Dietary Effects of Chromium Picolinate and Chromium Nanoparticles in Wistar Rats Fed with a High-Fat, Low-Fiber Diet: The Role of Fat Normalization

Abstract

Citation: Majewski, M.; Gromadziński, L.; Cholewińska, E.; Ognik, K.; Fotschki, B.; Juśkiewicz, J.

Full text

Citation: Majewski, M.;
Gromadzi´nski, L.; Cholewi´nska, E.;
Ognik, K.; Fotschki, B.; Ju´skiewicz, J.
Dietary Effects of Chromium
Picolinate and Chromium
Nanoparticles in Wistar Rats Fed
with a High-Fat, Low-Fiber Diet: The
Role of Fat Normalization. Nutrients
2022,14, 5138. https://doi.org/
10.3390/nu14235138
Academic Editor: Arrigo Cicero
Received: 19 October 2022
Accepted: 29 November 2022
Published: 2 December 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
nutrients
Article
Dietary Effects of Chromium Picolinate and Chromium
Nanoparticles in Wistar Rats Fed with a High-Fat, Low-Fiber
Diet: The Role of Fat Normalization
Michał Majewski 1, * , Leszek Gromadzi ´nski 2, Ewelina Cholewi ´nska 3, Katarzyna Ognik 3,
Bartosz Fotschki 4and Jerzy Ju´skiewicz 4, *
1
Department of Pharmacology and Toxicology, Faculty of Medicine, University of Warmia and Mazury in Olsztyn,
10-082 Olsztyn, Poland
2Department of Cardiology and Internal Medicine, Faculty of Medicine,
University of Warmia and Mazury in Olsztyn, 10-082 Olsztyn, Poland
3Department of Biochemistry and Toxicology, Faculty of Animal Sciences and Bioeconomy,
University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland
4Institute of Animal Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10,
10-748 Olsztyn, Poland
*Correspondence: michal.majewski@uwm.edu.pl (M.M.); j.juskiewicz@pan.olsztyn.pl (J.J.)
Abstract:
We aimed to evaluate how feeding a high-fat–low-fiber (F) diet to rats and dietary in-
tervention with the implementation of a standard-fat-and-fiber (S) diet affects the response of the
cardiovascular system to chromium (III) picolinate (Cr–Pic) and, alternatively, chromium nanoparti-
cles (Cr–NPs). Young male Wistar Han rats (n/group = 12) from either the fatty group (18 weeks on F
diet) or the intervention group (9 weeks on F diet + 9 weeks on S diet) received a pharmacologically
relevant dose of 0.3 mg Cr/kg body weight in the form of Cr–Pic or Cr–NPs for 9 weeks. Our study
on rats confirmed the pro-inflammatory effect of an F diet administered for 18 weeks. In the inter-
vention group, both Cr–Pic and Cr–NPs decreased heart glutathione ratio (GSH+GSSG), enhanced
participation of nitric oxide (NO) derived from inducible NO synthase (iNOS) in vascular relaxation
to acetylcholine (ACh), increased the vasodilator net effect of cyclooxygenase-2 (COX-2)-derived
prostanoids, and increased the production of superoxide anion (O
2.−
) in aortic rings. Meanwhile,
in the fatty group, there was increased heart superoxide dismutase (SOD), decreased heart catalase
(CAT), and reduced sensitivity in pre-incubated aortic rings to endogenous prostacyclin (PGI
2
). The
factors that significantly differentiated Cr–NPs from Cr–Pic were (i) decreased blood antioxidant
capacity of water-soluble compounds (0.75-fold, p= 0.0205), (ii) increased hydrogen peroxide (H
2
O
2
)
production (1.59-fold, p= 0.0332), and (iii) modified vasodilator response due to PGI
2
synthesis inhi-
bition (in the intervention group) vs. modified ACh-induced vasodilator response due to (iv) COX
inhibition and v) PGI
2
synthesis inhibition with thromboxane receptor blockage after 18 weeks on F
diet (in the fatty group). Our results show that supplementation with Cr–Pic rather than with Cr–NPs
is more beneficial in rats who regularly consumed an F diet (e.g., for 18 weeks). On the contrary,
in the intervention group (9 weeks on F diet + 9 weeks of dietary fat normalization (the S diet)),
Cr–Pic and Cr–NPs could function as pro-oxidant agents, initiating free-radical reactions that led to
oxidative stress.
Keywords:
1400 W; acetylcholine; chromium (III); dietary intervention; indomethacin; nanoparticles;
NS-398; obesity; thoracic aorta; tranylcypromine
1. Introduction
Excessive fat intake, together with obesity, have a negative impact on the cardiovas-
cular system, leading to atherosclerosis with an increased risk of blood clot formation,
vascular dysfunction, hypertension, and damage to arteries and many organs such as the
Nutrients 2022,14, 5138. https://doi.org/10.3390/nu14235138 https://www.mdpi.com/journal/nutrients

Nutrients 2022,14, 5138 2 of 20
heart, brain, kidneys, and eyes [
1
,
2
]. Consumption of a high-fat diet, especially over a
prolonged period, impairs antioxidant mechanisms, which in turn induces oxidative stress
in the body [
3
,
4
]. This leads to the overproduction of reactive oxygen and nitrogen species
(ROS and RNS), which damage lipids, proteins, and DNA [5].
Vascular relaxation of conduit arteries is regulated by several factors, of which nitric
oxide (NO) and arachidonic acid (AA) metabolites are the key mediators [
6
]. NO-induced
vasorelaxation is mediated by soluble guanylate cyclase stimulation, with a further increase
in the intracellular cyclic guanosine monophosphate (cGMP) level in the smooth muscle
cells of arteries. Prostanoids are derived from AA through the cyclooxygenase (COX)
pathway. Among prostanoids, thromboxane A
2
(TXA
2
) has recently received intensive
study due to its pivotal role in cardiovascular disorders [
6
]. Prostacyclin I
2
(PGI
2
) is another
prostanoid with either vasodilator or vasoconstrictor properties under certain conditions
that is widely investigated in vascular disorders [7].
Chromium (Cr) (III) is a popular ingredient in weight-reducing remedies widely sold
to the public and is freely available as a preparation for the treatment of type 2 diabetes.
However, the European Food Safety Authority (EFSA) concluded that Cr supplementa-
tion is not necessary for the proper functioning of the organism [
8
]. Recent studies have
indicated that intake of excessive doses of Cr (III) for a prolonged time period may lead to
undesirable processes that modulate the immune response [
9
–
11
]. Chromium as picolinate
(Cr–Pic) is an organic compound of Cr connected to picolinic acid, a tryptophan derivative
on the kynurenine pathway [
12
,
13
], and is the most popular form available due to its
higher absorption (2.8%) compared with inorganic forms (0.9%). Nonetheless, Cr–Pic is
poorly absorbed; thus, other chemical compounds are being intensively studied to find
a better preparation and to reduce the concentration of Cr. Recently, metal nanoparticles
are of interest due to their small size, lack of electric charge, and specific physicochemical
properties. On the other hand, these properties make nanoparticles a potential hazard for
living organisms, as they easily penetrate biological membranes [
5
,
10
–
15
]. There is also a
risk that Cr nanoparticles (Cr–NPs) increase oxidative processes in the body and that Cr
(with no electric charge) can be transformed into the highly toxic Cr (VI). Metal nanopar-
ticles, such as copper nanoparticles, were recently found to induce a pro-inflammatory
response [
16
–
19
], which negatively interfered with the vasodilation of isolated rat thoracic
arteries through the modulation of vasoconstrictor prostanoids and their receptors [14].
In our previous studies conducted on rats, supplementation with 0.3 mg of Cr (III)
per kg of body weight (BW), particularly in the form of nanoparticles, had a negative
impact
in vivo
, which was reflected in impaired DNA repair and increased DNA oxidation
processes in the heart, liver, and brain [
1
,
5
]. Cr–NPs reduced the number of white blood
cells, which impaired the immune response. Moreover, Cr–Pic and Cr–NPs favor a pro-
apoptotic environment with increased content of caspase 3 and 8 in the blood of rats [5].
Bearing all of this in mind, we have postulated that Cr (III), particularly Cr–NPs,
may have a negative impact on the heart and vasculature. We aimed to study whether
supplementation with Cr–Pic and Cr–NPs (0.3 mg/kg BW) for 9 weeks indeed possesses
a harmful effect. The further aim was to evaluate how feeding a high-fat–low-fiber (F)
diet to rats for either 9 or 18 weeks and dietary intervention with the implementation of a
standard-fat-and-fiber (S) diet affect the redox state of cardiac tissue and the responses of
the vascular system to Cr–Pic and Cr–NPs. The antioxidant status of blood plasma and
heart was studied together with the participation of arachidonic acid metabolites in the
vasodilator response of rat thoracic aorta to acetylcholine ex vivo.
2. Materials and Methods
2.1. Drugs and Chemicals
The following drugs were used: acetylcholine (ACh) chloride, noradrenaline (NA)
hydrochloride, 1400 W, indomethacin, NS-398, SQ-29,548, tranylcypromine (Sigma-Aldrich,
Schnelldorf, Germany). Stock solutions were prepared as directed: NA in a mixture of
sodium chloride + ascorbic acid (0.9% and 0.01% w/v), 1400 W in methanol, SQ-29,548 and

