Some dietary factors could inhibit lead toxicity. The aim of this study was to evaluate the effect of dietary compounds rich in unsaturated fatty acids (FA) on blood lead level, lipid metabolism, and vascular reactivity in rats. Serum metallothionein and organs’ lead level were evaluated with the aim of assessing the possible mechanism of unsaturated FA impact on blood lead level. For three months, male Wistar rats that were receiving drinking water with (100 ppm Pb) or without lead acetate were supplemented
Populations exposed to lead, the most ubiquitous xenobiotic in human environment, display increased cardiovascular morbidity and mortality [
Epidemiological studies on the association between cardiovascular disease and the body lead burden, led since the 1970’s, have showed a gradual decrease in blood lead levels in the world population [
On the other hand, many experimental studies very well documented that omega-3 PUFA, such as DHA and EPA, contained in marine algae, fatty fish, and fish oils, exert many positive effects on the circulatory system. In experimental models, a long-term consumption of lipid modified formula diet with DHA and EPA improved blood lipid pattern [
The aim of this study was to evaluate the effect of dietary compounds rich in omega-3 and omega-6 fatty acids, of animal or vegetable origin, on blood lead level, lipid metabolism, and vascular reactivity to norepinephrine in rats. Since only exposure to low doses of lead is associated with increased cardiovascular risk, part of the study was performed using a model corresponding to the environmental exposure to lead. Apart from drinking water and chow, containing some amounts of lead, some rats did not receive any additional lead compounds. At the same time, the study was carried out on a group of rats poisoned with lead (corresponding to model of occupational exposure), in order to conduct in-depth analysis of the possible impact of PUFAs on the toxicological, metabolic, and functional effects of lead.
Two vegetable sources of unsaturated fatty acids: virgin olive oil (premium extravirgin olive oil, Angel Camacho Alimentacion S.L., Moron de la Frontera, Spain) and linseed oil (Vis Nature Linseed Oil, Aleksander Nowak, Wroclaw, Poland), were used. One animal source of omega-3 and omega-6 FA: egg yolk phospholipid fraction (super lecithin) obtained in Department of Animal Products Technology and Quality Management, Wroclaw University of Environmental and Life Sciences, from Lohmann Brown hens line [
Fatty acid content in the used ingredients was analyzed by gas chromatography. Samples (ca. 50 mg) of linseed oil, virgin olive oil, and super lecithin were dissolved in 4 mL of 0.5 M methanolic NaOH solution and heated under reflux for 2 min. After that 4 mL of BF3 (14% solution in methanol) was added and the mixtures were heated once again under reflux for 2 min. Solution after methylation was cooled and extracted with 6 mL of hexane. Hexane extracts were dried using anhydrous magnesium sulphate and evaporated under reduced pressure and residues dissolved in 1.5 mL of hexane. Prepared fatty acid methyl esters (FAME) were analyzed by gas chromatography (GC Agilent 6890N Series), using a 88% cyanopropyl, and 12% aryl polysiloxane column (HP-88, 100 m 0.25 mm 0.25
Male Wistar rats, 4–6 weeks of age and
Ninety three rats were randomly divided into eight groups: untreated rats (12 animals); virgin olive oil treated rats (olive oil, 12 animals); linseed oil treated rats (linseed oil, 12 animals); super lecithin treated rats (super lecithin, 12 animals); lead poisoned rats (Pb group, 10 animals); lead poisoned rats treated by virgin olive oil (Pb plus olive oil, 11 animals); lead poisoned rats treated by linseed oil (Pb plus linseed oil, 12 animals); lead poisoned rats treated by super lecithin (Pb plus super lecithin, 12 animals). For three months animals poisoned with lead were receiving drinking water with the addition of lead acetate at a final concentration of 100 ppm Pb (the solution was prepared daily), whereas rats nonpoisoned with lead were receiving standard drinking water. Simultaneously, depending on the group, rats were given virgin olive oil or linseed oil or super lecithin or were not given any fatty acids. Oils were given every day, orally, by pipette, at a volume of 0.2 mL/kg b.w. Super lecithin was given with chow, at a dose of 50 g/kg b.w. daily.
