Selenium and zinc supplementation mitigates metals-(loids) mixture- mediated cardiopulmonary toxicity via attenuation of antioxidant, anti-inflammatory and antiapoptotic mechanisms in female Sprague Dawley rats

Mfoniso Antia; Anthonet N Ezejiofor; Chinna N Orish; Ana Cirovic; Aleksandar Cirovic; Orish E Orisakwe

doi:10.1093/toxres/tfad119

. 2023 Dec 9;13(1):tfad119. doi: 10.1093/toxres/tfad119

Selenium and zinc supplementation mitigates metals-(loids) mixture- mediated cardiopulmonary toxicity via attenuation of antioxidant, anti-inflammatory and antiapoptotic mechanisms in female Sprague Dawley rats

Mfoniso Antia ¹, Anthonet N Ezejiofor ², Chinna N Orish ³, Ana Cirovic ⁴, Aleksandar Cirovic ⁵, Orish E Orisakwe ^6,^✉

PMCID: PMC10762678 PMID: 38179003

Abstract

This study evaluated the cardiopulmonary protective effects of essential elements (Zn and Se) against heavy metals mixture (HMM) exposure. Twenty five female Sprague Dawley albino rats, divided in to five groups: controls were orally treated only with distilled water; next, group 2 was exposed to HMM with the following concentrations: 20 mg/kg of Pb body weight, 0.40 mg/kg of Hg, 0.56 mg/kg of Mn, and 35 mg/kg of Al. Groups 3, 4 and 5 were exposed to HMM and co-treated with zinc chloride (ZnCl₂; 0.80 mg/kg), sodium selenite (Na₂SeO₃;1.50 mg/kg) and both zinc chloride and sodium selenite, respectively. The experiment lasted for 60 days. Afterwards animals were sacrificed, and we conduced biochemical and histopathological examination of the heart and lungs. HMM only exposed animals had an increased levels of malondialdehyde (MDA) and nitric oxide (NO), increased IL-6 and TNF-α, attenuated SOD, GPx, CAT and GSH and caspase 3 in the heart and lungs. HMM affected NF-kB and Nrf2 in the heart muscle with histomorphological alterations. Zn and Se attenuated adverse effects of HMM exposure. Essential element supplementation ameliorated heavy metal cardiopulmonary intoxication in rats.

Keywords: heavy metals, metalloids, essential trace elements, oxido-inflammatory, heart, lungs

Graphical Abstract

Schematic approach to experimental design (used heavy metals mixture and duration of exposure), examined organs and main outcomes of this study.

Introduction

Proper and synchronous heart and lungs actions are necessary for an adequate function of all other organs. The pulmonary and cardiovascular systems are closely knitted together with the primary role of adequate delivering oxygen to the tissues. This interwoven relationship implies imply that a defect of either system inimically affects the other, hampering cardiopulmonary function, systemic oxygen supply, and activity of other major organs¹ i.e. decreased function of either of those (the heart and lungs) leads to suffering of other organs.

Lead Pb, Mercury Hg, Manganese Mn and Aluminum Al, ranked 2^nd, 3^rd, 140^th and 183^rd on the Agency for Toxic Substances and Disease Registry ATSDR Substance Priority List.² These metals, especially Pb and Hg, are of profound public health concern because of their inadvertent bioaccumulation with high toxicity in different organs and tissues.³^,⁴ The toxicity of these metals is premised on the disruption of cellular homeostatic condition via molecular mimicry, oxidative damage, and adduct formation with DNA or protein.⁵ Pb has toxicological effects on many organs including the lungs and heart.⁶ Pb creates reactive radicals which damage cell structures including DNA and cell membrane, interferes with the enzymes that maintain the integrity of the cell membrane, interferes with DNA transcription and increases endoplasmic reticulum stress.⁷ Exposure to Hg may damage lungs, change mucous membrane and may cause vomiting, nausea, skin rashes, increased blood pressure, renal dysfunction and severe neurological abnormalities.⁸ Hg induces genotoxic effects through oxidative stress, disturbs calcium homeostasis, and disrupts neurotransmitter systems and cytoskeletal interactions.⁹ Moreover, we have to keep in mind that for examples heart is incomparably more sensitive to heavy metals toxicity than some other organs such as the liver or kidneys¹⁰ and therefore less concentrations of heavy metals could induce more devastating effects.

According to WHO Pb is one of the ten chemicals of utmost public health concern given it ubiquity, toxicity even at diminutive levels and now touted as having no known safe blood lead concentration for human WHO 2017. The diverse anthropogenic activities (mining, smelting, manufacturing, and recycling activities, continued use of leaded paint, leaded gasoline, and leaded aviation fuel) add to the environmental burden of metals especially Pb, Al, Hg, Mn are rife in the Niger Delta, Nigeria.¹¹^,¹² Drinking water supply, diets, soil and air are the major sources of metals The pathway of exposure includes ingestion,¹³ inhalation,¹⁴ and dermal absorption, and pathway varies depending on their sources.¹⁵ Metal exposure especially Pb can cause anemia, hypertension, immunotoxicity etc. (WHO, 2017). Pb-toxicity impedes normal cardiovascular processes.¹⁶^,¹⁷ Manganese Mn is an essential transition element which plays an important role as cofactor of many metabolic and antioxidant enzymes like Mn superoxide dismutase (Mn-SOD), the principal mitochondrial antioxidant enzyme.^18–20 Mn exposure is associated with toxicity in lung and heart.²¹ Mn can cause an inflammatory response in the lungs, with clinical symptoms including cough, acute bronchitis, and decreased lung functions.²² Aluminum can induce pulmonary injuries through increasing oxidative stress, reducing total antioxidant capacity, triggering inflammatory response, and impairing lung mechanics.^23–25 Many studies have shown that occupational exposure to aluminum is associated with the increased risks of pneumoconiosis,²⁶ asthma,²⁷and non-malignant respiratory disease.^28–30