Nutrients 2022,14, 5138 3 of 20
indomethacin in ethanol, and TCP and NS-398 in DMSO. Other drugs were prepared in
distilled water. The stock solutions (10 mM) were kept at
−
20
◦
C. All dilutions were made
in a Krebs–Henseleit buffer (KH buffer: mM; NaCl 115, CaCl
2
2.5, KCl 4.6, KH
2
PO
4
1.2,
MgSO
4
1.2, NaHCO
3
25, and glucose 11.1 at pH 7.4) on the day of the experiment. The
maximal solvent concentration in organ baths was less than 0.01% (v/v).
Chromium nanoparticles/nanopowder of 99.9% trace metals basis purity, particle size
(APS) 60–80 nm, specific surface area (SSA) 6–8 m
2
/g, spherical morphology, bulk density
~0.15 g/cm
3
, and true density 8.9 g/cm
3
were purchased from SkySprings Nanopowders
(Houston, TX, USA). Chromium picolinate (Cr–Pic) and chromium nanoparticles (Cr–NPs)
were added to the diet as an emulsion together with dietary rapeseed oil.
2.2. Animal Protocol and Dietary Treatment
Male Wistar Han rats from the Institute of Animal Reproduction and Food Research
PAS, Olsztyn, Poland were randomly divided at 7 weeks of age into 7 groups of 12 animals
each, see Figure 1.
Nutrients 2022, 14, x FOR PEER REVIEW 3 of 21
2. Materials and Methods
2.1. Drugs and Chemicals
The following drugs were used: acetylcholine (ACh) chloride, noradrenaline (NA)
hydrochloride, 1400W, indomethacin, NS-398, SQ-29,548, tranylcypromine (Sigma-Al-
drich, Schnelldorf, Germany). Stock solutions were prepared as directed: NA in a mixture
of sodium chloride + ascorbic acid (0.9% and 0.01% w/v), 1400W in methanol, SQ-29,548
and indomethacin in ethanol, and TCP and NS-398 in DMSO. Other drugs were prepared
in distilled water. The stock solutions (10 mM) were kept at −20 °C. All dilutions were
made in a Krebs–Henseleit buffer (KH buffer: mM; NaCl 115, CaCl2 2.5, KCl 4.6, KH2PO4
1.2, MgSO4 1.2, NaHCO3 25, and glucose 11.1 at pH 7.4) on the day of the experiment. The
maximal solvent concentration in organ baths was less than 0.01% (v/v).
Chromium nanoparticles/nanopowder of 99.9% trace metals basis purity, particle
size (APS) 60–80 nm, specific surface area (SSA) 6–8 m2/g, spherical morphology, bulk
density ~0.15 g/cm3, and true density 8.9 g/cm3 were purchased from SkySprings Na-
nopowders (Houston, TX, USA). Chromium picolinate (Cr–Pic) and chromium nanopar-
ticles (Cr–NPs) were added to the diet as an emulsion together with dietary rapeseed oil.
2.2. Animal Protocol and Dietary Treatment
Male Wistar Han rats from the Institute of Animal Reproduction and Food Research
PAS, Olsztyn, Poland were randomly divided at 7 weeks of age into 7 groups of 12 animals
each, see Figure 1.
Figure 1. Animal feeding groups and experimental timeline. 0.3 mg Cr/kg of body weight as chro-
mium picolinate (Cr-Pic) and chromium nanoparticles (Cr-NPs) were added to the diet as an emul-
sion together with dietary rapeseed oil for 9 weeks of supplementation after the initial 9 week period
of experimental feed without Cr supplementation. Rats at 7 weeks of age were fed with two types
of diet: a standard-fat-and-fiber (S) diet and a high-fat–low-fiber (F) diet for either 9 weeks or 18
weeks (9 + 9 weeks). The following groups of rats were studied: Control S: the S diet plus the S diet;
Control FS: the F diet and the S diet; Cr–Pic FS: the F diet and the Cr–Pic S diet; Cr–NPs FS: the F
diet and the Cr–NPs S diet; Control FF: the F diet and the F diet; Cr–Pic FF: the F diet and the Cr–
Pic F diet; Cr–NPs FF: the F diet and the Cr–NPs F diet.
Experimental feeding consisted of two 9-week periods, i.e., initial and experimental,
wherein 0.3 mg Cr/kg of body weight as chromium picolinate (Cr–Pic) and chromium
nanoparticles (Cr-NPs) were added for 9 weeks of supplementation after the initial 9 week
period of experimental feed without Cr supplementation. Two types of diet were applied
(see Table 1): a standard-fat-and-fiber (S) diet and a high-fat–low-fiber (F) diet. The latter
Figure 1.
Animal feeding groups and experimental timeline. 0.3 mg Cr/kg of body weight as
chromium picolinate (Cr-Pic) and chromium nanoparticles (Cr-NPs) were added to the diet as an
emulsion together with dietary rapeseed oil for 9 weeks of supplementation after the initial 9 week
period of experimental feed without Cr supplementation. Rats at 7 weeks of age were fed with two
types of diet: a standard-fat-and-fiber (S) diet and a high-fat–low-fiber (F) diet for either 9 weeks or
18 weeks (9 + 9 weeks). The following groups of rats were studied: Control S: the S diet plus the S
diet; Control FS: the F diet and the S diet; Cr–Pic FS: the F diet and the Cr–Pic S diet; Cr–NPs FS: the F
diet and the Cr–NPs S diet; Control FF: the F diet and the F diet; Cr–Pic FF: the F diet and the Cr–Pic
F diet; Cr–NPs FF: the F diet and the Cr–NPs F diet.
Experimental feeding consisted of two 9-week periods, i.e., initial and experimental,
wherein 0.3 mg Cr/kg of body weight as chromium picolinate (Cr–Pic) and chromium
nanoparticles (Cr-NPs) were added for 9 weeks of supplementation after the initial 9 week
period of experimental feed without Cr supplementation. Two types of diet were applied
(see Table 1): a standard-fat-and-fiber (S) diet and a high-fat–low-fiber (F) diet. The latter
diet was a modification of the S diet, with 17% lard replacing maize starch and with only
3% cellulose content instead of 8%. Rats were fed with the S diet or F diet for either 9 weeks
or 18 weeks.

Nutrients 2022,14, 5138 4 of 20
Table 1. Composition of diets fed to rats (%) *.
Ingredient/Group S F
Casein 14.8 14.8
DL-methionine 0.2 0.2
Cellulose 8.0 3.0
Choline chloride 0.2 0.2
Cholesterol 0.3 0.3
Vitamin mix 1.0 1.0
Mineral mix 3.5 3.5
Maize starch 64 52
Rapeseed oil 8.0 8.0
Lard - 17.0
* Chromium (III) picolinate (Cr–Pic) and chromium nanoparticles (Cr–NPs) were emulgated in dietary rapeseed
oil, not in the mineral mixture (MX), and added into the diet.
This study was conducted on two main groups:
(A)
Control, which was further subdivided into:
1. Control S (the S diet and the S diet)
2. Control FS (the F diet and the S diet)
3. Control FF (the F diet and the F diet)
(B)
Supplemented, which was further subdivided into:
1. Cr–Pic FS (the FS diet supplemented with Cr–Pic)
2. Cr–NPs FS (the FS diet supplemented with Cr–NPs)
3. Cr–Pic FF (the FF diet supplemented with Cr–Pic)
4. Cr–NPs FF (the FF diet supplemented with Cr–NPs)
The concentration of Cr in the S diet was 1.26 mg/kg, which, in terms of Cr–Pic and
Cr–NPs, corresponded to concentrations of 4.01 and 4.05 mg/kg as measured with ASA
(AA-7000, Shimadzu). Each rat received chromium in the amount of 0.3 mg/kg of BW,
which was calculated daily from the amount of daily food intake and the body weight. For
safety reasons, Cr preparations were prepared in the form of emulsion in rapeseed oil and
added into the diet [10].
Rats were kept in separate metabolic cages under the following conditions: tempera-
ture of 21–22
◦
C, relative humidity of 60%
±
10%, and a ventilation rate of 15 air changes
during one hour. Rats had free access to a fresh diet and water on a daily basis.
2.3. Experimental Procedures
Intraperitoneal injection of ketamine/xylazine (100/10 mg/kg BW) was used for
anesthesia. Rats were killed by decapitation and exsanguinated. Blood samples were
kept in vials containing heparin + EDTA as an anticoagulant and centrifuged at 3000
×
g
for 10 min to separate the blood plasma. The hearts were carefully dissected, weighed,
and immediately placed in liquid nitrogen (
−
196
◦
C) for 30 min and then stored at low
temperature (
−
80
◦
C) for further analyses. The thoracic aorta was dissected with care,
cleaned of adherent tissue, cut into 6–8 rings, and kept on ice.
For the extraction and quantification of proteins, rat tissue was homogenized with
the RIPA buffer (Santa Cruz Biotechnology, Heidelberg, Germany), with absorbance set at
595 nm. Bovine serum albumin was used as a control [20].
2.4. Blood Pressure
Heart rate (bpm) and mean arterial pressure (mmHg) were measured one day before
the blood collection with the noninvasive tail-cuff method (LE5001, Panlab, Harvard
Apparatus, Barcelona, Spain) [21].

Nutrients 2022,14, 5138 5 of 20
2.5. The Antioxidant Capacity of Blood Plasma
The antioxidant capacity of water (ACW)- and lipid (ACL)-soluble compounds of
the blood plasma (in
µ
g/mL) was determined with Photochem (Analytik Jena AG, Jena,
Germany). This photo-chemiluminescence detection method generates free radicals that are
removed with the antioxidants presented in blood plasma, and the remaining radicals are
quantified. The calibration curve was prepared with ascorbic acid and Trolox as standards
for ACW and ACL [21].
2.6. Markers of Antioxidant Status in the Heart
Heart malondialdehyde (MDA, nmol/g), the total sum of reduced and oxidized
glutathione (GSH+GSSG, nmol/g), superoxide dismutase (SOD, U/g), and catalase (CAT,
U/g) were analyzed according to a previously described method [
2
,
14
]. The MDA reacts
with thiobarbituric acid (TBA), which gives an MDA–TBA adduct that was quantified
with a fluorometric assay kit (ab118970) at Ex/Em = 532/553 nm. GSH+GSSG and SOD
activities were determined with Ransel and Ransod colorimetric diagnostic kits from
Randox (Warsaw, Poland), following the manufacturer’s instructions. CAT activity was
determined with an Oxis International, Inc., (Portland, OR, USA) diagnostic kit following
the manufacturer’s instructions.
2.7. Vascular Reactivity Studies
Aortic rings from the thoracic segment of the aorta (4–5 mm length) were mounted
in stagnant 5 mL chambers (Graz Tissue Bath System, Barcelona, Spain) and aerated with
carbogen gas for 60 min. Two parallel stainless steel wires were implemented through the
lumen of the aortic rings: one fixed to the bath wall and the other connected to a force
transducer (FT20, TAM-A, Hugo Sachs Elektronik, March, Germany). Pre-load tension
of 1 cN was applied to the tissue (measured with LabChart, ADInstruments, New South
Wales, Australia). The functional integrity of aortic rings was checked with high KCl
(75 mM) and ACh (10
µ
M). Next, aortic rings were pre-incubated for 30 min with either the
inducible nitric oxide synthase (iNOS) inhibitor (1400 W at 1
µ
M), the non-selective COX
inhibitor (indomethacin at 10
µ
M), the selective cyclooxygenase-2 (COX-2) inhibitor (NS-
398 at 10
µ
M), the thromboxane-A
2
receptor (TP) antagonist (SQ-29,548 at 1
µ
M), the PGI
2
synthesis inhibitor (tranylcypromine, TCP at 10
µ
M), or TCP (10
µ
M) plus SQ-29,548 (1
µ
M)
and contracted with noradrenaline (0.1
µ
M). Afterwards, the cumulative concentrations of
ACh (0.1 nM–10
µ
M) were added into the incubation chambers, and this was done once
only. In another set of experiments, the cumulative concentrations of NA (0.1 nM–10
µ
M)
and SNP (0.1 nM–10 µM) were constructed.
2.8. Biochemical Studies of Aortic Rings
2.8.1. TXA2and PGI2Production
First, we stabilized the aortic rings from each group of rats in KHS at +37
◦
C for 30 min
at pH = 7.4. Next, aortic rings were washed twice with 200
µ
L of KHS for 10 min. Further,
we exposed aortic rings to noradrenaline (0.1
µ
M, 2 min) and to increasing concentrations
of ACh (0.1 nM–10
µ
M, at 1-min intervals). Production of TXA
2
and PGI
2
were monitored
by measuring the release of their stable metabolites, TxB
2
and 6-keto-PGF1
α
, with the
appropriate enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA). Results
were expressed as pg prostanoid/mg of protein [6].
2.8.2. Detection of Superoxide Anion
This was measured using lucigenin chemiluminescence, as previously described [
20
].
Aortic rings without endothelium were equilibrated in HEPES buffer at 37
◦
C for 30 min
and moved to test tubes containing lucigenin (5
µ
mol/L) + 1 mL HEPES buffer (
pH = 7.4
,
at 37
◦
C). Tiron (10 mmol/L), a superoxide anion (O
2.−
) scavenger, was added for chemilu-
minescence detection. For background emission, blank samples without aortic rings were
prepared. Results were expressed as chemiluminescence units CU/minute/mg tissue.