All reagents were of analytical grade. All aqueous solutions were prepared with deionized water obtained by using ultrapure water system. All drugs concentrations were expressed in terms of freebase and prepared
After the three-month exposition to lead and/or supplementation with dietary unsaturated FA, experiments
Lead levels in blood and mineralizated organs were determined by the atomic absorption spectrophotometer in an acetylate flame on a SOLAAR M6 (Thermo Elemental, Solaar House, Cambridge, UK). The accuracy and repeatability of the method were checked using control blood samples BCR produced by European Commission Joint Research Centre Institute for Reference Materials and Measurements and two level controls (ClinChec-Control I and II, Recipe Chemichals-Instruments, Munich, Germany) were used. Mineralization of samples obtained from kidney, liver, and heart was performed using High Performance Microwave Digestion System (Milestone, Ethos One, Sorisole, Italy, tube SK-12T). The serum total cholesterol, triglycerides, high density lipoprotein (HDL) cholesterol, HDL2, and HDL3 cholesterol levels were determined using enzymatic assay (SPINREACT, S.A. Ctra Santa Coloma, 7 E-17176 SantEsteve de Bas, Spain) by the Beckman DU 650 spectrophotometer (Beckman Instruments, Inc. 4300 Harbor Boulevard, Fullerton, CA California, US 92834). To measure precipitation of HDL2 and HDL3 cholesterol in serum, QUANTOLIP HDL (HDL2/HDL3) test (Technoclone GmbH, Vienna, Austria) was applied. Serum non-HDL cholesterol was calculated as a difference between total- and HDL-cholesterol concentrations. The serum cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) were determined using CETP Activity Assay Kit and PLTP Activity Assay Kit (BioVision Research Products, 2455-D Old Middlefield Way, Mountain View, CA 94043, USA) by the HITACHI F-2500 fluorescence spectrophotometer.
The concentration of serum metallothionein was determined using Rat MT ELISA Kit (Wuhan EIAab Science Co., Ltd., East Lake Hi-Tech Development Zone, Wuhan, China) by the BIOTEK-EPOCH 200–1000 spectrophotometer.
Values are presented as a mean ± standard deviation and analyzed by analysis of variance (ANOVA/MANOVA) followed by
Fatty acid content in the used ingredients is presented in Table
Fatty acid content in the used ingredients.
Fatty acid profile | Olive oil (%) | Linseed oil (%) | Super lecithin (%) |
---|---|---|---|
C14:0 | ND | ND | 0.40 |
C14:1 | ND | ND | 0.16 |
C16:0 | 17.82 | 8.18 | 26.36 |
C16:1 | 1.23 | ND | 2.52 |
C17:0 | 0.16 | ND | 0.24 |
C18:0 | 6.47 | 7.54 | 14.05 |
C18:1 | 63.96 | 27.15 | 29.66 |
C18:2 | 8.43 | 17.16 | 13.08 |
C18:3 | 1.19 | 39.52 | 3.12 |
C20:2 | 0.23 | 0.29 | 0.18 |
C20:4 | 0.51 | 0.16 | 2.41 |
C20:5 | ND | ND | 0.58 |
C22:6 | ND | ND | 7.12 |
1.19 | 39.52 | 10.82 | |
9.17 | 17.61 | 15.67 | |
7.71 | 0.45 | 1.45 | |
Saturated FA | 24.45 | 15.72 | 41.05 |
Unsaturated FA | 75.55 | 84.28 | 58.83 |
MUFA | 65.19 | 27.15 | 32.34 |
PUFA | 10.36 | 57.13 | 26.49 |
MUFA = monounsaturated fatty acids.
ND = not detected.