The long period of crude oil exploration and other anthropogenic activities in Niger Delt, Nigeria have led to heavy metal especially Pb, Hg, Al and Mn contamination of foods, water and air.¹¹^,¹² All in all, metals elicit toxicity via disruption of cellular metabolism, alterations in cell adhesion, intra- and inter-cellular signaling, protein folding, maturation, protein kinase C, apoptosis, ionic transportation, enzyme regulation, and release of neurotransmitters even at low concentration mainly due to its ability to replace other divalent cations like Ca²⁺, Mg²⁺, Fe²⁺ and monovalent cations like Na+. This leads to various disorders and can also result in excessive damage due to oxidative stress induced by free radical generation.³¹ There is ample evidence from both experimental and epidemiological studies that the combined effects of multi-heavy metals might be quite different from that induced by the individual metals.^32–34

Anti-oxidant and anti-inflammatory agents are known to be beneficial in tissue injury.³⁵^,³⁶ Zinc Zn a cofactor of several metabolic enzyme and selenium Se, which occupies the active centers of some proteins in the form of selenocysteine, are essential microelements considered vital in the maintenance of cellular homeostasis. The protective role of Zn and Se in cardiopulmonary injury, is largely unknown, therefore, this study has evaluated the anti-oxidant and anti-inflammatory effects of Zn, Se and Zn plus Se combination in heavy metal mixture mediated cardiopulmonary injury. This study hypothesized that Zn, Se and Zn plus Se combination supplementation might be beneficial in Pb, Al, Hg, Mn mixture-induced injury in the cardiopulmonary system of rat via attenuating oxidative stress, activating antioxidant enzymes, and reducing apoptosis and inflammatory response.

Materials and methods

Chemicals

All used chemicals, Pb(C₂H₃O₂)₂, AlCl₃, HgCl₂ and MnCl₂ were bought from Sigma Chemical Co. (St. Louis, MO, USA). Inflammatory cytokines (tumor necrosis factor alpha; TNF—α, interleukin 6; IL—6), transcription factors and apoptotic marker [(nuclear factor kappa B; NF-kB), (nuclear factor erythroid 2-related factor 2; Nrf2) and (Caspase 3)] ELISA Kit (for rats) were obtained from Elabscience science Biotechnology Company, (Beijing, China). All other reagents used were of analytical grade and they were obtained from the British Drug Houses (Poole, Dorset, UK).

Animals and treatments

Female Sprague Dawley albino rats (n = 25; 6–8 weeks old) were provided by the Department of Pharmacology, Animal House, University of Port Harcourt, Rivers State, Nigeria. All the rats were kept in polypropylene cages at the room temperature of 25 ± 2 °C with 12-h light/dark cycles throughout the experiment. Before the beginning of the study, the animals had been acclimatized for fourteen days. The experimental animals (n = 25) were divided into five groups and each group consisted of five animals. Controls (group 1) were orally treated with distilled water for 60 days; group 2 was exposed to heavy metal mixture (HMM) with the following concentrations:20 mg/kg of Pb body weight,³⁷ 0.40 mg/kg of Hg,³⁷ 0.56 mg/kg of Mn, and 35 mg/kg of Al.³⁸ Groups 3, 4 and 5 were exposed to HMM and co-treated with zinc chloride (ZnCl₂; 0.80 mg/kg),³⁹ sodium selenite (Na₂SeO₃;1.50 mg/kg)⁴⁰ and both zinc chloride and sodium selenite, respectively. The heavy-metal compounds were separately dissolved as a stock solution before use to avoid precipitation and they were diluted to the working concentration using ultrapure water. Environmentally relevant doses of metal mixtures according to some recently conducted studies from various matrices of the Niger Delta, Nigeria, were used in this study.¹² The exposure doses of metals mixture in this study were considered low³⁷; moreover, they are in the lower range when compared to the doses of metals used in similar in vivo studies.⁴¹^,⁴²

The rats were weighed weekly, and their feed and fluid intake were recorded. After 60 days of exposure, the animals were euthanized by intraperitoneal administration of pentobarbital (50 mg/kg). The heart and lungs of each rat were dissected and rinsed in cold saline water, weighed, and used for both biochemical parameters and heavy metal analyses.

The ethical approval was obtained from the University of Port Harcourt institutional Centre for Research Management and Development Animal Care and Use Research Ethics Committee (UPH/PUTOR/REC/12). The experiment was conducted in accordance with the “Guide for the Care of Laboratory Animals” approved by the National Academy of Science (NAS). The animals received standard feed and deionized water ad libitum.

Sample collection and heart and lung tissues preparation

The rats were euthanized using pentobarbital (intraperitoneal administration; 50 mg/kg dose), and the heart and lungs samples were quickly dissected and stored at −80 °C for metal and biochemical analyses.

