 2Fe3+ + 8OH → Fe3O4 + 4H2O] (1). Under an oxygen-free environment, complete precipitation of Fe3O4 is likely between pH 9 and 14, maintaining a molar ratio of Fe3+: Fe2+ (2:1). Fe3O4 might also be oxidized as [Fe3O4 + 0.25O2 + 4.5H2O → 3Fe (OH)3] (2). Magnetic iron oxide nanoparticles (Fe3O4NPs) were prepared by using aqueous leaf extracts of Petroselinum crispum as described earlier [22,37]. Briefly, 0.01 M FeCl3·6H2O solution was added to the Petroselinum crispum extract in a 1:1 volume ratio. Fe3O4NPs were immediately obtained by the reduction process. To obtain a colloidal suspension, the liquid was agitated for 30 min and then allowed to stand at room temperature for another 60 min. The mixture was centrifuged and washed several times with ethanol before being dried at 40 °C under vacuum to yield the Fe3O4NPs. Compared to other sections of the plant, Petroselinum crispum leaves have the best reduction potential against ferric chloride, as seen by the external color change. Leaves were chosen for additional procedures based on this observation. The prepared iron oxide nanoparticles (Fe3O4NPs) were characterized for their shape, size and dispersity using a transmission electron microscope operated at 100 KV and connected with a CD camera (TEM; Joel 1230, Japan). A Zetasizer system (Nano ZS, Malvern, UK) was used for the measurement of average hydrodynamic diameters and polydispersity indexes. The zeta potential range (mV) was 200:200 mV. Each sample was analyzed in triplicate at 25 °C at a scattering angle of 173°. The size distribution of the particles was estimated from laser particle analyzer (LPA) images by measurement of the diameters.

2.3. Experimental Design

Twenty rats were divided into four groups (5 rats each). Throughout the experimental period (8 weeks), the rats in group 1 were not given any therapy (negative control). The rats in groups 2, 3 and 4 were given 400 ppm lead acetate trihydrate ((CH3COO)2Pb·3H2O [38]; Al Gomhoria company, Cairo, Egypt) orally for 2 weeks to make them anemic. Following that, these rats were either left untreated (positive control), given 27 ppm of chemical IONPs orally [39] or given 27 ppm of natural IONPs orally for the remaining 6 weeks of the experiment [39]. Oral administration of lead acetate was conducted via a stomach tube [38]. Hematological indices were evaluated every week to ensure the induction of microcytic hypochromic anemia.

2.4. Collection of Blood and Serum Samples

Blood samples were collected from the medial canthus of the eye weekly in heparinized capillary tubes during the anemia induction experiment (2 weeks). To confirm the induction of anemia, blood samples were immediately transferred into tubes containing EDTA for estimation of hematological indices. At the end of the experiment, blood samples were collected by cardiac puncture in vacutainers with and without EDTA for analysis of either whole blood or serum, respectively.

2.5. Hematological Analysis

Total erythrocyte count, hemoglobin concentration (Hb), packed cell volume% (PCV%), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were estimated using an electronic cell counter (VetScan HM5 Hematology system, Abaxis, Inc., Union City, CA, USA).

2.6. Serum Biochemical Analysis

Harvested sera were used for determination of the activities of aspartate transaminase (AST) and alanine transaminase (ALT) [40] and the concentration of urea [41] and creatinine [42]. In addition, serum values of ferritin [43] and iron [44] and total iron-binding capacity (TBIC) [45] were determined.

2.7. Histopathological Analysis

At the end of the experiment, samples from liver, kidney and spleen tissues were excised from the sacrificed animals of each group before being fixed in 10% neutral buffered formalin for 24 h. The fixed tissues were properly dehydrated and cleared before being embedded in paraffin wax. Sections of about 5 μm in thickness were prepared and stained with hematoxylin and eosin (H&E) for histopathological examinations. All histopathological procedures were performed using the standard techniques [46]. H&E-stained sections were examined under a light microscope for evaluation of pathological changes. The severity of the microscopic lesions was classified as described earlier [46]. Degree 1 represents mild pathological changes, such as circulatory disturbances, including congestion, hemorrhage and edema, as well as mild degenerative changes. Degree 2 represents moderate pathological alterations, including degenerative changes, in association with cellular deposits with some inflammatory cell infiltrations. Degree 3 represents severe alterations, such as necrotic areas with leukocytic cellular aggregation.