In groups of rats that drank water without added lead, in comparison to untreated animals, olive oil decreased serum non-HDL cholesterol (
Influence of dietary supplementation with olive oil or linseed oil or super lecithin on serum lipids and lipid transfer proteins in nonpoisoned rats and rats poisoned with lead.
Group of rats | Lipids | Proteins | ||||||
---|---|---|---|---|---|---|---|---|
Chol |
TG |
HDL-C |
HDL3-C |
HDL2-C |
Non |
PLTP |
CETP | |
Untreated |
1.0 ± 0.23 | 1.04 ± 0.51 | 0.88 ± 0.23 | 0.45 ± 0.16 | 0.45 ± 0.07 | 0.62 ± 0.14 | 70.2 ± 23.1 | 28.7 ± 3.9 |
Olive oil |
1.35 ± 0.25 | 1.02 ± 0.3 | 0.95 ± 0.21 | 0.50 ± 0.12 | 0.45 ± 0.18 | 0.4 ± 0.14b | 82.3 ± 18.9 | 27.9 ± 5.1 |
Linseed oil |
1.32 ± 0.15c | 0.81 ± 0.29 | 0.8 ± 0.09 | 0.52 ± 0.07 | 0.28 ± 0.07a | 0.53 ± 0.1 | 61.7 ± 10.9 | 31.8 ± 5.6 |
Super lecithin |
1.57 ± 0.19 | 0.95 ± 0.25 | 0.95 ± 0.15 | 0.56 ± 0.10c | 0.39 ± 0.10 | 0.62 ± 0.21 | 61.4 ± 11.2 | 34.4 ± 3.6b |
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Pb |
1.43 ± 0.16 | 0.99 ± 0.64 | 0.93 ± 0.11 | 0.51 ± 0.06 | 0.42 ± 0.08 | 0.50 ± 0.12 | 74.3 ± 17.7 | 29.7 ± 8.3 |
Pb + olive oil |
1.42 ± 0.15 | 1.11 ± 0.46 | 0.88 ± 0.25 | 0.44 ± 0.14 | 0.44 ± 0.16 | 0.54 ± 0.2 | 85.4 ± 14.2 | 28.7 ± 2.6 |
Pb + linseed oil |
1.33 ± 0.12 | 0.81 ± 0.18 | 0.87 ± 0.13 | 0.51 ± 0.07 | 0.33 ± 0.13 | 0.42 ± 0.18 | 57.5 ± 13.6 | 27.9 ± 6.7 |
Pb + super lecithin |
1.62 ± 0.26 | 1.11 ± 0.20 | 0.91 ± 0.17 | 0.57 ± 0.10 | 0.34 ± 0.10 | 0.70 ± 0.2y | 60.1 ± 6.8z | 34.1 ± 2.2 |
Results are presented as mean ± SD. Statistically significant differences: a
Chol: cholesterol; TG: triglycerides; HDL-C: high density cholesterol; HDL3-C: HDL3 cholesterol; HDL2-C: HDL2 cholesterol; non HDL-C: non HDL cholesterol; PLTP: phospholipid transfer protein; CETP: cholesteryl ester transfer protein.
Multiple regression analysis showed the existence of the linear correlation between CETP and total cholesterol (
In rats which drank water without lead, super lecithin slightly reduced pressor responses to NE injected at doses from 0.1 to 5.0
Influence of virgin olive oil, linseed oil, and super lecithin (lecithin) on changes of perfusion pressure (in mmHg) induced by NE, and on the D50NE.