The heart and lungs were separately homogenized in 9 vol of cold phosphate buffer (0.1 M: pH 7.4) using homogenizer. The tissue homogenates were centrifuged at 3,000 rpm for 20 min at 4 °C to separate the nuclear debris. The heart and lungs lysates were used for assays of MDA, NO, GSH, GPx, GST, SOD and for ELISA assays [(Tumour necrosis factor alpha (TNF-α), Interleukin-6 (IL-6), Nuclear factor E2-related factor 2 (NRF2), Factor Kappa B (NF-kB), Casp-3).

ELISA analysis

The assay that determined the cytokine levels, transcription factors, and biomarkers of apoptosis in the heart and lungs were described in another study.⁴³ Pro-inflammatory cytokines (IL-6 and TNF-α), transcription factors (NF-kB and Nrf2), (Hmox1), and a biomarker of apoptosis (Caspase 3) were analysed using the instructions provided by the manufacturer (Elabcience Biotechnology Company (Beijing, China)). An ELISA kit (Elabscience Biotechnology Company, (Beijing, China) was used to measure supernatant concentrations in pro-inflammatory cytokines (IL-6 and TNF-α), NFKB and Nrf2, (Hmox1), and the biomarker of apoptosis (Caspase 3) according to the manufacturer’s instructions.

Antioxidant and oxidative stress markers

Glutathione related antioxidants (GPx), reduced glutathione (GSH), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase, lipid peroxidation, nitric oxide

In order to measure activity of various antioxidants we used following methodology Paglia and Valentine⁴⁴ for the GPx measurement (412 nm), Jollow et al.’s method⁴⁵ for the GSH measurements (412 nm), Habig et al.’s method⁴⁵ for the GST activity (310 nm), using the following equation: GST activity (μmol min⁻¹ mg⁻¹ protein) = At/(1.9 × time × mg protein). SOD activity was assayed using 20 μL of the heart and lungs (separately) supernatant sample (test) or buffer (reference) and 10 μL pyrogallol (20 mM in 10 mM HCl) to which, 1 mL buffer solution was added.⁴⁶ The absorbance of test (At) or reference (Ar) was measured at 420 nm against the air after 30 s and 90 s. The percentage inhibition of pyrogallol autoxidation was calculated according to the following equation: the percentage inhibition = [100 – (At min⁻¹ mL⁻¹ sample)/(Ar min⁻¹ mL⁻¹ reference)] × 100. Catalase activity was estimated by monitoring the rate of H₂O₂ breakdown at 240 nm according to Aebi’s method.⁴⁷ Briefly, 990 μL of catalase buffer (0.036% H₂O₂ prepared in 50 mM phosphate buffer, pH 7.0) was added to 10 μL of the heart and lungs lysates separately in a cuvette. Catalase activity was assayed immediately at 240 nm for 3 min and expressed as μmol/min/mg protein.

Lipid peroxidation was analyzed as thiobarbituric acid reactive substances (TBARS) by the adaptation of Esterbauer and Cheeseman method.⁴⁸ In a nutshell, 500 μL of heart and lungs supernatant, separately, was added to one ml TCA (20%) and mixed thoroughly. The mixture was centrifuged at 3,000 rpm for 10 min. One ml of the supernatant was added to 0.5 mL of 0.7% TBA and allowed to boil for 10 min. After cooling, the absorbance was read at 532 nm against blank.

Nitric oxide (NO): this assay adapted the Griess reaction technique.⁴⁹^,⁵⁰ One microliter 100 μL of heart and lungs (separately) supernatant was added to 100 μL acidic Griess reagent (1% sulfanilamide and 0.1% naphthlethylenediamine dihydrochloride in 2.5% phosphoric acid). The absorbance was read at 540 nm against blank.

Determination of heavy metals

The harvested heart and lungs were dried for 48 h, then weighed and placed in 10 mL conical flasks with polypropylene lids containing 3 mL of HNO₃ at room temperature until the solution became clear. Then, 1 mL of 30% H₂O₂ was added to the samples. At the end of effervescence, the samples were heated at 80 °C to remove the HNO₃, cooled to room temperature and the final volume was made up to 10 mL with 2% HNO₃. The samples were brought to a constant volume.

In order to ensure reproducible and reliable data the instrument was recalibrated after every10 runs. Chemical reagents and solvents were of picograde quality. The calibration curves for each element were obtained with high purity multi-cathode lamp (1,000 mg/kg) (Cambridge CB5 8BZ, UK) and multi element calibration curves were verified with a multi-element certified material (1,000 mg/kg) (Cambridge). Different concentrations (0.5, 1.0, 2.0, 5.0, and 10.0 mg/L) of trace elements were used for calibration of standard graphs.⁵¹The elements were measured at the stipulated wavelengths Pb: 216.9 nm, Al: 309.3 nm, Hg: 253.7 nm, and Mn: 530 nm. The limits of detection (LoD) were 0.001 for Al, Hg and Mn and 0.01 mg/kg for Pb, while the limits of quantification (LoQ) were 0.0033 for Al, Hg and Mn and 0.033 mg/kg for Pb. Pb, Al, Hg and Mn (Certified Reference Materials) standard solutions (1,000 mg/L) were obtained from the National Institute of Standards and Technology (NIST, USA) and diluted appropriately to obtain working standards.