2.8. Statistical Analysis

All data are presented as the mean ± standard deviation (SD) of five replicates. All tests were performed in IBM SPSS 15.0 statistical software (SPSS Inc., Chicago, IL, USA) using one-way analysis of variance (ANOVA). The Duncan multiple comparison test was used to show the difference between means. The magnetic properties of IONPs are expressed as mean ± standard deviation (SD) of the volume of occupation of nanomagnets in 10 mL volume test tubes. Each experiment was repeated 10 times. IONPs’ size is expressed as mean ± standard deviation (SD) using data provided by the statistical computer system of the TEM. The size distribution of the particles was estimated from laser particle analyzer (LPA) images by measurement of diameters provided by the computer programming system of Zetasizer (Nano ZS, Malvern, UK).

3. Results

The current findings indicate that chemically synthesized IONPs have a better magnetic property than those made with Petroselinum crispum leaf extract (Figure 1).

Figure 1

The magnetic property of IONPs prepared using chemical method (A) and Petroselinum crispum leaf extract (B). Values are expressed as mean ± standard deviation (SD; n = 10).

Figure 2 shows TEM images of the IONPs prepared with either Petroselinum crispum leaf extract or the chemical method. The TEM analysis indicated that the size of the IONPs prepared by the chemical method was 6.22–9.7 nm (Figure 2A), whereas the size of the IONPs prepared using Petroselinum crispum leaf extract was 64–68 nm (Figure 2B). In the same context, IONPs were well dispersed in the solution, with oval, cubic and spherical shapes (Figure 2).

Figure 2

TEMs image of iron oxide nanoparticles biosynthesized using (A) chemical method and (B) green biosynthesis method with Petroselinum crispum leaf extract. Values are expressed as mean ± standard deviation (SD; n = 5). Data provided by statistical computer system of TEM.

The size distributions of IONPs prepared by either the chemical method or using Petroselinum crispum leaf extract in colloids are shown in Figure 3. Chemically produced IONPs had a size distribution ranging from 38.71 to 133.74 nm. Constituting 42.527 percent of the entire colloidal solution, the majority of them had a diameter of roughly 68.22 nm (Figure 3A). However, the size distribution of IONPs biosynthesized by Petroselinum crispum leaf extract in colloids ranged from 68.71 to 146.74 nm. Constituting 67.44 percent of the entire colloidal solution, the majority of them had a diameter of about 88.22 nm (Figure 3B). Particle sizes bigger than 100 nm, on the other hand, accounted for barely 10% of all solutions. The polydispersity index (Pdi) was 0.7 and 0.4 in the chemical and natural methods, respectively.

Figure 3

Size distributions of IONPs synthesized chemically (A) or using Petroselinum crispum leaf extract (B) in colloids. Size distribution of the particles was estimated from laser particle analyzer (LPA) images by measurement of diameters provided by the computer programming system of Zetasizer (Nano ZS, Malvern, UK).

Table 1 shows the iron profile in lead-acetate-induced anemic rats treated with chemically or naturally biosynthesized IONPs. The concentrations of serum ferritin and iron were significantly lower in non-treated lead-acetate-induced anemic rats than in the negative control. TIBC was significantly higher in the non-treated lead-acetate-induced anemic rats than in the negative control. Compared to anemic rats treated with chemical IONPs and non-treated anemic rats, ferritin concentrations in the anemic rats treated with IONPs prepared from Petroselinum crispum leaf extract restored to normal levels. Anemic rats treated with chemical or natural IONPs had significantly higher iron concentrations than non-treated anemic rats did, but they remained lower than the negative control values. TIBC returned to the normal level in anemic rats treated with either natural or chemical IONPs.

Table 1

Iron profile in lead-acetate-induced anemic rats treated with chemical or green biosynthesized magnetic iron oxide nanoparticles.