Nutrients 2022,14, 5138 6 of 20
2.8.3. Detection of Hydrogen Peroxide
This was measured with a fluorescence assay kit (Cayman Chemicals, Ann Arbor,
MI, USA) at 530/590 nm excitation/emission wavelengths following the manufacturer’s
instructions. To ensure the specificity of the method, some of the rings were subjected to
CAT, a hydrogen peroxide (H
2
O
2
) scavenger. Results were expressed as nmol/
µ
g protein.
2.9. Data Analysis and Statistics
Results are given as means
±
SD or means
±
SEM (for vascular reactivity studies)
and were prepared in GraphPad Prism 8.4. Vascular relaxation to ACh was calculated
as a percentage of the response to NA and analyzed with a nonlinear regression model.
This determined the area under the curve (AUC), the maximal response (Emax, %), and
the potency (the negative logarithm of the concentration causing a half-maximum effect,
pEC
50
). The obtained data were checked with Grubbs’ test and were analyzed for Gaussian
distribution of residuals and homoscedasticity of variance. The group comparison was
performed by two-way ANOVA with an appropriate post hoc test. Differences were
considered significant when p≤0.05.
3. Results
3.1. General Characteristics
Blood pressure did not differ between the studied groups (data not presented).
3.2. The Antioxidant Capacity of Blood Plasma
3.2.1. Blood Plasma ACW
When the F diet was administered for 9 weeks, ACW did not change in the control FS
group (S group vs. FS control, p= 0.7225). However, Cr–NPs decreased the ACW compared
to the S group (0.71-fold, p= 0.0016), see Figure 2. A significant decrease was observed in
the Cr–NPs group vs. the Cr–Pic group (0.75-fold, p= 0.0205).
When the F diet was administered for 18 weeks, ACW decreased in the control FF
group (S group vs. FF control, 0.76-fold, p= 0.0173). Cr–NPs, but not Cr–Pic, decreased the
ACW compared to the S group (0.71-fold, p= 0.0014).
We did not observe any statistically significant change due to the dietary intervention
between 9 weeks and 18 weeks of F diet for Cr-supplemented or non-supplemented
(control) rats.
3.2.2. Blood Plasma ACL
ACL did not differ between the studied groups (5.339
±
1.446, p
≥
0.8818, data
not presented).
3.3. Markers of Antioxidant Status in the Heart
3.3.1. Heart Malondialdehyde
When the F diet was administered for 9 weeks, MDA did not change in the control FS
group (S group vs. FS control, p= 0.9191). Neither Cr–NPs nor Cr–Pic changed the MDA,
see Figure 3A.
When the F diet was administered for 18 weeks, MDA did not change in the control
FF group (S group vs. FF control, p= 0.9758). However, it was Cr–NPs, not Cr–Pic, that
decreased MDA compared to the control FF group (0.60-fold, p= 0.0036) and the S group
(0.65-fold, p= 0.0378).
We did not observe any significant change due to dietary intervention between 9 weeks
and 18 weeks of F diet in Cr-supplemented or non-supplemented (control) rats.

Nutrients 2022,14, 5138 7 of 20
Nutrients 2022, 14, x FOR PEER REVIEW 7 of 21
Control S
Control FS
Cr−Pic FS
Cr−NPs FS
Control FF
Cr−Pic FF
Cr−NPs FF
0
2
4
6
ACW in blood
(μg/mL ascorbic acid)
0.0173
0.0016
0.0014
0.0205
0.0200
FFFSS
Figure 2. Blood plasma ACW of either chromium (Cr)-supplemented (9 weeks of supplementation)
or non-supplemented (Control S, Control FS, and Control FF) rats. 0.3 mg of Cr per kg of body
weight as chromium picolinate (Cr–Pic) or chromium nanoparticles (Cr–NPs) were added after the
initial 9 weeks of experimental feed without Cr supplementation. Two types of diet were applied: a
standard-fat-and-fiber (S) diet and a high-fat–low-fiber (F) diet. Rats at 7 weeks of age were fed with
an S diet or an F diet for either 9 weeks or 9 + 9 weeks. The following groups of rats were studied:
Control S, the S diet plus the S diet; Control FS, the F diet and the S diet; Cr–Pic FS, the F diet and
the Cr–Pic S diet; Cr–NPs FS, the F diet and the Cr–NPs S diet; Control FF, the F diet and the F diet;
Cr–Pic FF, the F diet and the Cr–Pic F diet; Cr–NPs FF, the F diet and the Cr–NPs F diet. Values are
means ± SD, n = 10, p ≤ 0.05 (two-way ANOVA/Tukey’s multiple comparisons test). ACW is a marker
that distinguishes Cr–NPs from Cr–Pic in rats fed with the F diet for 9 weeks. Abbreviations: ACW,
antioxidant capacity of water-soluble compounds.
3.2.2. Blood Plasma ACL
ACL did not differ between the studied groups (5.339 ± 1.446, p ≥ 0.8818, data not
presented).
3.3. Markers of Antioxidant Status in the Heart
3.3.1. Heart Malondialdehyde
When the F diet was administered for 9 weeks, MDA did not change in the control
FS group (S group vs. FS control, p = 0.9191). Neither Cr–NPs nor Cr–Pic changed the
MDA, see Figure 3A.
Figure 2.
Blood plasma ACW of either chromium (Cr)-supplemented (9 weeks of supplementation)
or non-supplemented (Control S, Control FS, and Control FF) rats. 0.3 mg of Cr per kg of body
weight as chromium picolinate (Cr–Pic) or chromium nanoparticles (Cr–NPs) were added after the
initial 9 weeks of experimental feed without Cr supplementation. Two types of diet were applied: a
standard-fat-and-fiber (S) diet and a high-fat–low-fiber (F) diet. Rats at 7 weeks of age were fed with
an S diet or an F diet for either 9 weeks or 9 + 9 weeks. The following groups of rats were studied:
Control S, the S diet plus the S diet; Control FS, the F diet and the S diet; Cr–Pic FS, the F diet and
the Cr–Pic S diet; Cr–NPs FS, the F diet and the Cr–NPs S diet; Control FF, the F diet and the F diet;
Cr–Pic FF, the F diet and the Cr–Pic F diet; Cr–NPs FF, the F diet and the Cr–NPs F diet. Values are
means
±
SD, n= 10, p
≤
0.05 (two-way ANOVA/Tukey’s multiple comparisons test). ACW is a
marker that distinguishes Cr–NPs from Cr–Pic in rats fed with the F diet for 9 weeks. Abbreviations:
ACW, antioxidant capacity of water-soluble compounds.
3.3.2. Heart GSH+GSSG
When the F diet was administered for 9 weeks, GSH+GSSG did not change in the
control FS group (S group vs. FS control, p= 0.8875). Cr–Pic decreased GSH+GSSG com-
pared to the control FS group (0.62-fold, p< 0.0001) and the S group (0.67-fold,
p= 0.0008
).
Meanwhile, Cr–NPs decreased GSH+GSSG compared to the control FS group (0.74-fold,
p= 0.0048), but not the S group (0.80-fold, p= 0.1074), see Figure 3B.
When the F diet was administered for 18 weeks, GSH+GSSG tended to increase in the
control FF group (S group vs. FF control, 1.2-fold, p= 0.0973). Cr–Pic tended to decrease
GSH+GSSG compared to the control FF group (0.82-fold, p= 0.0704).
However, we did observe a significant increase in GSH + GSSG during 18 weeks of F
diet fed Cr–Pic and Cr–NPs rats by 1.46-fold, p= 0.0031 and 1.43-fold, p= 0.0004, respec-
tively. This was not observed in non-supplemented controls (p= 0.6796), see Figure 3B.