Group | Changes of perfusion pressure (in mmHg) in response to NE at dose of: | D50NE ( | |||||
---|---|---|---|---|---|---|---|
0.01 |
0.1 |
0.5 |
1.0 |
3.0 |
5.0 | ||
Control |
13.0 ± 10.3 | 44.0 ± 24.4 | 76.3 ± 48.5 | 80.5 ± 49.2 | 89.5 ± 56.3 | 90.4 ± 49.8 | 0.20 ± 0.18 |
Olive oil |
20.3 ± 27.0 | 34.2 ± 19.9 | 79.7 ± 35.8 | 89.7 ± 43.9 | 96.2 ± 41.7 | 96.3 ± 49.3 |
|
Linseed oil |
15.4 ± 11.8 | 34.2 ± 11.8 | 57.7 ± 19.9 | 66.5 ± 20.7 | 69.1 ± 23.3 | 72.1 ± 27.7 |
|
Lecithin |
12.2 ± 12.5 |
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Pb |
24.0 ± 25.3 | 35.1 ± 25.4 | 67.0 ± 27.4 | 80.2 ± 34.9 | 89.9 ± 42.7 | 95.4 ± 43.2 | 0.34 ± 0.21 |
Pb + olive oil |
30.0 ± 30.2 | 45.1 ± 37.3 | 84.1 ± 42.4 | 89.5 ± 44.9 | 100.9 ± 44.5 | 109.4 ± 53.0 | 0.28 ± 0.21 |
Pb + linseed oil |
10.7 ± 12.8 | 30.4 ± 16.3 | 59.9 ± 30.9 | 68.8 ± 36.7 | 68.7 ± 33.8 | 88.1 ± 62.8 | 0.32 ± 0.21 |
Pb + lecithin |
8.83 ± 3.71 | 24.7 ± 7.61 | 50.4 ± 12.7 | 68.5 ± 13.5 | 75.3 ± 16.8 | 78.0 ± 15.9 | 0.40 ± 0.18 |
Results are presented as mean ± SD. Differences statistically significant:
Dose-response curve to norepinephrine in rats treated with olive oil, or linseed oil, or super lecithin or not treated with any fatty acids.
Dose-response curve to norepinephrine in lead poisoned rats treated with olive oil, or linseed oil, or super lecithin or not treated with any fatty acids.
In rats nonpoisoned with lead, two sources of FA: linseed oil and super lecithin, decreased the Pb-B (
In rats not poisoned with lead, the decrease in Pb-B induced by linseed oil (but not by super lecithin) was associated with the increase in serum MT concentration (Table
Lead and metallothionein (MT) concentrations in tissues of rats poisoned with lead and/or treated with PUFA.
Group of rats | Lead concentration | MT | |||
---|---|---|---|---|---|
Blood |
Liver |
Kidney |
Heart |
Serum | |
Untreated |
78.6 ± 93.1 | 0.048 ± 0.061 | 1.09 ± 2.02 | 0.016 ± 0.022 | 6.55 ± 1.63 |
Olive oil |
34.9 ± 22.8 | 0.079 ± 0.107 | 0.459 ± 0.55 | 0.074 ± 0.068 | 7.23 ± 2.44 |
Linseed oil |
5.94 ± 5.56c | 0.038 ± 0.076 | 0.281 ± 0.69 | 0.019 ± 0.018 | 8.46 ± 2.82a |
Super lecithin |
4.76 ± 4.53c | 0.034 ± 0.036 | 0.175 ± 0.19 | 0.045 ± 0.061 | 7.16 ± 1.74 |
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Pb |
678.7 ± 429.2 | 0.251 ± 0.312 | 46.01 ± 38.63 | 0.101 ± 0.122 | 6.56 ± 0.47 |
Pb + olive oil |
582.4 ± 284.9 | 0.192 ± 0.275 | 18.72 ± 18.70y | 0.233 ± 0.172x | 7.18 ± 0.77 |
Pb + linseed oil |
214.8 ± 73.0x | 0.342 ± 0.252 | 46.66 ± 29.6 | 0.032 ± 0.035 | 9.17 ± 4.31x |
Pb + lecithin |
288.8 ± 51.4x | 1.135 ± 1.08x | 26.34 ± 15.4z | 0.089 ± 0.041 | 7.33 ± 2.12 |
Results are presented as means ± SD. Statistically significant differences: a
In rats treated with lead and super lecithin, lead concentration in the liver was greater (
Rats treated with lead and virgin olive oil also had lower, in comparison to Pb rats, lead concentration in the kidney (
Multivariate analysis of variance showed an existence of interaction between exposure to lead and treatment with unsaturated FA in their influence on Pb-B: F (1; 89) = 7.2023;
In various experimental models, long-term consumption of lipid modified formula diet with DHA and EPA decreased non-HDL cholesterol and impacted HDL [
On the contrary, a three-month dietary supplementation with olive oil or linseed oil in rats poisoned with lead did not significantly affect serum lipids or lipid transfer proteins. The relationship between blood lead level and lipid metabolism, studied in many experimental models, is described in a very varied, sometimes contradictory manner, from proatherogenic action of lead on lipid metabolism to a lack of connection between this metal and lipids [