Groups	Ferritin (ng/dL)	Iron (µg/dL)	TIBC (mcg/dL)
1	0.70 ± 1.5 a	2.31 ± 1.8 a	6.01 ± 1.4 b
2	0.47 ± 0.2 b	1.29 ± 0.1 c	8.53 ± 0.2 a
3	0.50 ± 0.1 b	1.89 ± 0.4 b	6.26 ± 0.7 b
4	0.70 ± 0.3 a	1.64 ± 0.2 b	6.96 ± 0.3 b

Values are expressed as mean ± standard deviation (SD) of five rats for each group. a–d Means with different superscripts in the same column differ significantly at p < 0.05. Group 1: Rats administered distilled water orally throughout the experimental period without any treatment (negative control). Group 2: Non-treated lead-acetate-induced anemic rats (positive control). Group 3: Lead-acetate-induced anemic rats treated with daily oral administration of 27 ppm of chemically prepared iron oxide nanoparticles until the end of the experiment. Group 4: Lead-acetate-induced anemic rats treated with daily oral administration of 27 ppm of plant iron oxide nanoparticles prepared from Petroselinum crispum leaf extract until the end of the experiment. TIBC: Total iron-binding capacity.

Hematological indices in lead-acetate-induced anemic rats treated with chemically or naturally biosynthesized IONPs are illustrated in Table 2. The values of Hb, RBCs, MCV, MCH, MCHC and PCV percentage reduced significantly in non-treated lead-acetate-induced anemic rats compared to those of the negative control. These parameters returned to the normal level in anemic rats treated with either natural or chemical IONPs. The improvement in Hb and RBCs was more pronounced in anemic rats treated with IONPs produced by Petroselinum crispum leaf extract compared to that of anemic rats treated with chemical IONPs. The liver and kidney functioning in lead-acetate-induced anemic rats treated with chemical or natural biosynthesized IONPs are illustrated in Table 3. The concentrations of serum urea and creatinine and the activities of ALT and AST increased significantly in non-treated lead-acetate-induced anemic rats compared to those of the negative control. These parameters improved significantly in anemic rats treated with either chemical or natural IONPs compared to non-treated anemic rats, but they remained lower than the negative control values. The improvement of these parameters was more pronounced in anemic rats treated with IONPs produced by Petroselinum crispum leaf extract compared to anemic rats treated with chemical IONPs.

Table 2

Hematological indices in lead-acetate-induced anemic rats treated with chemical or green biosynthesized magnetic iron oxide nanoparticles.

Groups	Hb (mg/dl)	RBCs (×10⁶ µL)	PCV (%)	MCV (fl/cell)	MCH (pg/cell)	MCHC (g/dL)
1	13.8 ± 2.7 a	6.07 ± 1.6 ab	36.6 ± 5.5 a	60.5 ± 3.5 a	20.43 ± 1.2 a	33.9 ± 3.1 a
2	8.73 ± 2.36 c	5.48 ± 1.2 c	28.3 ± 3.4 b	51.7 ± 1.6 b	19.07 ± 1.5 b	30.7 ± 1.5 b
3	11.7 ± 1.3 b	6.09 ± 1.7 ab	36.83 ± 1.8 a	60.8 ± 1.5 a	20.8 ± 1.0 a	32.67 ± 1.3 a
4	12.66 ± 2.5 a	6.57 ± 1.5 a	39.53 ± 2.0 a	60.13 ± 2.8 a	20.73 ± 1.1 a	32.5 ± 0.3 a

Values are expressed as mean ± standard error of means (SD) of five rats for each group. a–d Means with different superscripts in the same column differ significantly at p < 0.05. Group 1: Rats administered distilled water orally throughout the experimental period without any treatment (negative control). Group 2: Non-treated lead-acetate-induced anemic rats (positive control). Group 3: Lead-acetate-induced anemic rats treated with daily oral administration of 27 ppm of chemically prepared iron oxide nanoparticles until the end of the experiment. Group 4: Lead-acetate-induced anemic rats treated with daily oral administration of 27 ppm of plant iron oxide nanoparticles prepared from Petroselinum crispum leaf extract until the end of the experiment.

Table 3

Liver and kidney function in lead-acetate-induced anemic rats treated with chemical or green biosynthesized magnetic iron oxide nanoparticles.