Histopathological examination

On day 60, the animals were sacrificed and submitted to transcardiac perfusion with heparinized saline 0.9% solution followed by 4% paraformaldehyde in 0.2 M phosphate buffer. Surgical manipulation was performed only after the removal of the corneal and paw reflexes. The heart and lungs were excised and postfixed for 6 h in Bouin’s solution. The specimens were then washed with 50% alcohol, dehydrated in alcohol solutions in progressive concentrations clarified in xylol and embedded in paraffin.

The heart and lungs were harvested at the end of the experiment, and immediately fixed with 4% paraformaldehyde. These were embedded in paraffin and sectioned into 5 μm slices for hematoxylin and eosin staining. The histological examination was blindly evaluated by two experienced histopathologists.

Statistical analysis

The data were expressed as mean ± standard deviation (SD). The Kolmogorov-Smirnov test revealed normal distribution of all the measured parameters. Microsoft Xlstat 2014 was used for performing the one-way analysis of variance (ANOVA) with the Tukey’s post-hoc test to check if the concentration of the biomarkers was significantly different between the groups. Pandas was used in obtaining descriptive statistical parameters (biomarkers and metal mean concentrations) for the various organs of the rats. Seaborn and Matplotlib were used in plotting all the graphs. The data analysis involved performing descriptive statistics of the metal and biomarker concentration before One-Way ANOVA was used to determine if there was a significant difference in the concentrations of heavy metals and biomarkers among the groups. All significant differences were at P < 0.05.

Results

Effect of essential trace elements Zn, Se, Zn + Se combination of bioaccumulation of heavy metal in heart and lungs of Sprague Dawley rats following exposure to metal mixture

Zn, Se, Zn + Se combination caused significant (P < 0.05) decrease in the concentration (25.78, 12.70 and 27.05% respectively) of Pb when compared with HMM only treated group. The essential trace elements Zn, Se, Zn + Se combination significantly (P < 0.05) decreased the concentration (20.78, 8.92 and 22.12% respectively) of Hg in comparison to the HMM only treated group. The essential trace elements Zn, Se, Zn + Se combination significantly decreased the concentration (18.42, 7.18 and 19.79% respectively) of Mn in comparison to the HMM only treated groups (P < 0.05). The essential trace elements Zn, Se, Zn + Se combination caused significant (P < 0.05) reduction in the percent concentration (25.00, 14.25 and 26.37% respectively) of Al when compared with the HMM only treated groups.

The essential trace elements Zn, Se, Zn + Se combination significantly (P < 0.05) decreased the concentration (28.62, 14.14 and 29.85% respectively) of Pb when compared with HMM only treated. Zn, Se, Zn + Se combination co-treatment significantly (P < 0.05) decreased the concentration (22.44, 8.05 and 23.44% respectively) of Hg in comparison to the HMM only treated groups. The essential trace elements Zn, Se, Zn + Se combination significantly reduced the concentration (25.78, 12.70 and 27.05% respectively) of Mn in comparison to the HMM only treated group (P < 0.05). The essential trace elements Zn, Se, Zn + Se combination significantly (P < 0.05) decreased the concentration (26.25, 12.60 and 27.60% respectively) of Al in comparison to the HMM only treated.

Table 1 shows the effect of essential elements on the body weight, absolute and relative weight of lungs and heart of female albino rats exposed to HMM. There were no significant differences in the absolute and relative weights of the lungs and heart between the HMM exposed groups from the control groups (P > 0.05); moreover, absolute and relative weights of the lungs and heart did not vary between HMM only exposed animals and HMM animals co-treated with Zn, Se and Zn combined with Se (P > 0.05). There was gain in body weight in all groups both the percent gain was higher in the essential element treated groups with zinc showing the highest percent gain.

Table 1.

Effect of essential elements on the body weight, absolute and relative weight of lungs and heart of female albino rats exposed to HMM.

Treatment	Absolute weight Lungs (g)	Relative weight Lungs (%)	Absolute weight Heart (g)	Relative weight Heart (%)	%Body weight gain	Feed intake	Fluid intake
Deionized H₂O (only)	1.30 ± 0.00^a	0.65 ± 0.00^a	0.70 ± 0.14^a	0.35 ± 0.09	58.9	154.10 ± 23.42^a	242.98 ± 35.36^a
Metal mixture (only)	1.30 ± 0.14^a	0.72 ± 0.03^a	0.60 ± 0.14^a	0.33 ± 0.13	54.5	148.27 ± 16.42^a	210.07 ± 33.46^a
Metal mixture + Zinc	0.98 ± 0.01^a	0.60 ± 0.08^a	0.52 ± 0.06^a	0.32 ± 0.05	172.6	147.94 ± 22.13^a	195.62 ± 43.20^a
Metal mixture + Selenium	0.99 ± 0.16^a	0.64 ± 0.02^a	0.52 ± 0.01^a	0.33 ± 0.01	105.5	127.63 ± 35.47^a	209.74 ± 51.27^a
Metal mixture + Zn + Se	1.01 ± 0.02^a	0.65 ± 0.03^a	0.51 ± 0.01^a	0.33 ± 0.14	126.5	127.82 ± 29.85^a	203.85 ± 53.26^a

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Values expressed as Mean ± standard deviation, N = 5. Different superscripts (a, b, c) are significantly different from each other (P < 0.05). HMM: Heavy metal mixture.