Nutrients 2022,14, 5138 8 of 20
Nutrients 2022, 14, x FOR PEER REVIEW 8 of 21
Control S
Control FS
Cr−Pic FS
Cr−NPs FS
Control FF
Cr−Pic FF
Cr−NPs FF
0
1
2
3
4
5
MDA (nmol/g)
0.0378
0.0036
FFFSS
A
Control S
Control FS
Cr−Pic FS
Cr−NPs FS
Control FF
Cr−Pic FF
Cr−NPs FF
0
20
40
60
80
GSH+GSSG (nmol/g)
0.0008
<0.0001
0.0048
<0.0001
0.0031
<0.0001
<0.0001
0.0004
FFFSS
B
Control S
Control FS
Cr−Pic FS
Cr−NPs FS
Control FF
Cr−Pic FF
Cr−NPs FF
0
200
400
600
SOD (U/g of protein)
0.0060
0.0008
0.0015
0.0073
0.0117
<0.0001
FFFSS
C
Control S
Control FS
Cr−Pic FS
Cr−NPs FS
Control FF
Cr−Pic FF
Cr−NPs FF
0
100
200
300
CAT (U/g of protein)
0.0038
0.0088
0.0295
0.0018
FFFSS
D
Figure 3. Heart MDA (A), GSH+GSSG (B), SOD (C) and CAT (D) of either chromium (Cr)-supple-
mented (9 weeks of supplementation) or nonsupplemented (Control S, Control FS, and Control FF)
rats. 0.3 mg Cr/kg of body weight as chromium picolinate (Cr–Pic) or chromium nanoparticles (Cr–
NPs) were added after the initial 9 weeks of experimental feed without Cr supplementation. Two
types of diet were applied: a standard-fat-and-fiber (S) diet and a high-fat–low-fiber (F) diet. Rats at
7 weeks of age were fed with an S diet or an F diet for either 9 weeks or 9 + 9 weeks. The following
groups of rats were studied: Control S, the S diet plus the S diet; Control FS, the F diet and the S
diet; Cr–Pic FS, the F diet and the Cr–Pic S diet; Cr–NPs FS, the F diet and the Cr–NPs S diet; Control
FF, the F diet and the F diet; Cr–Pic FF, the F diet and the Cr–Pic F diet; Cr–NPs FF, the F diet and
the Cr–NPs F diet. Values are means ± SD, n = 8, p ≤ 0.05 (two-way ANOVA/Tukey’s multiple com-
parisons test). In rats fed with the F diet for 9 weeks, Cr–NPs and Cr–Pic decreased the GSH+GSSG.
In rats fed with F diet for 18 weeks Cr–NPs decreased both the MDA and CAT and increased SOD;
meanwhile, Cr–Pic decreased CAT and increased SOD. No significant difference between Cr–NPs
Figure 3.
Heart MDA (
A
), GSH+GSSG (
B
), SOD (
C
) and CAT (
D
) of either chromium (Cr)-
supplemented (9 weeks of supplementation) or nonsupplemented (Control S, Control FS, and Control
FF) rats. 0.3 mg Cr/kg of body weight as chromium picolinate (Cr–Pic) or chromium nanoparticles
(Cr–NPs) were added after the initial 9 weeks of experimental feed without Cr supplementation.
Two types of diet were applied: a standard-fat-and-fiber (S) diet and a high-fat–low-fiber (F) diet.
Rats at 7 weeks of age were fed with an S diet or an F diet for either 9 weeks or 9 + 9 weeks. The
following groups of rats were studied: Control S, the S diet plus the S diet; Control FS, the F diet
and the S diet; Cr–Pic FS, the F diet and the Cr–Pic S diet; Cr–NPs FS, the F diet and the Cr–NPs S
diet; Control FF, the F diet and the F diet; Cr–Pic FF, the F diet and the Cr–Pic F diet; Cr–NPs FF, the
F diet and the Cr–NPs F diet. Values are means
±
SD, n = 8, p
≤
0.05 (two-way ANOVA/Tukey’s
multiple comparisons test). In rats fed with the F diet for 9 weeks, Cr–NPs and Cr–Pic decreased
the GSH+GSSG. In rats fed with F diet for 18 weeks Cr–NPs decreased both the MDA and CAT and
increased SOD; meanwhile, Cr–Pic decreased CAT and increased SOD. No significant difference
between Cr–NPs and Cr–Pic was observed in MDA, GSH+GSSG, SOD, and CAT. Abbreviations:
CAT, catalase; GSH+GSSG, total glutathione; MDA, malondialdehyde; SOD, superoxide dismutase.

Nutrients 2022,14, 5138 9 of 20
3.3.3. Heart SOD
When the F diet was administered for 9 weeks, SOD did not change in the control FS
group (S group vs. FS control, p= 0.9939). Neither Cr–NPs nor Cr–Pic changed the SOD,
see Figure 3C.
When the F diet was administered for 18 weeks, SOD decreased in the control FF group
(S group vs. FF control, 0.39-fold, p= 0.0060), and both Cr–NPs and Cr–Pic increased SOD
compared to the control FF group (3.44-fold, p< 0.0001 and 2.52-fold,
p= 0.0117
, respectively).
We did not observe any significant change due to the dietary intervention between
9 weeks and 18 weeks of F diet in Cr-supplemented rats. However, a significant decrease
was observed in non-supplemented control FF rats (0.35-fold, p= 0.0008).
3.3.4. Heart CAT
When the F diet was administered for 9 weeks, CAT did not change in the control FS
group (S group vs. FS control, p= 0.8968). Neither Cr–NPs nor Cr–Pic changed the CAT,
see Figure 3D.
When the F diet was administered for 18 weeks, CAT increased in the control FF group
(S group vs. FF control, 1.40-fold, p= 0.0038), and both Cr–NPs and Cr–Pic decreased
CAT compared to the control FF group (0.70-fold, p= 0.0018 and 0.75-fold, p= 0.0295,
respectively).
We did not observe any significant change due to the dietary intervention between
9 weeks and 18 weeks of F diet in Cr-supplemented or non-supplemented (control) rats.
3.4. Vascular Studies
3.4.1. Vascular Reactivity Studies
Vasoconstrictor response to high KCl (75 mM) and NA (0.1 nM–10
µ
M) did not differ
between the studied groups (data not presented).
Vasodilator response to SNP did not differ between the studied groups (data not
presented), as opposed to the ACh-induced response, see Figure 4A–D.
When the F diet was administered for 9 weeks, the vasodilator response to ACh did
not change in the control FS group (Figure 4C). Neither Cr–NPs nor Cr–Pic changed the
vasodilation (Figure 4A).
When the F diet was administered for 18 weeks, the vasodilator response to ACh
shifted to the left in the control FF group. However, it was Cr–Pic, not Cr–NPs that shifted
the vasodilation back to the right (Figure 4B). We did not observe any significant change due
to the dietary (F) intervention (9 weeks vs. 18 weeks) in Cr–Pic- and Cr–NPs-supplemented
rats (Figure 4D). However, a significant change was observed between non-supplemented
controls (control FS vs. control FF, Figure 4C).
Pre-incubation of the aortic rings with the inducible nitric oxide synthase (iNOS)
inhibitor 1400 W did not change the vasodilator response to ACh in arteries compared to
the control groups of rats (control S, control FS, and control FF, Figure 5A–C).
When the F diet was administered for 9 weeks, pre-incubation with 1400 W shifted
the ACh-induced response to the right in Cr–Pic and Cr–NPs groups (Figure 5D,F).
When the F diet was administered for 18 weeks, pre-incubation with 1400 W did not
change vasodilation in all studied groups (Figure 5E,G).
Pre-incubation of the aortic rings with the specific COX-2 inhibitor NS-398 shifted the
vasodilator response to the right in ACh in arteries from the control S rats. A non-selective
COX inhibitor indomethacin did not modify that response (Figure 6A).
When the F diet was administered for 9 weeks, pre-incubation with NS-398 and
indomethacin shifted the ACh-induced response to the right in Cr–Pic- and Cr–NPs-
supplemented rats (Figure 6D,F), but not in the control group (Figure 6B). Moreover, the
maximal response (Emax) decreased in arteries from the Cr–Pic group (Figure 6D).
When the F diet was administered for 18 weeks, pre-incubation with NS-398 and
indomethacin shifted vasodilation to the right in the control group (Figure 6C) and in the

Nutrients 2022,14, 5138 10 of 20
two Cr groups (Cr–Pic and Cr–NPs, Figure 6E,G). Moreover, the maximal response (Emax)
decreased in arteries from Cr–Pic-supplemented rats only (Figure 6E).
Pre-incubation of the aortic rings with the TXA
2
(TP) receptor antagonist SQ-29,548
(1 mmol/L, 30 min) did not modify the response to ACh in arteries from all studied groups
of rats (Figure 7A–G).
Pre-incubation of the aortic rings with the specific PGI
2
synthesis inhibitor TCP shifted
the vasodilator response to ACh in arteries to the right in all three control groups: control S,
control FS and, control FF (Figure 8A–C).
When the F diet was administered for 9 weeks, pre-incubation with TCP shifted the
ACh-induced response to the right in the arteries from Cr–NPs rats, but not in the Cr–Pic
group (Figure 8B,D,F).
Nutrients 2022, 14, x FOR PEER REVIEW 10 of 21
When the F diet was administered for 9 weeks, the vasodilator response to ACh did
not change in the control FS group (Figure 4C). Neither Cr–NPs nor Cr–Pic changed the
vasodilation (Figure 4A).
When the F diet was administered for 18 weeks, the vasodilator response to ACh
shifted to the left in the control FF group. However, it was Cr–Pic, not Cr–NPs that shifted
the vasodilation back to the right (Figure 4B). We did not observe any significant change
due to the dietary (F) intervention (9 weeks vs. 18 weeks) in Cr–Pic- and Cr–NPs-supple-
mented rats (Figure 4D). However, a significant change was observed between non-sup-
plemented controls (control FS vs. control FF, Figure 4C).
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
Relaxation (%)
A9 weeks
aControl FS
bCr−Pic FS
cCr−NPs FS
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
*
*
a–b:
ANOVA,p≤ 0.05
B18 weeks
aControl FF
bCr−Pic FF
cCr−NPs FF
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
aControl S
bControl FS
cControl FF
a–c,b–c:
ANOVA,p ≤ 0.05
C
*
*
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
aCr−Pic FS
bCr−Pic FF
cCr−NPs FS
dCr−NPs FF
D
Figure 4. The vasodilator response to acetylcholine (ACh) in aortic rings from either chromium (Cr)
supplemented (9 weeks of supplementation) or non-supplemented (Control S, Control FS, and Con-
trol FF) rats. 0.3 mg Cr/kg of body weight as chromium picolinate (Cr–Pic) or chromium nanoparti-
cles (Cr–NPs) were added after the initial 9 weeks of experimental feed without Cr supplementation.
Two types of diet were applied: a standard-fat-and-fiber (S) diet and a high-fat–low-fiber (F) diet.
Rats at 7 weeks of age were fed with an S diet or an F diet for either 9 weeks or 9 + 9 weeks. The
following groups of rats were studied: Control S, the S diet plus the S diet; Control FS, the F diet
and the S diet; Cr–Pic FS, the F diet and the Cr–Pic S diet; Cr–NPs FS, the F diet and the Cr–NPs S
diet; Control FF, the F diet and the F diet; Cr–Pic FF, the F diet and the Cr–Pic F diet; Cr–NPs FF, the
F diet and the Cr–NPs F diet. FS group (A), FF group (B), Control groups (C), FS vs. FF group (D).
Results (means ± SEM) are expressed as a percentage of the inhibition of the contraction induced by
noradrenaline (0.1 μM), n = 8, * p ≤ 0.05 (two-way ANOVA/Šídák’s). F diet given for 18 weeks de-
creased vasodilation to ACh compared to either S diet (18 weeks) or F diet (9 weeks), and it was Cr–
Pic that modified vasodilation.
Figure 4.
The vasodilator response to acetylcholine (ACh) in aortic rings from either chromium (Cr)
supplemented (9 weeks of supplementation) or non-supplemented (Control S, Control FS, and Control
FF) rats. 0.3 mg Cr/kg of body weight as chromium picolinate (Cr–Pic) or chromium nanoparticles
(Cr–NPs) were added after the initial 9 weeks of experimental feed without Cr supplementation.
Two types of diet were applied: a standard-fat-and-fiber (S) diet and a high-fat–low-fiber (F) diet.
Rats at 7 weeks of age were fed with an S diet or an F diet for either 9 weeks or 9 + 9 weeks. The
following groups of rats were studied: Control S, the S diet plus the S diet; Control FS, the F diet
and the S diet; Cr–Pic FS, the F diet and the Cr–Pic S diet; Cr–NPs FS, the F diet and the Cr–NPs S
diet; Control FF, the F diet and the F diet; Cr–Pic FF, the F diet and the Cr–Pic F diet; Cr–NPs FF, the
F diet and the Cr–NPs F diet. FS group (
A
), FF group (
B
), Control groups (
C
), FS vs. FF group (
D
).
Results (means
±
SEM) are expressed as a percentage of the inhibition of the contraction induced
by noradrenaline (0.1
µ
M), n= 8, * p
≤
0.05 (two-way ANOVA/Šídák’s). F diet given for 18 weeks
decreased vasodilation to ACh compared to either S diet (18 weeks) or F diet (9 weeks), and it was
Cr–Pic that modified vasodilation.