Groups	Urea (mg/dL)	Creatinine (mg/dL)	AST (U/mL)	ALT (U/mL)
1	24.89 ± 4.4 d	0.71 ± 0.1 c	64.1 ± 6.1 d	27.9 ± 3.4 d
2	125.87 ± 5.1 a	3.21 ± 0.9 a	145.7 ± 6.4 a	60.00 ± 1.7 a
3	55.50 ± 3.3 b	1.61 ± 0.3 b	111.9 ± 8.8 b	44.67 ± 3.1 b
4	42.98 ± 6.1 c	0.97 ± 0.3 c	84.62 ± 9.1 c	37.34 ± 3.0 c

Values are expressed as mean ± standard deviation (SD) of five rats for each group. a–d Means with different superscripts in the same column differ significantly at p < 0.05. Group 1: Rats administered distilled water orally throughout the experimental period without any treatment (negative control). Group 2: Non-treated lead-acetate-induced anemic rats (positive control). Group 3: Lead-acetate-induced anemic rats treated with daily oral administration of 27 ppm of chemically prepared iron oxide nanoparticles until the end of the experiment. Group 4: Lead-acetate-induced anemic rats treated with daily oral administration of 27 ppm of plant iron oxide nanoparticles prepared from Petroselinum crispum leaf extract until the end of the experiment.

The examined kidneys of non-treated lead-intoxicated rats revealed an extensive congestion of the renal blood vessels and inter-tubular and glomerular blood capillaries. Hemorrhage in the glomeruli and thrombosis of the renal blood vessels (Figure 4a) were observed in non-treated lead-intoxicated rats. Additionally, vacuolation of the glomerular endothelial cells and necrosis and atrophy of the glomerular tuft with widening of Bowman’s space were demonstrated in non-treated lead-intoxicated rats (Figure 4b). Furthermore, the lining epithelium of the convoluted tubules in the renal cortex showed extensive degenerative changes such as vacuolation, hydropic degeneration, desquamation and necrosis with pyknotic nuclei and the presence of eosinophilic hyaline casts in the lumen of some renal tubules (Figure 4b). Inter-tubular hemorrhage as well as mononuclear leukocytic cellular infiltration in interstitial tissue (Figure 4c) were observed in the renal cortex of non-treated lead-intoxicated rats. Interestingly, clumps of amorphous blue staining lead pigment were precipitated in variable quantities in the cytoplasm of the degenerated tubules (Figure 4d).

Figure 4

Histopathological features of kidneys in lead-acetate-induced anemic rats (a–d) treated with 27 ppm/day iron oxide nanoparticles (e) prepared chemically or (f) green biosynthesized with Petroselinum crispum leaf extract for 6 weeks. (a) Thrombosis of the renal blood vessels (T letter) with degenerative changes of the lining epithelium of the convoluted tubules (arrow). (b) Necrosis and atrophy of the glomerular tuft (N letter) with extensive hydropic degeneration of the lining epithelium of some renal tubules (D letter) and its necrosis with pyknotic nuclei in other tubules (arrow). (c) Inter-tubular mononuclear leukocytic cellular infiltration, mainly lymphocytes (arrow). (d) Precipitation of lead pigment either in the lining epithelium of the convoluted tubules or in the lumen of renal tubules (arrow ×1000). (e) Vacuolation of the lining epithelium of some renal tubules (arrow) and (f) fibrous connective tissue proliferation around the degenerated renal tubules (arrow).

Treatment of anemic rats with either chemical or natural IONPs (27 ppm/day) improved the pathological alterations produced by lead acetate in the kidneys to variable degrees. However, the microscopic examination of the kidneys of anemic rats treated with chemically prepared IONPs displayed mild improvements in the congestion of the renal blood vessels and glomerular blood capillaries induced by lead acetate (Figure 4e). Degenerative changes in the glomeruli that manifested by vacuolation of the endothelial cell lining of the glomerular tuft were also noticed (Figure 4e). Additionally, vacuolar degeneration of some proximal and distal convoluted tubules characterized by swollen pale vacuolated cytoplasm was also observed (Figure 4e).