The effects of essential elements (Zn and Se) on an antioxidant (SOD, GPx, CAT, GSH) and the oxidative stress markers MDA (μmol/mL) and NO (μM/L) levels in the heart and the lungs of the female albino rats following HMM exposure is shown in Table 2. HMM significantly decreased activity of the SOD, GPx, CAT and GSH in comparison to the control group in the heart and lungs (P < 0.05). Zn, Se and Zn plus Se combination significantly increased the SOD, GPx, CAT and GSH when compared with the HMM exposed rats (P < 0.05) Table 2. The MDA and NO levels of the HMM treated rats were significantly higher than the control group in the heart and lungs (P < 0.05). Zn, Se and Zn plus Se combination significantly decreased concentrations of the MDA and NO when compared with the HMM treated rats in both examined organs (P < 0.05).

Table 2.

Effects essential elements (Zn and Se) on antioxidants (SOD, GPx, CAT, GSH), MDA (μmol/mL) and NO (μM/L) levels in heart and lungs of female albino rats after exposure to heavy metal mixtures.

Treatment	SOD		GPx		CAT		GSH		MDA		NO
Treatment	Heart	Lungs	Heart	Lungs	Heart	Lungs	Heart	Lungs	Heart	Lungs	Heart	Lungs
Control	0.36 ± 0.06^a	0.60 ± 0.04^a	0.06 ± 0.01^a	0.07 ± 0.01^a	0.84 ± 0.08^bc	2.69 ± 0.13^a	1.08 ± 0.04^b	1.45 ± 0.12^a	0.46 ± 0.01^b	0.25 ± 0.07^bc	3.58 ± 0.42^c	4.30 ± 0.03^c
HMM only	0.20 ± 0.02^b	0.36 ± 0.06^b	0.03 ± 0.001^c	0.04 ± 0.001^a	0.41 ± 0.02^d	0.82 ± 0.08^a	0.72 ± 0.02^c	0.82 ± 0.02^c	0.56 ± 0.02^a	0.495 ± 0.03^a	8.05 ± 0.09^a	8.63 ± 0.92^a
HMM + Zn	0.45 ± 0.03^a	0.52 ± 0.02^bc	0.03 ± 0.001^c	0.06 ± 0.004^a	0.67 ± 0.08^cd	1.63 ± 0.72^a	0.78 ± 0.05^c	1.16 ± 0.19^a	0.43 ± 0.01^b	0.28 ± 0.000^b	6.21 ± 0.06^ab	7.225 ± 0.11^ab
HMM + Se	0.56 ± 0.06^c	0.66 ± 0.05^ab	0.05 ± 0.001^ab	0.07 ± 0.01^a	1.02 ± 0.14^b	1.89 ± 0.79^a	1.20 ± 0.01^a	1.47 ± 0.21^a	0.23 ± 0.03^c	0.17 ± 0.02^bc	5.53 ± 0.25^bc	5.90 ± 0.28^bc
HMM + Zn + Se	0.65 ± 0.02^a	0.74 ± 0.01^a	0.049 ± 0.001^b	0.07 ± 0.01^a	1.41 ± 0.098^a	2.52 ± 0.38^a	1.09 ± 0.01^ab	1.52 ± 0.33^a	0.16 ± 0.01^c	0.11 ± 0.01^c	4.78 ± 0.18^bc	6.03 ± 0.18^bc

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Values are presented as Mean ± standard deviation values with different superscripts are significantly different from each other at P < 0.05, while values with the same superscripts are not significantly different.

Table 3 shows the effect of essential elements on the inflammatory cytokines (IL-6, (pg/mL)), (TNF-α, (pg/mL)), transcription factors (NRF2, (pg/mL)), (NFkB, (pg/mL)), and Caspase 3 (μmol/mL) in the heart and the lungs of Sprague Dawley female albino rats after heavy metal mixtures (Pb, Hg, Mn and Al) exposure.

Table 3.

Effect of essential elements on the pro-inflammatory cytokines (IL-6, (pg/mL)), (TNF-α, (pg/mL)), transcription factors (Nrf2, (pg/mL)), (NFkb, (pg/mL)), and caspase 3 (μmol/mL) in heart and lungs of female albino rats after heavy metal mixtures (Pb, hg, Mn and Al) exposure.

Treatment	IL-6		TNF-α		Nrf2		Nf-kβ		Casp 3
Treatment	Heart	Lungs	Heart	Lungs	Heart	Lungs	Heart	Lungs	Heart	Lungs
Control	115.25 ± 2.80^c	349.00 ± 2.68^b	122.50 ± 2.33^b	39.950 ± 2.24^b	71.65 ± 8.98^d	512.50 ± 8.27^a	1.02 ± 0.02^b	2.58 ± 0.02^a	0.54 ± 0.01^b	0.67 ± 0.04^b
HMM only	364.00 ± 5.66^a	613.00 ± 17.40^a	321.50 ± 9.97^a	109.00 ± 1.41^a	719.50 ± 13.44^a	1030.000 ± 7.07^ab	2.38 ± 0.17^a	2.60 ± 0.04^a	2.60 ± 0.11^a	5.06 ± 0.43^a
HMM + Zn	132.50 ± 7.78^c	251.50 ± 7.14^c	184.50 ± 10.61^ab	34.10 ± 1.83^b	221.50 ± 2.12^b	1046.500 ± 13.44^b	1.56 ± 0.16^b	2.10 ± 0.68^a	0.65 ± 0.13^b	1.98 ± 0.50^b
HMM + Se	215.50 ± 1.91^b	401.00 ± 1.97^d	113.50 ± 2.12^b	22.00 ± 7.78^ab	70.50 ± 6.36^d	802.50 ± 3.12^c	1.28 ± 0.13^b	2.54 ± 0.05^a	0.80 ± 0.04^b	1.92 ± 0.60^b
HMM + Zn + Se	211.00 ± 5.66^b	342.00 ± 4.80^a	141.50 ± 1.20^ab	31.60 ± 2.29^b	127.50 ± 7.78^c	513.55 ± 7.12^a	1.190 ± 0.31^b	2.56 ± 0.01^a	0.82 ± 0.09^b	1.17 ± 0.06^b