Nutrients 2022,14, 5138 11 of 20
When the F diet was administered for 18 weeks, pre-incubation with TCP did not
modify the vasodilation in Cr–NPs and Cr–Pic arteries (Figure 8C,E,G).
Pre-incubation with TCP plus SQ-29,548 normalized the ACh-induced response to the
control level in arteries from the control S and control FS groups but not in the control FF
group (Figure 8A–C).
Nutrients 2022, 14, x FOR PEER REVIEW 11 of 21
Pre-incubation of the aortic rings with the inducible nitric oxide synthase (iNOS) in-
hibitor 1400W did not change the vasodilator response to ACh in arteries compared to the
control groups of rats (control S, control FS, and control FF, Figure 5A–C).
When the F diet was administered for 9 weeks, pre-incubation with 1400W shifted
the ACh-induced response to the right in Cr–Pic and Cr–NPs groups (Figure 5D,F).
When the F diet was administered for 18 weeks, pre-incubation with 1400W did not
change vasodilation in all studied groups (Figure 5E,G).
Control S
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
C
C
1400W
A
Control FS
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
CC
1400W
B
Control FF
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
CC
1400W
C
Cr–Pic FS
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
CC
1400W
D
ANOVA,p ≤ 0.05
**
Cr–Pic FF
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
CC
1400W
E
Cr–NPs FS
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
CC
1400W
F
ANOVA,p ≤ 0.05 *
*
Cr–NPs FF
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
CC
1400W
G
Figure 5. Effects of 1400W (1 µM) on the concentration–response curves to acetylcholine (ACh) in
aortic rings from the following groups of rats: control S (A), control FS (B), control FF (C), Cr–Pic FS
(D), Cr–Pic FF (E), Cr–NPs FS (F), and Cr–NPs FF (G). 0.3 mg Cr/kg of body weight as chromium
picolinate (Cr–Pic) and chromium nanoparticles (Cr–NPs) were added for 9 weeks of supplementa-
tion after the initial 9-week period of experimental feed without Cr supplementation. Two types of
diet were applied: a standard-fat-and-fiber (S) diet and a high-fat–low-fiber (F) diet. Rats at 7 weeks
of age were fed with an S diet or an F diet for either 9 weeks or 9 + 9 weeks. The following groups
of rats were studied: Control S, the S diet plus the S diet; Control FS, the F diet and the S diet; Cr–
Pic FS, the F diet and the Cr–Pic S diet; Cr–NPs FS, the F diet and the Cr–NPs S diet; Control FF, the
F diet and the F diet; Cr–Pic FF, the F diet and the Cr–Pic F diet; Cr–NPs FF, the F diet and the Cr–
NPs F diet. Results (means ± SEM) are expressed as percentage of inhibition of contraction induced
by noradrenaline (0.1 µM). n = 8, * p ≤ 0.05 (two-way ANOVA/Šídák’s) compared with control con-
ditions. In arteries from rats fed with the F diet for 9 weeks, both Cr–Pic and Cr–NPs enhanced
participation of NO derived from iNOS in vascular relaxation to ACh.
Figure 5.
Effects of 1400 W (1
µ
M) on the concentration–response curves to acetylcholine (ACh) in
aortic rings from the following groups of rats: control S (
A
), control FS (
B
), control FF (
C
), Cr–Pic FS
(
D
), Cr–Pic FF (
E
), Cr–NPs FS (
F
), and Cr–NPs FF (
G
). 0.3 mg Cr/kg of body weight as chromium
picolinate (Cr–Pic) and chromium nanoparticles (Cr–NPs) were added for 9 weeks of supplementation
after the initial 9-week period of experimental feed without Cr supplementation. Two types of diet
were applied: a standard-fat-and-fiber (S) diet and a high-fat–low-fiber (F) diet. Rats at 7 weeks of age
were fed with an S diet or an F diet for either 9 weeks or 9 + 9 weeks. The following groups of rats
were studied: Control S, the S diet plus the S diet; Control FS, the F diet and the S diet; Cr–Pic FS, the F
diet and the Cr–Pic S diet; Cr–NPs FS, the F diet and the Cr–NPs S diet; Control FF, the F diet and the
F diet; Cr–Pic FF, the F diet and the Cr–Pic F diet; Cr–NPs FF, the F diet and the Cr–NPs F diet. Results
(means
±
SEM) are expressed as percentage of inhibition of contraction induced by noradrenaline
(0.1
µ
M). n= 8, * p
≤
0.05 (two-way ANOVA/Šídák’s) compared with control conditions. In arteries
from rats fed with the F diet for 9 weeks, both Cr–Pic and Cr–NPs enhanced participation of NO
derived from iNOS in vascular relaxation to ACh.

Nutrients 2022,14, 5138 12 of 20
When the F diet was administered for 9 weeks, pre-incubation with TCP plus SQ-29,548
did not modify the vasodilator response to ACh in arteries from Cr–Pic rats (Figure 8D),
while it was not normalized in arteries from Cr–NPs rats (Figure 8F).
When the F diet was administered for 18 weeks, pre-incubation with TCP plus SQ-
29,548 did not modify the vasodilation in Cr–NPs and Cr–Pic arteries (Figure 8E,G).
For AUC, Emax (%), and pEC50, see Table 2.
Nutrients 2022, 14, x FOR PEER REVIEW 12 of 21
Pre-incubation of the aortic rings with the specific COX-2 inhibitor NS-398 shifted
the vasodilator response to the right in ACh in arteries from the control S rats. A non-
selective COX inhibitor indomethacin did not modify that response (Figure 6A).
When the F diet was administered for 9 weeks, pre-incubation with NS-398 and in-
domethacin shifted the ACh-induced response to the right in Cr–Pic- and Cr–NPs-sup-
plemented rats (Figure 6D,F), but not in the control group (Figure 6B). Moreover, the max-
imal response (Emax) decreased in arteries from the Cr–Pic group (Figure 6D).
When the F diet was administered for 18 weeks, pre-incubation with NS-398 and in-
domethacin shifted vasodilation to the right in the control group (Figure 6C) and in the
two Cr groups (Cr–Pic and Cr–NPs, Figure 6E,G). Moreover, the maximal response
(Emax) decreased in arteries from Cr–Pic-supplemented rats only (Figure 6E).
Control S
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
aCC
bINDO
cNS-398
*
a–c:
ANOVA,p ≤ 0.05
A
*
*
Control FS
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
aCC
bINDO
cNS-398
B
Control FF
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
aCC
cNS-398
bINDO
a–b,a–c:
ANOVA,p ≤ 0.05
C
**
*
**
Cr–Pic FS
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
aCC
cNS-398
bINDO
a–b,a–c:
ANOVA,p ≤ 0.05
D**
*
*
*
Cr–Pic FF
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
aCC
cNS-398
bINDO
a–b,a–c:
ANOVA,p ≤ 0.05
E
*
***
*
*
*
Cr–NPs FS
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
aCC
cNS-398
bINDO
a–b,a–c:
ANOVA,p ≤ 0.05
F**
**
**
Cr–NPs FF
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
aCC
cNS-398
bINDO
a–b,a–c:
ANOVA,p ≤ 0.05
G
**
**
*
Figure 6. Effects of indomethacin (10 µM) and NS-398 (10 µM) on the concentration–response curves
to acetylcholine (ACh) in aortic rings from the following groups of rats: control S (A), control FS (B),
control FF (C), Cr–Pic FS (D), Cr–Pic FF (E), Cr–NPs FS (F), and Cr–NPs FF (G). 0.3 mg Cr/kg of
body weight as chromium picolinate (Cr–Pic) and chromium nanoparticles (Cr–NPs) were added
for 9 weeks of supplementation after the initial 9-week period of experimental feed without Cr sup-
plementation. Two types of diet were applied: a standard-fat-and-fiber (S) diet and a high-fat–low-
fiber (F) diet. Rats at 7 weeks of age were fed with an S diet or an F diet for either 9 weeks or 9 + 9
weeks. The following groups of rats were studied: Control S, the S diet plus the S diet; Control FS,
the F diet and the S diet; Cr–Pic FS, the F diet and the Cr–Pic S diet; Cr–NPs FS, the F diet and the
Cr–NPs S diet; Control FF, the F diet and the F diet; Cr–Pic FF, the F diet and the Cr–Pic F diet; Cr–
NPs FF, the F diet and the Cr–NPs F diet. Results (means ± SEM) are expressed as percentage of
Figure 6.
Effects of indomethacin (10
µ
M) and NS-398 (10
µ
M) on the concentration–response curves
to acetylcholine (ACh) in aortic rings from the following groups of rats: control S (
A
), control FS (
B
),
control FF (
C
), Cr–Pic FS (
D
), Cr–Pic FF (
E
), Cr–NPs FS (
F
), and Cr–NPs FF (
G
). 0.3 mg Cr/kg of
body weight as chromium picolinate (Cr–Pic) and chromium nanoparticles (Cr–NPs) were added
for 9 weeks of supplementation after the initial 9-week period of experimental feed without Cr
supplementation. Two types of diet were applied: a standard-fat-and-fiber (S) diet and a high-fat–
low-fiber (F) diet. Rats at 7 weeks of age were fed with an S diet or an F diet for either 9 weeks or
9 + 9 weeks
. The following groups of rats were studied: Control S, the S diet plus the S diet; Control
FS, the F diet and the S diet; Cr–Pic FS, the F diet and the Cr–Pic S diet; Cr–NPs FS, the F diet and the
Cr–NPs S diet; Control FF, the F diet and the F diet; Cr–Pic FF, the F diet and the Cr–Pic F diet; Cr–NPs
FF, the F diet and the Cr–NPs F diet. Results (means
±
SEM) are expressed as percentage of inhibition
of contraction induced by noradrenaline (0.1
µ
M). n = 8, * p
≤
0.05 (two-way ANOVA/Šídák’s)
compared with control conditions. In arteries from rats fed with the F diet for 9 weeks, both Cr–Pic
and Cr–NPs attenuated vasodilation.