Interestingly, the microscopic examination of the kidneys of anemic rats treated with naturally prepared IONPs revealed distinct improvements in renal tissue histology compared to the control group (Figure 4f). Mild congestion of the renal blood vessels and glomerular blood capillaries with mild vacuolation of the endothelial cell lining of the glomerular tuft was seen in some examined cases (Figure 4f). Meanwhile, hyper-cellularity of the glomerular tuft to the extent of its adhesion with the parietal layer of Bowman’s capsule and the absence of Bowman’s space were also demonstrated in other cases (Figure 4f). However, mild degenerative changes in the epithelial cell lining of the renal tubules in the form of cloudy swelling in association with small amounts of eosinophilic debris were noticed in the lumen of some renal tubules (Figure 4f). Interestingly, blue staining of the lead substance was not observed in the cytoplasm of the degenerated tubules of both the renal cortex and medulla. The microscopic examination of the renal tissue taken from negative control rats revealed a normal histological structure of renal tissues.

The microscopic examination of livers obtained from non-treated lead-intoxicated rats showed congestion of the hepatic blood vessels and blood sinusoids with distension of the portal area with mononuclear leukocytic cells—mainly lymphocytes and macrophages (Figure 5a). Additionally, there was marked damage of hepatocytes in the form of diffuse hydropic degeneration with apoptotic bodies in some degenerated hepatocytes (Figure 5b). Multifocal areas of coagulative necrosis of hepatocytes with pyknotic nuclei and diffuse hemorrhage in the hepatic parenchyma (Figure 5c) were demonstrated. The variable degrees of lesion score in renal tissues obtained from different groups are summarized in Table 4.

Figure 5

Histopathological features of liver in lead-acetate-induced anemic rats (a–c) treated with 27 ppm/day iron oxide nanoparticles (d,e) prepared chemically or (f) green biosynthesized with Petroselinum crispum leaf extract for 6 weeks. (a) Congestion of the portal veins with mild hyperplasia of the biliary epithelium (arrow). (b) Focal area of lytic necrosis characterized by loss of hepatocytes and replaced by aggregates of mononuclear leukocytes (arrow). (c) Apoptotic bodies in some degenerated hepatocytes (arrow, ×1000). (d) Degenerative changes of hepatocytes in combination with fibrous connective tissue proliferation around the bile duct (arrow). (e) Portal areas were mildly expanded by aggregates of mononuclear inflammatory cells—mainly lymphocytes and macrophages—in association with mild hyperplasia of the lining epithelium of the bile duct. (f) Improvement in the hepatocellular architecture with more regular and less altered hepatocytes.

Table 4

Effect of chemical and natural iron oxide nanoparticles on the pathological grading of lead toxicity in renal tissues of different treated groups.

Groups	Number	Pathological Grading
		0	I	II	III
1	5	5	0	0	0
2	5	0	1	1	3
3	5	0	1	3	1
4	5	3	1	1	0

Group 1: Rats administered distilled water orally throughout the experimental period without any treatment (negative control). Group 2: Non-treated lead-acetate-induced anemic rats (positive control). Group 3: Lead-acetate-induced anemic rats treated with daily oral administration of 27 ppm of chemically prepared iron oxide nanoparticles until the end of the experiment. Group 4: Lead-acetate-induced anemic rats treated with daily oral administration of 27 ppm of plant iron oxide nanoparticles prepared from Petroselinum crispum leaf extract until the end of the experiment.

However, the microscopic examination of hepatic tissues obtained from anemic rats treated with chemically prepared IONPs (27 ppm/day) revealed mild amelioration in the histopathological alterations induced by lead acetate. The examined livers of these rats showed mild congestion of central and portal veins and blood sinusoids (Figure 5d). Occasionally, mild perivascular mononuclear inflammatory cells, mainly lymphocytes and macrophages, infiltrating in association with centrilobular hydropic degeneration of hepatocytes were observed. This degeneration was characterized by swollen, pale, vacuolated cytoplasm as well as a small focal area of mononuclear leukocytes, mainly lymphocytes and macrophage aggregations that replaced the hepatic parenchyma. Multifocally, the portal areas were mildly expanded by aggregates of mononuclear inflammatory cells, mainly lymphocytes and macrophages, in association with mild hyperplasia of the lining epithelium of the bile duct (Figure 5e).