Simultaneously, the effect of super lecithin on non-HDL-C in rats was related to some increase in activity of cholesteryl ester transfer protein. Lecithin-induced CETP stimulation may explain the lecithin-induced increase in non-HDL cholesterol in lead-poisoned rats, as an existence of a positive correlation between CETP and non-HDL cholesterol was shown [
Obtained results indicate also that the effect of unsaturated fatty acids on lipid metabolism in rats poisoned with lead is less beneficial or even adverse, in comparison to one observed in nonpoisoned animals. For example, in rats treated with lead, neither non-HDL cholesterol reduction by virgin olive oil, nor decrease in total cholesterol induced by linseed oil, nor increase in the HDL3 cholesterol induced by super lecithin was observed, in contrast to the effect of unsaturated FA in nonpoisoned rats. In rats poisoned with lead, and lecithin induced increase in nonHDL cholesterol, which can be recognized as an adverse effect of the FA supplementation (Table
Other studies have shown that the beneficial effect of omega-3 FA in healthy rats was associated with PUFAs’ inhibitory impact on cardiac [
Recently, it has also been described that DHA can inhibit gene expression of cyclooxygenase-2, decreasing tension and causing an easier relaxation of rat’s aorta [
The most important observation in this study, as it has been noted probably for the first time, is a significant reduction of blood lead concentration, induced by ingredients rich in omega-3 and omega-6 fatty acids, such as super lecithin and linseed oil. This effect was observed in lead-poisoned rats, as well as in nonpoisoned animals. The effect of virgin olive oil on Pb-B was much weaker (not statistically significant). Of course, the difference between actions of various preparations could be related to various contents of lead and other toxic or essential metals and to interactions between them on the level of intestinal absorption, intra- and extracellular transport, and mechanisms of elimination. However, the difference could also be related to various omega-6 to omega-3 fatty acid ratio. This ratio was 1,45 in super lecithin, 0,45 in linseed oil, and 7,71 in olive oil (Table
It is quite possible that super lecithin enhanced also biliary secretion of lead. Super lecithin increased lead concentration in the liver, as well as CETP activity in blood, which was associated with a slightly reduced PLTP activity. Most long-chain polyunsaturated fatty acids cause an increase in bile production and secretion in the liver [
There are more questions, such as whether the lead reduction in blood results from reduced absorption of this metal in the gastrointestinal tract, chelating action of omega-3 and omega-6 FA, or increased accumulation of lead in tissues. In rats, lead cumulates mainly in kidneys, liver, lungs, spleen, and cardiac muscle (as an intermediately fast exchangeable pool) and in bone (as a slowly exchangeable pool). In our study, distribution of lead (maximal in kidneys and minimal in the heart) was typical for a compartment that is characterized by a slower turnover rate and transit time, that is, for intracellular space (soft tissues and erythrocytes) [
In summary, in rats nonpoisoned with lead, the mean blood lead level was lower than 100
The authors declare that there is no conflict of interests regarding the publication of this paper.
The study was supported by the Programme of Innovative Economy (Grant no. 01.03.01-00-133080) and was funded from the resources available to a Wrocław Medical University (statutory activities, nr 729). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.