Nutrients 2022,14, 5138 13 of 20
Nutrients 2022, 14, x FOR PEER REVIEW 13 of 21
inhibition of contraction induced by noradrenaline (0.1 µ M). n = 8, * p ≤ 0.05 (two-way
ANOVA/Šídák’s) compared with control conditions. In arteries from rats fed with the F diet for 9
weeks, both Cr–Pic and Cr–NPs attenuated vasodilation.
Pre-incubation of the aortic rings with the TXA2 (TP) receptor antagonist SQ-29,548
(1 mmol/L, 30 min) did not modify the response to ACh in arteries from all studied groups
of rats (Figure 7A–G).
Control S
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
CC
SQ-29,548
A
Control FS
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
CC
SQ-29,548
B
Control FF
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
CC
SQ-29,548
C
Cr–Pic FS
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
CC
SQ-29,548
D
Cr–Pic FF
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
CC
SQ-29,548
E
Cr–NPs FS
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
CC
SQ-29,548
F
Cr–NPs FF
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
CC
SQ-29,548
G
Figure 7. Effects of SQ-29,548 (1 µM) on the concentration–response curves to acetylcholine (ACh)
in aortic rings from the following groups of rats: control S (A), control FS (B), control FF (C), Cr–Pic
FS (D), Cr–Pic FF (E), Cr–NPs FS (F), and Cr–NPs FF (G). 0.3 mg Cr/kg of body weight as chromium
picolinate (Cr–Pic) and chromium nanoparticles (Cr–NPs) were added for 9 weeks of supplementa-
tion after the initial 9-week period of experimental feed without Cr supplementation. Two types of
diet were applied: a standard-fat-and-fiber (S) diet and a high-fat–low-fiber (F) diet. Rats at 7 weeks
of age were fed with an S diet or an F diet for either 9 weeks or 9 + 9 weeks. The following groups
of rats were studied: Control S, the S diet plus the S diet; Control FS, the F diet and the S diet; Cr–
Pic FS, the F diet and the Cr–Pic S diet; Cr–NPs FS, the F diet and the Cr–NPs S diet; Control FF, the
F diet and the F diet; Cr–Pic FF, the F diet and the Cr–Pic F diet; Cr–NPs FF, the F diet and the Cr–
NPs F diet. Results (means ± SEM) are expressed as percentage of inhibition of contraction induced
by noradrenaline (0.1 µM). n = 8, p ≤ 0.05 (two-way ANOVA/Šídák’s) compared with control condi-
tions. No significant difference was observed between the analyzed groups.
Figure 7.
Effects of SQ-29,548 (1
µ
M) on the concentration–response curves to acetylcholine (ACh) in
aortic rings from the following groups of rats: control S (
A
), control FS (
B
), control FF (
C
), Cr–Pic FS
(
D
), Cr–Pic FF (
E
), Cr–NPs FS (
F
), and Cr–NPs FF (
G
). 0.3 mg Cr/kg of body weight as chromium
picolinate (Cr–Pic) and chromium nanoparticles (Cr–NPs) were added for 9 weeks of supplementation
after the initial 9-week period of experimental feed without Cr supplementation. Two types of diet
were applied: a standard-fat-and-fiber (S) diet and a high-fat–low-fiber (F) diet. Rats at 7 weeks of
age were fed with an S diet or an F diet for either 9 weeks or 9 + 9 weeks. The following groups of
rats were studied: Control S, the S diet plus the S diet; Control FS, the F diet and the S diet; Cr–Pic
FS, the F diet and the Cr–Pic S diet; Cr–NPs FS, the F diet and the Cr–NPs S diet; Control FF, the F
diet and the F diet; Cr–Pic FF, the F diet and the Cr–Pic F diet; Cr–NPs FF, the F diet and the Cr–NPs
F diet. Results (means
±
SEM) are expressed as percentage of inhibition of contraction induced by
noradrenaline (0.1
µ
M). n = 8, p
≤
0.05 (two-way ANOVA/Šídák’s) compared with control conditions.
No significant difference was observed between the analyzed groups.
3.4.2. TXA2and PGI2Production
Neither Cr–Pic nor Cr–NPs modified the production of TXA
2
and PGI
2
over 9 and
18 weeks of F diet administration (data not presented).
3.4.3. Detection of Superoxide Anion
In arteries from rats fed the F diet for 9 weeks, relative O
2.−
production did not change
in the control FS group. However, both Cr–Pic (1.22-fold) and Cr–NPs (1.20-fold) increased
the relative O
2.−
production. This was not observed when the F diet was administered for
18 weeks (Figure 9A).

Nutrients 2022,14, 5138 14 of 20
Nutrients 2022, 14, x FOR PEER REVIEW 14 of 21
Pre-incubation of the aortic rings with the specific PGI2 synthesis inhibitor TCP
shifted the vasodilator response to ACh in arteries to the right in all three control groups:
control S, control FS and, control FF (Figure 8A–C).
When the F diet was administered for 9 weeks, pre-incubation with TCP shifted the
ACh-induced response to the right in the arteries from Cr–NPs rats, but not in the Cr–Pic
group (Figure 8B,D,F).
When the F diet was administered for 18 weeks, pre-incubation with TCP did not
modify the vasodilation in Cr–NPs and Cr–Pic arteries (Figure 8C,E,G).
Pre-incubation with TCP plus SQ-29,548 normalized the ACh-induced response to
the control level in arteries from the control S and control FS groups but not in the control
FF group (Figure 8A–C).
When the F diet was administered for 9 weeks, pre-incubation with TCP plus SQ-
29,548 did not modify the vasodilator response to ACh in arteries from Cr–Pic rats (Figure
8D), while it was not normalized in arteries from Cr–NPs rats (Figure 8F).
When the F diet was administered for 18 weeks, pre-incubation with TCP plus SQ-
29,548 did not modify the vasodilation in Cr–NPs and Cr–Pic arteries (Figure 8E,G).
Control S
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
*
aCC
bTCP
cTCP+SQ-29,548
A
a–b:
ANOVA,p ≤ 0.05
Control FS
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
*
aCC
bTCP
cTCP+SQ-29,548
B
a–b:
ANOVA,p ≤ 0.05
Control FF
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
*
aCC
bTCP
cTCP+SQ-29,548
C
a–b, a–c:
ANOVA,p ≤ 0.05
*
Cr–Pic FS
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
aCC
bTCP
cTCP+SQ-29,548
D
Cr–Pic FF
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
aCC
bTCP
cTCP+SQ-29,548
E
Cr–NPs FS
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
*
*
aCC
bTCP
cTCP+SQ-29,548
F
a–b, a–c:
ANOVA,p ≤ 0.05
*
*
Cr–NPs FF
-10 -9 -8 -7 -6 -5
-100
-80
-60
-40
-20
0
ACh, log M
Relaxation (%)
aCC
bTCP
cTCP+SQ-29,548
G
Figure 8. Effects of TCP (10 µM) and TCP (10 µM) plus SQ-29,548 (1 µM) on the concentration–
response curves to acetylcholine (ACh) in aortic rings from the following groups of rats: control S
(A), control FS (B), control FF (C), Cr–Pic FS (D), Cr–Pic FF (E), Cr–NPs FS (F), and Cr–NPs FF (G).
0.3 mg Cr/kg of body weight as chromium picolinate (Cr–Pic) and chromium nanoparticles (Cr–
Figure 8.
Effects of TCP (10
µ
M) and TCP (10
µ
M) plus SQ-29,548 (1
µ
M) on the concentration–
response curves to acetylcholine (ACh) in aortic rings from the following groups of rats: control
S (
A
), control FS (
B
), control FF (
C
), Cr–Pic FS (
D
), Cr–Pic FF (
E
), Cr–NPs FS (
F
), and Cr–NPs FF
(
G
). 0.3 mg Cr/kg of body weight as chromium picolinate (Cr–Pic) and chromium nanoparticles
(Cr–NPs) were added for 9 weeks of supplementation after the initial 9-week period of experimental
feed without Cr supplementation. Two types of diet were applied: a standard-fat-and-fiber (S) diet
and a high-fat–low-fiber (F) diet. Rats at 7 weeks of age were fed with an S diet or an F diet for either
9 weeks or 9 + 9 weeks. The following groups of rats were studied: Control S, the S diet plus the
S diet; Control FS, the F diet and the S diet; Cr–Pic FS, the F diet and the Cr–Pic S diet; Cr–NPs FS,
the F diet and the Cr–NPs S diet; Control FF, the F diet and the F diet; Cr–Pic FF, the F diet and the
Cr–Pic F diet; Cr–NPs FF, the F diet and the Cr–NPs F diet. Results (means
±
SEM) are expressed
as a percentage of inhibition of contraction induced by noradrenaline (0.1
µ
M). n= 8, * p
≤
0.05
(two-way ANOVA/Šídák’s) compared with control conditions. In arteries from rats fed with the F
diet for 9 weeks, Cr–NPs decreased the sensitivity to ACh in arteries pre-incubated with TCP and
TCP+SQ-29,548.
3.4.4. Detection of Hydrogen Peroxide
In arteries from rats fed the F diet for 9 weeks, H
2
O
2
production did not change in the
control FS group (p= 0.5951). Neither Cr–Pic nor Cr–NPs modified that response. However,
a significant increase (1.59-fold) was observed in the Cr–NPs group vs. the Cr–Pic group
(Figure 9B).