Interestingly, improvements in the hepatocellular architecture with more regular and less altered hepatocytes were noticed in the hepatic tissue obtained from anemic rats treated with naturally prepared IONPs compared to that of non-treated anemic rats (Figure 5f). The hepatic tissue reconditioned its normal histological structure in comparison to the negative control group. Most of the hepatic parenchyma showed a certain degree of recovery, except for the portal area, which appeared normal and contained a normal bile duct with a mildly congested portal vein only (Figure 5f). The livers of these treated rats showed mild congestion and dilatation of the central and portal veins, and blood sinusoids with activation of Von Kuepfer cells (Figure 5f). The microscopic examination of the hepatic tissue taken from negative control rats revealed ordinary histological structures of hepatic parenchyma, the portal area and hepatic blood vessels. The lesion scores in hepatic tissue obtained from different groups are summarized in Table 5.

Table 5

Effect of chemical and natural iron oxide nanoparticles on the pathological grading of lead toxicity in hepatic tissue of different treated groups.

Groups	Number	Pathological Grading
		0	I	II	III
1	5	5	0	0	0
2	5	0	1	1	3
3	5	0	2	3	0
4	5	2	2	1	0

Group 1: Rats administered distilled water orally throughout the experimental period without any treatment (negative control). Group 2: Non-treated lead-acetate-induced anemic rats (positive control). Group 3: Lead-acetate-induced anemic rats treated with daily oral administration of 27 ppm of chemically prepared iron oxide nanoparticles until the end of the experiment. Group 4: Lead-acetate-induced anemic rats treated with daily oral administration of 27 ppm of plant iron oxide nanoparticles prepared from Petroselinum crispum leaf extract until the end of the experiment.

The splenic tissues of anemic non-treated rats showed ill-defined histological structures such as congested blood vessels and sinusoids, with extensive diffuse areas of hemorrhages observed in the red pulp (Figure 6a). Additionally, expansion of the interstitium by aggregates of mononuclear inflammatory cells, mainly lymphocytes and macrophages, was detected in some lymphoid follicles of the white pulp. Marked necrotic foci were detected in some lymphoid follicles of the white pulp and were accompanied by lymphoid depletion (Figure 6b).

Figure 6

Histopathological features of spleen in lead-acetate-induced anemic rats (a,b) treated with 27 ppm/day iron oxide nanoparticles (c) prepared chemically or (d) green biosynthesized with Petroselinum crispum leaf extract for 6 weeks. (a) Extensive diffuse areas of hemorrhages in the red pulp. (b) Lymphoid depletion of the white pulp. (c) Mild lymphoid depletion of the white pulp. (d) Mild congestion of blood vessels of red pulp and normal lymphoid follicles of white pulp.

In anemic rats treated with chemically prepared IONPs, spleen samples revealed a mild reduction in the pathological alterations induced by lead acetate. In addition, congested blood vessels and sinusoids of the red pulp with mild lymphoid depletion of the white pulp were detected (Figure 6c). In anemic rats treated with naturally prepared IONPs, splenic tissue revealed marked improvements. The splenic tissue appeared well defined with mild congestion of blood vessels of the red pulp and normal lymphoid follicles of the white pulp (Figure 6d). The spleen tissue obtained from the negative control group showed a normal histological structure of the white and red pulps. The variable degrees of lesion score in spleens obtained from different groups are summarized in Table 6.

Table 6

Effect of chemical and natural iron oxide nanoparticles on the pathological grading of lead toxicity in splenic tissue of different treated groups.

Groups	Number	Pathological Grading
		0	I	II	III
1	5	5	0	0	0
2	5	0	1	2	2
3	5	0	2	3	0
4	5	3	1	1	0

Group 1: Rats administered distilled water orally throughout the experimental period without any treatment (negative control). Group 2: Non-treated lead-acetate-induced anemic rats (positive control). Group 3: Lead-acetate-induced anemic rats treated with daily oral administration of 27 ppm of chemically prepared iron oxide nanoparticles until the end of the experiment. Group 4: Lead-acetate-induced anemic rats treated with daily oral administration of 27 ppm of plant iron oxide nanoparticles prepared from Petroselinum crispum leaf extract until the end of the experiment.