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The inflammatory cytokines (IL-6, (pg/mL)), (TNF-α, (pg/mL)) in the HMM exposed rats were significantly higher than the control groups (P < 0.05). Zn, Se and Zn plus Se combination caused significant decrease in the levels of IL-6 and TNF-α in comparison to the HMM exposed rats (P < 0.05). The levels of NRF2 and NFkB in the heart and lungs of HMM treated rats were significantly higher than the control group (P < 0.05). The essential elements Zn, Se and Zn + Se caused significant decrease in the NRF2 of the heart while Se and Zn + Se caused significant decrease in the NRF2 of the lungs in comparison to the HMM exposed rats. There were significant difference between NFkB (heart) levels between the HMM group 2.38 ± 0.17 pg/mL and control group 1.02 ± 0.02 pg/mL. The NFkB (heart) after Zn, Se and Zn + Se co-treatment with HMM were significantly (P < 0.05) lower than HMM only groups. There were no significant differences in the NFkb levels in the lungs among the control, HMM and the Zn, Se and Zn + Se co-treated groups. The caspase 3 activity was significantly (P < 0.05) increased from 0.54 ± 0.01 and 0.67 ± 0.04 μmol/mL to 2.60 ± 0.11 and 5.06 ± 0.43 μmol/mL in heart and lungs respectively. Co-treatment with Zn, Se and Zn + Se caused significant (P < 0.05) decrease in the caspase 3 μmol/mL) in heart and lungs of Sprague Dawley female albino rats.

Histopathological examination (heart)

In controls we found interstitial oedema (IO) and perivascular oedema (PVO) Fig. 1A–E. Histopathological examination of the HMM rats revealed multifocal interstitial oedema focal myofiber waviness, with associated (arrows). As for the HMM plus Zn animals, we found myocardial inflammation with multifocal areas of inflammatory cells infiltration (arrows). The photomicrograph of the HMM plus Se rats revealed normal myocardium showing the syncytium of myocardial fibers with central nuclei. Finally, HMM and Zn + Se photomicrographs revealed a myocardium with a focal area of interstitial oedema (IO), showing a large vein (LV).

Histopathological examination (lungs)

In controls we found lungs showing diffuse alveolar septal necrosis with septal hypertrophy Fig. 1A1–E1. Histopathological examination of the HMM rats showing a reversible degenerative change with diffuse cytoplasmic vacuolation (arrows) of the alveolar septal wall and multi-necrotic foci (NF). Group 3: As for the HMM plus Zn animals, lungs showing lung regeneration, reformation of normal lung architecture with necrotic debris (arrows) in epithelial wall of the respiratory bronchiole. The photomicrograph of the HMM plus Se rats revealed lung degeneration. There is distortion of lung architecture associated with multi-foci necrosis (arrows) and sloughing of epithelial cells into alveolar lumen (ES). Finally, HMM and Zn + Se photomicrographs revealed lung regeneration. There is normal lung architecture associated with few necrosis foci (arrows).

Discussion

In the current study, toxicological evaluation of quaternary metal mixture (Pb, Hg, Al and Mn) and in combination essential metal (loid) Zn and Se on different parameters of the cardiopulmonary system in Sprague female rats have been evaluated. Usually, metals are not biodegradable and not digested in humans and animals. In an exposed organism, metals are poorly metabolized resulting in increased body burden with chronic exposure.⁵² Although the hepatic system is largely the target site for accumulation, these metals can also accumulate in the cardiac and pulmonary systems.⁵³^,⁵⁴

Our results demonstrated that heart and lung tissues have tendencies to accumulate Pb, Hg, Al and Mn in high concentrations after exposure to these metals. This accumulation of non-essential metals in the heart may induce cardiovascular toxicity. The non-essential metals reach the vascular walls by metal-laden monocytes and accumulate in the walls of the aorta where it also accumulates in vascular smooth muscles and induce apoptosis of endothelial cells. The suggested cardiotoxicity mechanism may include direct myocardial structure damage, disruption of calcium channels, inhibition of vasodilators like NO, and/or direct vasoconstriction.⁵³^,⁵⁵ Accumulation of metal in the artery can induce aortic weakness via enhancing vascular remodeling which leads to increased arterial stiffening, causing adversarial effects on the metabolism of smooth muscle cells.⁵⁶ On exposure to various metal(loid), chances of hypertension and lung disease are exaggerated.⁵⁷ Exposure of Hg and other metals predispose the heart to increased oxidative stress or decreased antioxidant enzymes e.g. GPx which culminate in increased lipoxidation.⁵⁸^,⁵⁹