Nutrients 2022,14, 5138 15 of 20
In arteries from rats fed the F diet for 18 weeks, H
2
O
2
production increased in the
control FF group (1.96-fold). Cr–Pic decreased (0.73-fold) the elevated H
2
O
2
production
(Figure 9B). This was not observed for Cr–NPs (0.87-fold, p= 0.6087).
Table 2.
The influence of inducible nitric oxide synthase (iNOS) inhibitor (1400 W at 1
µ
M), the non-
selective COX inhibitor (indomethacin at 10
µ
M), the selective cyclooxygenase-2 (COX-2) inhibitor
(NS-398 at 10
µ
M), the thromboxane-A2 receptor (TP) antagonist (SQ-29,548 at 1
µ
M), the PGI2 syn-
thesis inhibitor (tranylcypromine, TCP at 10
µ
M), or TCP plus SQ-29,548 on the vasorelaxant effects to
acetylcholine of thoracic arteries of either chromium (Cr)-supplemented (9 weeks of supplementation)
or not-supplemented (Control) rats. 0.3 mg Cr/kg of body weight as chromium picolinate (Cr–Pic)
and chromium nanoparticles (Cr–NPs) were added after 9 weeks of experimental feed without Cr
supplementation. Rats were fed a standard (S) diet for 18 weeks, and a high-fat–low-fiber (F) diet for
either 9 weeks (FS group) or 18 weeks (FF group).
Control Conditions * 1400 W Indomethacin NS-398 SQ-29,548 Tranylcypromine TCP plus SQ-29,548
AUC Emax
(%) pEC50 AUC Emax
(%) pEC50 AUC Emax
(%) pEC50 AUC Emax
(%) pEC50 AUC Emax
(%) pEC50 AUC Emax
(%) pEC50 AUC Emax
(%) pEC50
SControl 305.3 96.16 7.967 308.0 96.38 7.950 287.2 92.37 7.921 254.9
*90.84 7.675 259.6 88.87 7.743 257.9
*−91.97 7.566
*280.0 93.47 7.815
FS
Control 310.4 98.78 8.002 305.2 96.47 7.943 278.0
*95.75 7.816 290.9 96.26 7.939 294.5 98.61 7.866 278.3
*−97.01 7.632
*311.3 97.54 7.996
Cr–Pic 300.4 95.60 7.940 285.2
*95.81 7.805 236.5
*86.90
*7.530
*223.0
*87.51
*7.384
*249.7 92.89 7.589 246.7 −91.48 7.597 247.5 89.71 7.536
Cr–NPs 315.6 98.77 8.032 283.5
*91.61 7.938 254.9
*92.52
*7.616
*228.2
*94.09 7.486
*276.5 96.21 7.759 267.3
*−94.70
*7.641
*253.8
*92.49
*7.571
*
FF
Control 331.5 97.63 8.238 343.9 96.77 8.285 298.6
*95.93 7.867
*261.2
*94.13 7.562
*301.5 97.05 7.904 276.9 −93.83 7.855 273.0
*96.29 7.661
*
Cr–Pic 302.3 98.81 7.943 278.3 94.88 7.710 233.9
*86.18
*7.605
*203.1
*83.53
*7.471
*296.5 97.64 7.858 294.9 −95.57 7.924 270.3 97.58 7.676
Cr–NPs 311.7 95.31 8.124 295.4 93.71 7.977 288.2
*95.10 7.805
*232.4
*86.34 7.503
*285.3 95.27 7.826 305.5 −97.65 7.937 313.7 95.52 8.017
Values are based on the concentration–response curves. Data are expressed as means, n= 8, * p
≤
0.05 compared
with the control conditions, as determined by two-way ANOVA followed by Tukey’s post hoc test.
Nutrients 2022, 14, x FOR PEER REVIEW 16 of 21
Control S
Control FS
Cr−Pic FS
Cr−NPs FS
Control FF
Cr−Pic FF
Cr−NPs FF
80
100
120
140
160
180
Relative O2.− production
(C.U./minute/mg tissue)
FFFSS
0.0009
0.0029
0.0048
0.0145
A
Control S
Control FS
Cr−Pic FS
Cr−NPs FS
Control FF
Cr−Pic FF
Cr−NPs FF
0
5
10
15
20
25
H2O2production
(nmol/μg protein)
FFFSS
0.0060
<0.0001
0.0041
0.0332 0.0293
B
Figure 9. Production of superoxide anion (A) and hydrogen peroxide (B) in aortic rings from either
chromium (Cr) supplemented (9 weeks of supplementation) or non-supplemented (Control) rats.
0.3 mg Cr/kg of body weight as chromium picolinate (Cr–Pic) and chromium nanoparticles (Cr–
NPs) were added for 9 weeks of supplementation after the initial 9-week period of experimental
feed without Cr supplementation. Two types of diet were applied: a standard-fat-and-fiber (S) diet
and a high-fat–low-fiber (F) diet. Rats at 7 weeks of age were fed with an S diet or an F diet for either
9 weeks or 9 + 9 weeks. The following groups of rats were studied: Control S, the S diet plus the S
diet; Control FS, the F diet and the S diet; Cr–Pic FS, the F diet and the Cr–Pic S diet; Cr–NPs FS, the
F diet and the Cr–NPs S diet; Control FF, the F diet and the F diet; Cr–Pic FF, the F diet and the Cr–
Pic F diet; Cr–NPs FF, the F diet and the Cr–NPs F diet. Values are means ± SD, n = 8, p ≤ 0.05 (two-
way ANOVA/Tukey’s multiple comparisons test). In arteries from rats fed with the F diet for 9
weeks, both Cr–Pic and Cr–NPs increased relative O2.− production. In arteries from rats fed with the
F diet for 18 weeks, Cr–Pic but not Cr–NPs decreased elevated H2O2 production.
3.4.4. Detection of Hydrogen Peroxide
In arteries from rats fed the F diet for 9 weeks, H2O2 production did not change in the
control FS group (p = 0.5951). Neither Cr–Pic nor Cr–NPs modified that response. How-
ever, a significant increase (1.59-fold) was observed in the Cr–NPs group vs. the Cr–Pic
group (Figure 9B).
In arteries from rats fed the F diet for 18 weeks, H2O2 production increased in the
control FF group (1.96-fold). Cr–Pic decreased (0.73-fold) the elevated H2O2 production
(Figure 9B). This was not observed for Cr–NPs (0.87-fold, p = 0.6087).
4. Discussion
We examined the cardiovascular effects of a pharmacologically relevant dose of 0.3
mg Cr/kg body weight [10] in the form of picolinate (Cr–Pic) and metal nanoparticles (Cr–
NPs) in male Wistar Han rats administered in 9- to 16-week-old rats together with imple-
mentation of a high-fat–low-fiber (F) diet for either 9 or 18 weeks to investigate the role of
dietary fat normalization.
It is well-known that a prolonged high-fat diet intake results in the development of
chronic systemic inflammation, as well as tissue and organ dysfunction, and our study
confirmed this effect in rats fed the F diet for 18 weeks.
Figure 9.
Production of superoxide anion (
A
) and hydrogen peroxide (
B
) in aortic rings from either
chromium (Cr) supplemented (9 weeks of supplementation) or non-supplemented (Control) rats.
0.3 mg Cr/kg of body weight as chromium picolinate (Cr–Pic) and chromium nanoparticles (Cr–NPs)
were added for 9 weeks of supplementation after the initial 9-week period of experimental feed
without Cr supplementation. Two types of diet were applied: a standard-fat-and-fiber (S) diet and a

Nutrients 2022,14, 5138 16 of 20
high-fat–low-fiber (F) diet. Rats at 7 weeks of age were fed with an S diet or an F diet for either
9 weeks or 9 + 9 weeks. The following groups of rats were studied: Control S, the S diet plus the S
diet; Control FS, the F diet and the S diet; Cr–Pic FS, the F diet and the Cr–Pic S diet; Cr–NPs FS, the F
diet and the Cr–NPs S diet; Control FF, the F diet and the F diet; Cr–Pic FF, the F diet and the Cr–Pic F
diet; Cr–NPs FF, the F diet and the Cr–NPs F diet. Values are means
±
SD, n = 8, p
≤
0.05 (two-way
ANOVA/Tukey’s multiple comparisons test). In arteries from rats fed with the F diet for 9 weeks,
both Cr–Pic and Cr–NPs increased relative O
2.−
production. In arteries from rats fed with the F diet
for 18 weeks, Cr–Pic but not Cr–NPs decreased elevated H2O2production.
4. Discussion
We examined the cardiovascular effects of a pharmacologically relevant dose of
0.3 mg Cr/kg
body weight [
10
] in the form of picolinate (Cr–Pic) and metal nanoparti-
cles (Cr–NPs) in male Wistar Han rats administered in 9- to 16-week-old rats together with
implementation of a high-fat–low-fiber (F) diet for either 9 or 18 weeks to investigate the
role of dietary fat normalization.
It is well-known that a prolonged high-fat diet intake results in the development of
chronic systemic inflammation, as well as tissue and organ dysfunction, and our study
confirmed this effect in rats fed the F diet for 18 weeks.
In the functional studies, we demonstrated that Cr–Pic and Cr–NPs supplementation
favor the COX-2 pathway (over 9 and 18 weeks of F-diet administration), which is also an
attribute of a prolonged and regular F diet intake (in this study, it only occurred in 18 weeks,
not 9 weeks, of F-diet administration). Surprisingly, Cr preparations induced more negative
changes in arteries over 9 weeks of F-diet intake compared to 18 weeks, which resulted
in (i) enhanced participation of NO derived from iNOS and (ii) an enhanced net effect
of vasodilator prostanoids derived from COX-2 in vascular relaxation. However, it was
Cr–NPs that induced an imbalance between the function of COX-2-derived vasodilator and
vasoconstrictor prostanoids over 9 weeks rather than 18 weeks of feeding, in such a way
that the net vasodilator effect of prostanoids other than PGI
2
predominated in this group.
A similar effect was seen in 18-week F-diet intake that had no added Cr (control FF group).
We also noticed that both Cr–Pic and Cr–NPs increased the relative production of O
2.−
in
arteries from rats fed the F diet for 9 weeks; however, it was Cr–Pic but not Cr–NPs that
decreased elevated H2O2production in arteries from rats fed the F diet for 18 weeks.
This undesirable effect of Cr, especially related to Cr–NPs, was also observed in
the blood and hearts of supplemented rats and was reflected in decreased blood plasma
antioxidant capacity of water-soluble compounds and heart GSH+GSSG over 9 weeks of
F-diet intake.
However, some beneficial effects of Cr–Pic and Cr–NPs were observed after 18 weeks
of F-diet administration. In this study, an 18-week F diet decreased heart SOD and increased
heart CAT, and both Cr–Pic and Cr–NPs modified that response to the level observed in
control rats fed standard-fat-and-fiber diet. This suggests some beneficial effects that can
be attributed to Cr; however, they are only seen when an F diet is consumed for a longer
period (18 weeks instead of 9 weeks). Moreover, Cr–NPs decreased heart MDA levels in
this group of rats, again pointing to some benefits of Cr–NPs, but only when an F diet is
administered for 18 weeks.
It is worth mentioning that in our study, Cr preparation did not modify the blood
pressure, the ACL in the blood, the vasodilator response to SNP, the contractile response to
noradrenaline and high KCl, or the production of TXA2and PGI2.
4.1. Vascular Effects of Experimental Feeding
Neither receptor-dependent nor independent mechanisms of contraction were in-
volved in this study (research with noradrenaline and KCl). Moreover, dietary supple-
mentation did not modify the relaxant response to SNP of endothelium-denuded rings,
which is an endothelium-independent mechanism, indicating that the sensitivity of the
smooth muscles to NO was not modified. This corresponds well with the fact that the blood