4. Discussion

TEM analysis indicated that IONPs’ sizes when produced either chemically or using Petroselinum crispum leaf extracts were 6.22–9.7  nm [47] and 64–68  nm [48,49], respectively. The prepared IONPs were well dispersed in the solution, with oval, cubic and spherical shapes [47,50]. The Zetasizer average diameters of the IONPs synthesized either chemically or using Petroselinum crispum leaf extracts were similar to those described earlier [51,52,53,54].

Lead is a multi-organ toxicant implicated in various cancers and diseases of the hepatic, renal, hematopoietic, nervous and reproductive systems [55]. Lead poisoning is associated with physiological, morphological and biochemical alterations, such as liver dysfunction [56] and hematological disorders [13]. Iron profile and blood indices are important tools that can be used as reliable and sensitive indices to monitor anemia. The current findings revealed a significant decrease in serum ferritin (iron store) and iron but an increase in TIBC in lead-acetate-induced anemic non-treated rats when compared to the negative control. The significant increase in TIBC could be due to the liver’s increased production of transferrin in an attempt to maximize the utilization of the low available iron [57,58,59]. A similar result [60] showed that lead toxicity induced iron deficiency anemia by impairing heme biosynthesis. Reduced or absent serum ferritin, decreased transferrin saturation and a significant increase in TIBC indicate iron deficiency anemia [61].

Iron absorption and oral bioavailability may be enhanced by IONPs [62]. In the current study, liver histopathology revealed an improvement in liver tissues in intoxicated rats treated with IONPs. In addition, ALT and AST improved in intoxicated rats treated with IONPs. Therefore, anemia and liver dysfunction were improved. This could explain why the ferritin concentrations in inebriated rats treated with IONPs (group 4) were not significantly different from those in the negative control group (group 1). An increase in serum iron and ferritin, as well as a decrease in TIBC, was observed in lead-acetate-induced anemic rats treated with chemical or natural IONPs. Iron absorption and oral bioavailability are improved by IONPs, which are markers of anemia relief [62]. Lead acetate toxicity can cause anemia by impairment of heme biosynthesis and subsequent induction of iron deficiency anemia [60]. Iron deficiency anemia is characterized by low serum iron concentration, decreased transferrin saturation and a marked increase in TIBC [61]. Treatment of lead-acetate-induced anemic rats with either chemical or natural IONPs improved the absorption and enhanced the bioavailability and cellular uptake of iron [16,17,18].

Microcytic hypochromic anemia leads to significant decreases in RBCs, Hb, PCV percentage, MCV, MCH and MCHC [63]. In the present study, lead acetate toxicity-induced microcytic hypochromic anemia was reflected in the decreased RBCs, Hb, PCV percentage, MCV, MCH and MCHC levels [13,64]. In addition, lead acetate inhibits several enzymes that are critical for heme biosynthesis [65]. The toxic effect of lead acetate on cell metabolism, which suppresses several enzymatic activities such as aminolevulinic dehydratase and ferrochelatase that play a key part in heme production, may be responsible for the decrease in blood indices [38,66,67]. This suppression may refer to the imperfection of iron metabolism [68].

In the current study, RBCs, Hb, PCV%, MCV, MCH and MCHC returned to the normal level in anemic rats treated with either natural or chemical IONPs. The improvement in these hematological indices may be attributed to the enhanced bioavailability, increased surface area and improved cellular uptake following the nanonization of iron [62,69,70]. The current histopathological results confirmed this mechanism. The livers of non-treated lead-acetate-induced anemic rats (positive control) showed congestion of the hepatic blood vessels and blood sinusoids with distension of the portal area with blood as well as diffuse hemorrhage. Moreover, the examined kidneys of non-treated lead-acetate-induced anemic rats (positive control) revealed extensive congestion of the renal blood vessels and inter-tubular and glomerular blood capillaries, with hemorrhage in the glomeruli with thrombosis of the renal blood vessels. In addition, the splenic tissue of the same group showed congested blood vessels and sinusoids with extensive diffuse areas of hemorrhages in the red pulp.