In this study the essential trace elements Zn, Se, Zn + Se combination significantly (P < 0.05) decreased the concentration of Pb, Hg, Al and Mn in heart and lungs when compared with untreated HMM only group. An antagonistic relationship between Se and Pb in the accumulation of both metal(loid) upon co-exposure has been reported.⁶⁰ Se in co-exposure with Pb significantly reduced the Pb concentration in comparison to only Pb exposure.⁶⁰ Therefore, the net Se-mediated detoxification potential has been reported against Pb toxicity. Furthermore, Se decreased the levels of other noxious metals. It has also been reported that with Se-insufficiency or deficiency exacerbates Pb-induced health risk even at miniscule amounts of Pb exposure.⁶¹

Zn has two-edged effect of on Pb toxicity according to Piao et al.⁶². According to this study Pb level in whole blood was not affected by Zn alone or Zn + Pb, but showed that Zn was significantly higher in the Zn alone treated group.⁶² In another study, Zn was observed to boost the efficacy of Pb chelation through lowering the blood, hepatic and renal Pb contents, and reversed the inhibited activity on blood ALAD.⁶³ Prasanthi et al., have also reported the antioxidant property of Zn against Pb-toxicity in small mammals study.⁶⁴ All in all, Se and Zn supplementation reduce Pb-toxicity, attenuates the oxidative stress and restores the essential metal homeostasis by decreasing the Pb accumulation in different organs.³¹ Many non-essential metals are known to form selenium-metal complexes via bonding to selenium and thiol (-SH)-containing molecules. These complexes hamper the efficiency of different antioxidant enzymes. For instance the diminution in the activity of catalase is due to the deficiency of selenium-binding protein.⁶⁵ Cardiotoxicity mediated by metals is multifaceted involving diminishing of antioxidant enzyme activity, amplification of reactive oxygen species ROS which have been implicated in myocardial infarction,⁶⁶ destruction of phospholipases, increased oxidation of low-density lipoproteins,⁶⁷ and the inactivation of paraoxonase implicated in atherosclerosis and acute myocardial infarction. Furthermore, metal activation of phospholipase A2 and the concomitant hydrolysis of glycerol phospholipids, formation of lysophosphatidic and arachidonic acid catabolic products such as thromboxanes and prostaglandins have also been associated with coronary artery disease.⁶⁸

Following oral exposure, aluminum is distributed throughout an organism, and its accumulation in many tissues. The exact mechanism of aluminum toxicity is poorly understood. Al amplifies oxidative stress and inflammatory responses which culminate into cellular injury.^69–71 The release of various cytokines into the circulation after metal exposure may increase both cardiac and vascular inflammation to mediate cardiovascular toxicity.

As a divalent metal, zinc (Zn) is one of the essential metals in which the mimicry function of all the divalent metal and metalloids has demonstrated. Zn mediates several physiological functions⁷² in addition to its protective biological role against free radical damage via ramping of enough metallothioneins (MT) levels, sustenance of being an essential component of Cu,Zn- superoxide dismutase (Cu,Zn-SOD), or as protective agent of thiols and other chemical groups.⁷³ Zn is also a crucial cofactor of several vital enzymes.⁷⁴ Another essential trace element selenium (Se) ameliorates toxicity exerted by many metals. It is now known that Se acts by the formation of selenoproteins in accentuating cellular antioxidant defense, and immune responses in various biological processes.⁷⁵^,⁷⁶ Se is an essential oligo element in humans with significant antioxidant, free radical scavenging, and protective roles from oxidative damage in the tissues and organs. It is also an enhancer of the body’s immune system. Whereas HMM only significantly (P < 0.05) decreased the SOD, GPx, CAT and GSH in comparison to the control group in the heart and lungs, Zn, Se and Zn plus Se combination significantly (P < 0.05) increased the SOD, GPx, CAT and GSH when compared with the HMM exposed rats.

In one study, Li et al.⁷⁷ reported an upregulation of TNF-α, iNOS and NF-κB following Pb exposure which was significantly reduced by Se and Pb co-administration. Another study found that, Se in combination with Pb exposure alleviated the Pb-induced downregulation of selenoproteins level Gpx1, Gpx2, Gpx4.⁷⁸ In another study, it was also reported that Se inhibited Pb mediated apoptosis^79–81 The mechanisms of aluminum mediated pulmonary toxicity is not fully understood, although Al is known to directly trigger lung mechanics impairment,⁸² and also cause oxidative stress. Epidemiological data suggest a positive association between Al and decreased total antioxidant capacity in Al exposed workers,²³ and a positive relationship of Al exposure with increased urinary MDA among schoolchildren.⁸³ Furthermore, inflammation might be implicated in Al pulmonary toxicity since in vitro, Al salt crystals have been observed to activate NLRP3 inflammasome and up-regulate the maturation and release of interleukin-1b in human peripheral blood mononuclear cells⁸⁴; and in vivo, inhalation of aluminum hydroxide has been reported to promote the recruitment of inflammatory cells to the lung and increases the levels of cytokine interferon-g and chemokine CCL2 in lung tissues.²⁵

Jin and coworkers reported increased oxidative stress (MDA), and pro-apoptotic genes (caspase 3) following Pb exposure which were significantly reversed by co-treatment with Se and Pb.⁷⁹ Similarly, Pb-mediated oxidative stress by decreasing total antioxidant activity, SOD, CAT, GSH, GPx and increase of MDA,⁷⁸ were significantly reversed by the co-treatment of Se with Pb in.⁸⁵ The MDA and NO of the HMM treated rats were significantly (P < 0.05) higher than the control group. Zn, Se and Zn plus Se combination significantly (P < 0.05) decreased MDA and NO when compared with the HMM treated rats in the present study.

