Dietary supplementation with copper nanoparticles influences the markers of oxidative stress and modulates vasodilation of thoracic arteries in young Wistar rats

Abstract

We aimed to study the physiological effects of diet supplemented with copper (Cu) nanoparticles (NPs). During the eight weeks of the experiment, young Wistar rats (at seven weeks of age, n = 9) were supplemented with 6.5 mg of Cu either as NPs or carbonate salt (Cu6.5). A diet that was not supplemented with Cu served as a negative control (Cu0). The impact of nano Cu supplementation on lipid (reflected as thiobarbituric acid reactive substances-TBARS) and protein peroxidation (thiol and carbonyl groups) in blood plasma as well as the influence on the vasodilatory mechanism(s) of isolated rat thoracic arteries were studied. Supplementation with Cu enhanced lipid peroxidation (TBARS) in NP6.5 (x2.4) and in Cu6.5 (x1.9) compared to the negative control. Significant increase in TBARS was also observed in NP6.5 (x1.3) compared to the Cu6.5 group. The level of thiol groups increased in NP6.5 (x1.6) compared to Cu6.5. Meanwhile, significant (x0.6) decrease was observed in the Cu6.5 group compared to the negative control. Another marker of protein oxidation, carbonyl groups increased in NP6.5 (x1.4) and Cu6.5 (x2.3) compared to the negative control. However significant difference (x0.6) was observed between NP6.5 and Cu6.5. Arteries from Cu supplemented rats exhibited an enhanced vasodilation to gasotransmitters: nitric oxide (NO) and carbon monoxide (CO). An enhanced vasodilation to NO was reflected in the increased response to acetylcholine (ACh) and calcium ionophore A23187. The observed responses to ACh and CO releasing molecule (CORM-2) were more pronounced in NP6.5. The activator of cGMP-dependent protein kinases (8-bromo-cGMP) induced similar vasodilation of thoracic arteries in NP6.5 and Cu0 groups, while an increased response was observed in the Cu6.5 group. Preincubation with the inducible nitric oxide (iNOS) synthase inhibitor- 1400W, decreased the ACh-induced vasodilation in NP6.5, exclusively. Meanwhile the eicosanoid metabolite of arachidonic acid (20-HETE) synthesis inhibitor-HET0016, enhanced vasodilation of arteries from Cu0 group. In conclusion, this study demonstrates that supplementation with nano Cu influences oxidative stress, which further has modified the vascular response.

Fig 1

Effects of experimental supplementation on (a) lipid peroxidation level–TBARS, (b) the thiol groups and (c) the carbonyl groups. Rats were supplemented with Cu (6.5 mg/kg of diet) either as Cu_6.5 or NP_6.5 (40 nm). A diet that was not supplemented with Cu served as a negative control–Cu₀. Results are mean ± SD from n = 9 rats *p ≤ 0.05 (one-way ANOVA with Tukey’s multiple comparisons test).

Fig 2. The cumulative concentration-response curves to acetylcholine (ACh, 10⁻¹¹–10⁻⁵ M) in the isolated rat thoracic arteries.

Rats were supplemented with Cu (6.5 mg/kg of diet) either as Cu_6.5 or NP_6.5 (40 nm). A diet that was not supplemented with Cu served as a negative control–Cu₀. (a) Control conditions. (b) Isolated rat arteries were pre-incubated with the inducible nitric oxide synthase (iNOS) inhibitor (1400W, 1 μM, 30 min). Results (mean ± SEM, n = 9) are expressed as a percentage of inhibition of the contraction induced by noradrenaline (0.1 μM) *p ≤ 0.05 (two-way ANOVA with Tukey’s multiple comparisons test).

Fig 3. The cumulative concentration-response curves to acetylcholine (ACh, 10⁻¹¹–10⁻⁵ M) in the isolated rat thoracic arteries.

Rats were supplemented with Cu (6.5 mg/kg of diet) either as (a) Cu_6.5 or (b) NP_6.5 (40 nm). (c) A diet that was not supplemented with Cu served as a negative control–Cu₀. ACh-induced vasodilation was analyzed in the absence and presence of the iNOS inhibitor (1400W, 1 μM, 30 min), and 20-HETE synthesis inhibitor (HET0016, 0.1 μM, 30 min). Results (mean ± SEM, n = 9) are expressed as a percentage of inhibition of the contraction induced by noradrenaline (0.1 μM), *p ≤ 0.05 (two-way ANOVA with Tukey’s multiple comparisons test).

Fig 4. The cumulative concentration-response curves to calcium ionophore (A23187, 10⁻¹¹–10⁻⁵ M) in the isolated rat thoracic arteries.

Rats were supplemented with Cu (6.5 mg/kg of diet) either as Cu_6.5 or NP_6.5 (40 nm). A diet that was not supplemented with Cu served as a negative control–Cu₀. Results (mean ± SEM, n = 9) are expressed as a percentage of inhibition of the contraction induced by noradrenaline (0.1 μM), *p ≤ 0.05 (two-way ANOVA with Tukey’s multiple comparisons test).

Fig 5. The cumulative concentration-response curves to a cell-permeable analog of cGMP (8-bromo-cGMP, 10⁻⁸–10⁻³ M) in the isolated rat thoracic arteries.

Rats were supplemented with Cu (6.5 mg/kg of diet) either as Cu_6.5 or NP_6.5 (40 nm). A diet that was not supplemented with Cu served as a negative control–Cu₀. Results (mean ± SEM, n = 9) are expressed as a percentage of inhibition of the contraction induced by noradrenaline (0.1 μM), *p ≤ 0.05 (two-way ANOVA with Tukey’s multiple comparisons test).

Fig 6. The cumulative concentration-response curves to carbon monoxide releasing molecule, (CORM-2, 10⁻⁶–10⁻⁴ M) in the isolated rat thoracic arteries.

Rats were supplemented with Cu (6.5 mg/kg of diet) either as Cu_6.5 or NP_6.5 (40 nm). A diet that was not supplemented with Cu served as a negative control–Cu₀. Results (mean ± SEM, n = 9) are expressed as a percentage of inhibition of the contraction induced by noradrenaline (0.1 μM), *p ≤ 0.05 (two-way ANOVA with Tukey’s multiple comparisons test).

Fig 7. A summary of the results.