However, lead-acetate-induced anemic rats treated with either chemical or natural IONPs showed a mildly congested portal vein only. The livers of these treated rats showed mild congestion and dilatation of central and portal veins and blood sinusoids with activation of Von Kuepfer cells. The splenic tissue of these groups revealed a marked improvement. This improvement manifested in the blood indices as well-defined splenic tissue with minor congestion of red pulp blood vessels, which improved the anemic picture of the blood analysis.

The current study investigated serum urea and creatinine to detect the renal function. The current findings indicate that concentrations of serum urea and creatinine and the activities of ALT and AST increased significantly in non-treated lead-acetate-induced anemic rats compared to the negative control. Similar results have been obtained in rats intoxicated with lead acetate [13,56]. These results confirmed the current histopathological findings. Vacuolation of the glomerular endothelial cells and necrosis and atrophy of the glomerular tuft with widening of Bowman’s space were demonstrated. Furthermore, the lining epithelium of the convoluted tubules in the renal cortex showed extensive degenerative changes such as vacuolation, hydropic degeneration, desquamation and necrosis with pyknotic nuclei and the presence of eosinophilic hyaline casts.

In the current study, serum urea and creatinine improved significantly in anemic rats treated with either chemical or natural IONPs compared to non-treated anemic rats. Similar findings [57] showed that the kidneys of rats treated with 10 and 30 ppm nano-iron showed normal histopathological structures of glomeruli and renal tubules, while the 60 ppm nano-iron-treated groups showed mild inflammatory cell infiltration in between renal tubules.

In the current study, ALT and AST activities were estimated to investigate the state of liver function. The findings indicate that the activities of ALT and AST increased significantly in non-treated lead-acetate-induced anemic rats compared to the negative control. This elevation may be attributed to the toxic effect of lead acetate, which increases hepatocytes’ permeability or damages the hepatocyte cell membrane with subsequent release of AST and ALT enzymes. Elevated serum AST and ALT levels have been estimated after lead hepatotoxicity [71]. The increase in ALT and AST after lead toxicity may be attributed to the increase in cellular basal metabolic rate, destructive alternation of liver cells and production of free radicals [56,72]. These findings are reinforced by the current histopathological picture, which indicated marked hepatocytic damage in the form of diffused hydropic degeneration with apoptotic bodies in some degenerated hepatocytes. Multifocal areas of coagulative necrosis of hepatocytes with pyknotic nuclei were observed in the non-treated lead-acetate-induced anemic rats compared to the control.

In the current study, serum ALT and AST significantly improved in anemic rats treated with either chemical or natural IONPs compared to non-treated anemic rats. This finding was confirmed by the current histopathological findings. Improvements in the hepatocellular architecture with more regular and less altered hepatocytes were noticed in the hepatic tissue obtained from anemic rats treated with naturally prepared IONPs compared to that of non-treated anemic rats. Previous research [57] showed that the livers of rats treated with 10 and 30 ppm nano-iron showed no abnormal histopathological alternation, with normal central veins and surrounding hepatocytes. In the same study, the 60 ppm nano-iron-treated group showed mild inflammatory cell infiltration, which explains the mild elevation in AST and ALT activities.

5. Conclusions

TEM analysis indicated that the sizes of IONPs prepared by either chemical or natural methods were 6.22–9.7 and 64–68 nm, respectively. Serum ferritin and iron concentrations were significantly reduced in non-treated lead-acetate-induced anemic rats compared to the control. Total iron-binding capacity (TIBC), ALT, AST, urea and creatinine were significantly increased in the non-treated lead-acetate-induced anemic rats compared to the control. Congestion, hemorrhage, necrosis, vacuolation and leukocytic infiltration in the kidneys, liver and spleen were observed in non-treated lead-acetate-induced anemic rats compared to the control. The effects of lead acetate were mitigated by IONPs, particularly the natural ones. The current study suggests that IONPs biosynthesized from Petroselinum crispum leaf extract can be used as an efficient, safe and eco-friendly anti-anemic therapy in lead-acetate-induced anemic rats.