As a component of glutathione peroxidase (GSH-Px), Se regulates pro-inflammatory cytokines and antioxidant defense.⁸⁶^,⁸⁷ Whereas systemic inflammatory response or oxidative stress conditions predispose individuals to the risk of Se deficiency,⁸⁸ Se supplementation has been observed to be beneficial.⁸⁹^,⁹⁰ Also, experimental studies have demonstrated increased oxidative stress in blood and impairment in vascular function in Se deficient rats.⁹¹ Conversely, Se administration could ameliorate chemical induced tissue injury via activation antioxidant enzyme GSH-Px, reducing inflammatory cytokines TNF-α, interleukin-1 beta (IL-1β),⁴³^,⁷⁸^,⁹² or decreasing oxidative product MDA in tissues.^93–96 As hypothesized, the pro-inflammatory cytokines IL-6, TNF-α, in the HMM exposed rats were significantly (P < 0.05) higher than the control groups but supplementation with Zn, Se and Zn plus Se combination caused significant decrease (P < 0.05) when compared with the HMM exposed rats. In the same vein, the levels of Nrf2 and NFkβ in the heart and lungs of HMM treated rats were significantly (P < 0.05) higher than the control group and co-treatment with the essential elements Zn, Se and Zn + Se significantly decreased in the Nrf2 of the heart while Se and Zn + Se caused significant decrease in the Nrf2 of the lungs in comparison to the HMM exposed rats. Also, in this study the significant increases in the caspase 3 activities in the heart and lungs following HMM exposure was attenuated by co-administration with Zn, Se and Zn + Se combination.

Taken together, it could be deduced from the present study that micronutrient Zn, Se and Zn plus Se combination supplementations might be beneficial in cocktail quaternary metal (Pb, Al, Hg, Mn) mixture-induced cardiopulmonary toxicity by augmentation of antioxidant status, abrogation of inflammatory processes and antiapoptotic mechanisms.

Author contributions

Orish Ebere Orisakwe and Chinna N Orish have designed the study, interpreted the data and drafted the manuscript. Mfoniso Antia and Anthonet N. Ezejiofor collected the data of the work. Ana Cirovic and Aleksander Cirovic proofread the manuscript. All the authors finally approved the version to be published.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest statement: All authors involved in the production of this research declared that they have no conflict of interest.

Data availability

Authors declare that all the data are original and they are available upon request.

Contributor Information

Mfoniso Antia, African Centre of Excellence for Public Health and Toxicological Research (ACE-PUTOR), University of Port Harcourt, PMB, East West Road, Choba, Port Harcourt, Rivers State, Choba 5323, Nigeria.

Anthonet N Ezejiofor, African Centre of Excellence for Public Health and Toxicological Research (ACE-PUTOR), University of Port Harcourt, PMB, East West Road, Choba, Port Harcourt, Rivers State, Choba 5323, Nigeria.

Chinna N Orish, Department of Anatomy, Faculty of Basic Medical Sciences, College of Health Sciences, University of Port Harcourt, PMB, East West Road, Choba, Port Harcourt, Rivers State, Choba 5323, Nigeria.

Ana Cirovic, Faculty of Medicine, The Institute of Anatomy, University of Belgrade, East West Road, Choba, Port Harcourt, Rivers State Belgrade, Serbia.

Aleksandar Cirovic, Faculty of Medicine, The Institute of Anatomy, University of Belgrade, East West Road, Choba, Port Harcourt, Rivers State Belgrade, Serbia.

Orish E Orisakwe, African Centre of Excellence for Public Health and Toxicological Research (ACE-PUTOR), University of Port Harcourt, PMB, East West Road, Choba, Port Harcourt, Rivers State, Choba 5323, Nigeria.

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Associated Data

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Data Availability Statement

Authors declare that all the data are original and they are available upon request.

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PERMALINK

Selenium and zinc supplementation mitigates metals-(loids) mixture- mediated cardiopulmonary toxicity via attenuation of antioxidant, anti-inflammatory and antiapoptotic mechanisms in female Sprague Dawley rats

Mfoniso Antia

Anthonet N Ezejiofor

Chinna N Orish

Ana Cirovic

Aleksandar Cirovic

Orish E Orisakwe

Abstract

Graphical Abstract

Graphical Abstract.

Introduction

Materials and methods

Chemicals

Animals and treatments

Sample collection and heart and lung tissues preparation

ELISA analysis

Antioxidant and oxidative stress markers

Glutathione related antioxidants (GPx), reduced glutathione (GSH), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase, lipid peroxidation, nitric oxide

Determination of heavy metals

Histopathological examination

Statistical analysis

Results

Effect of essential trace elements Zn, Se, Zn + Se combination of bioaccumulation of heavy metal in heart and lungs of Sprague Dawley rats following exposure to metal mixture

Table 1.

Table 2.

Table 3.

Histopathological examination (heart)

Fig. 1.

Histopathological examination (lungs)

Discussion

Author contributions

Funding

Data availability

Contributor Information

References

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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