Nutrients 2022,14, 5138 17 of 20
pressure was also not modified. Comparable results were obtained by Abebe et al. [
22
], who
reported no influence of Cr–Pic on vascular contraction and blood pressure; however, this
study was conducted on spontaneously hypertensive rats (SHR) instead of normotensive
Wistar Han rats. Nonetheless, Kopilas et al. [
23
] reported that Cr (III) reduced systolic
blood pressure in mesenteric resistance arteries of sucrose-induced hypertension in SHR.
Next, we studied the endothelium-dependent relaxation of ACh. Our results showed
that arteries from non-supplemented rats fed an F diet for 18 weeks (control FF group)
responded with increased relaxation of ACh, and Cr–Pic decreased that response to a level
corresponding with non-supplemented rats fed a standard-fat-and-fiber diet (control S
group). This indicates that it was 18 weeks rather than 9 weeks of F-feeding that resulted in
modified vascular relaxation, possibly through enhanced production of H
2
O
2
and Cr–Pic.
These functional results with Cr–Pic are in contrast with the results of Abebe et al. [
22
], who
reported increased ACh-induced vasodilation in spontaneously hypertensive rats (SHR)
supplemented with Cr–Pic. However, in these specific animal strains, vascular relaxation is
already decreased due to the inherent hypertension.
As there was no change in response to NA, we could rule out the influence of vascular
contraction on ACh-induced relaxation. Similarly, as the vasodilation to SNP was also
not altered, we could rule out modification of the sensitivity of the smooth muscles to
exogenous NO. Since NO is a major vasodilator agent in the rat aorta, which is a large
elastic conduit artery, we concluded that the observed effect might be associated with
increased NO production/release rather than modified sensitivity to NO.
Next, we observed that the specific iNOS inhibitor 1400 W reduced the vasodilation
to ACh in Cr–Pic- and Cr–NPs-supplemented groups of rats fed an F diet for 9 weeks.
Surprisingly, 1400 W did not modify this response over 18 weeks of F-diet intake. These
results show that the sensitivity to NO derived from iNOS increased during these 9 weeks of
feeding, and these same findings also point to a possible increase of NO production/release
as a response to the proinflammatory mediators such as O
2.−
, whose production was
elevated in our study. Since the sensitivity to NO derived from iNOS was not altered over
18 weeks of F-diet administration, there may be a possible compensatory effect when both
Cr and an F diet are taken for prolonged periods; a conclusion that merits further study.
As there is an interplay between NO and COX-derived prostanoids in vascular tone
regulation [
6
,
14
], our next step was to analyze the participation of COX derivates in that
response. The non-selective COX inhibitor indomethacin had no effect on the vasodilatory
response in control rats fed a standard-fat-and-fiber diet or an F diet for 9 weeks (control S
and control FS group, respectively), which indicates that COX derivates do not participate
in that response. Another possible explanation is that the sensitivity of the arteries to
prostanoids was lost in these two controls.
Interestingly, vasodilation to both indomethacin and selective COX-2 inhibitor NS-
398 was attenuated in preincubated arteries from Cr–Pic and Cr–NPs supplemented rats,
which indicates that COX-2-derived prostanoids modulate the endothelium-dependent
vasodilation in thoracic arteries of rats fed an F diet for 9 weeks. Since the same attenuation
in vascular response was reported in control rats fed an F diet for 18 weeks (control FF) and
Cr-supplemented FF rats, this only adds to our observation that raised COX-2 expression is
the factor that characterizes the pro-inflammatory status and that Cr supplementation only
enhances this process. An increased production of ROS further adds to this conclusion. It
is worth mentioning that Cr–Pic further decreased the maximal vasodilator response to
ACh during the 9 and 18 weeks of an F diet, but this was not observed with Cr–NPs. This
can be partially explained by the fact that H
2
O
2
production was not modified by Cr–NPs
as it was by Cr–Pic (18 weeks of F diet).
Since both the non-selective COX inhibitor and the selective COX-2 inhibitor atten-
uated the ACh-induced response to the same degree, we hypothesized that increased
participation of a strong vasodilator derived from COX-2 seems to play a key role in this
response in Cr-supplemented rats. The available literature indicates that PGI
2
is the most
potent vasodilator in rat conduit arteries. However, TCP, which is a PGI
2
synthesis inhibitor,

Nutrients 2022,14, 5138 18 of 20
did not modify that response in the two Cr-supplemented groups of rats fed an F diet
for 18 weeks as it did for the control (control FF), which indicates that the sensitivity of
these arteries to PGI
2
had been lost. The same effect was observed in arteries from Cr–Pic-
supplemented rats fed an F diet for 9 weeks. Surprisingly, Cr–NPs during the 9 weeks of
feeding with an F diet increased sensitivity to PGI
2
, but this was not modified with TCP
plus SQ-29,548 (a TP receptor inhibitor), possibly due to the increased H
2
O
2
production
also observed in aortas from affected controls fed a pro-inflammatory diet (an F diet) for
18 weeks.
Since other prostanoids modify vascular reactivity through TP receptor activation,
pre-incubation with TP receptor antagonist SQ-29,548 was studied. However, SQ-29,548
did not modify that response in supplemented rats or the control rats, indicating that TP
receptors did not participate in that response, which agrees with other reports [24,25].
Complex interactions have been reported between COX derivates, since the synthesis
of one prostanoid is usually accompanied by changes in the synthesis of others, which
can be shifted to the compensatory responses [
26
]. As there is crosstalk between the PGI
2
and TXA
2
systems, we analyzed vascular relaxation after the inhibition of PGI
2
synthesis
and TP receptors with TCP plus SQ-29,548. In the two control groups (standard-fat-and-
fiber diet (control S) and F diet for 9 weeks (control FS)), pre-incubation with TCP plus
SQ-29,548 reversed the decreased response to ACh caused by TCP, demonstrating a balance
between PGI
2
and TXA
2
in these arteries. However, in arteries from rats fed the F diet
for 18 weeks (control FF group), TCP plus SQ-29,548 did not modify the decreased ACh
response caused by TCP. A similar effect to that described for the control FF group was
also observed in Cr–NPs-supplemented rats fed a high-fat diet for 9 weeks, indicating the
imbalance between these two prostanoids and the possible participation of prostanoids
other than PGI
2
and TXA
2
through their specific receptors, rather than on TP receptors.
This has already been confirmed in a functional study with NS-398, in which the vasodilator
effect of prostanoid was decreased and neither TCP nor TCP plus SQ-29,548 reached the
vasodilator level observed for NS-398. Surprisingly, in the other three experimental groups
supplemented with Cr, sensitivity to endogenous PGI
2
was lost (study with TCP and TCP
plus SQ-29,548), which points to some imbalance between PGI
2
and TXA
2
. This might be
explained by the fact that the production of O
2.−
and H
2
O
2
was increased in the control
arteries (control FF), while Cr supplementation decreased that production.
4.2. Cardiac Effects of Experimental Feeding
It is well-known that a high-fat diet modifies the metabolic pathway, leading to
the generation of ROS that modulate the antioxidant defense mechanisms. Our results
indicate that the hearts of rats fed an F diet for 18 weeks, contrary to 9 weeks, exhibited
decreased SOD (an enzyme that degrades O
2.−
) and increased CAT (an enzyme that
degrades H
2
O
2
), with a tendency to increase GSH+GSSG (ROS scavenger). The decreased
blood-plasma antioxidant capacity of water-soluble compounds further supports our
results. Surprisingly, the MDA level, which is a marker of lipid peroxidation, was not
modified; this is in contrast with our previous results [
5
] and Noeman et al. [
27
]. Overall,
our results have shown that a prolonged and regular F diet significantly modified the
antioxidant defense mechanisms that are crucial for the development and progression
of metabolic syndrome [
28
]. Irrespective of its form, supplementation with Cr–Pic and
Cr–NPs increased SOD and decreased CAT in compromised rats over 18 weeks of a
proinflammatory diet to the same level observed in rats fed with a standard-fat-and-fiber
diet. Surprisingly, Cr–NPs decreased MDA; meanwhile, Cr–Pic exhibited a tendency to
lower GSH+GSSG. This is in contrast with our previous results, which showed an increased
level of MDA in the hearts of rats supplemented with Cr–Pic and Cr–NPs [5].
However, this prior experiment lasted for 9 weeks instead of 18 weeks; in our 9-week
experiment, MDA did not change, and rats were fed with a different type of a high-fat diet.
These results point to some beneficial effects of Cr preparations on the redox status, but only
when an F diet is taken for a longer and regular period. Surprisingly, dietary intervention

Nutrients 2022,14, 5138 19 of 20
and reduction of F diet duration to 9 weeks completely modified the effects described
above. At some point, 9 weeks of an F diet intake caused this, despite not modifying MDA,
GSH+GSSG, SOD, CAT, or ACW. That is why neither MDA, SOD, nor CAT were changed
by these two forms of Cr, as opposed to GSH+GSSG, which was reduced. However, it
was Cr–NPs that further reduced antioxidant capacity in the blood. Our results agree
with research conducted by Chen et al. [
29
], which suggested a positive effect of Cr on
redox status in individuals that regularly consume a high-fat diet, and who are often obese.
However, in healthy individuals within body mass guidelines, Cr preparations can function
as pro-oxidant agents, initiating free-radical reactions that lead to oxidative stress [10].
5. Conclusions
Our study on rats confirmed the pro-inflammatory effect of an F diet administered for
18 weeks. Our results show that supplementation with Cr–Pic rather than with Cr–NPs is
more beneficial in rats who regularly consume an F diet (18 weeks). On the contrary, in
healthy organisms (9 weeks of an F followed by dietary fat normalization), both Cr–Pic
and Cr–NPs can function as pro-oxidant agents, initiating free-radical reactions that lead to
oxidative stress.
Author Contributions:
Conceptualization, K.O. and J.J.; data curation, M.M. and J.J.; formal analysis,
M.M., E.C., and K.O.; funding acquisition, J.J.; investigation, M.M., L.G., E.C., K.O., B.F., and J.J.;
methodology, M.M., E.C., and K.O.; project administration, J.J.; resources, J.J.; software, M.M.; su-
pervision, J.J.; validation, M.M.; visualization, M.M.; writing—original draft, M.M.;
writing—review
and editing, M.M. All authors have read and agreed to the published version of the manuscript.
Funding:
This work was supported by the National Science Centre, Grant No. 2020/39/B/NZ9/00674.
Institutional Review Board Statement:
Written consent was provided by the National Ethics Com-
mittee for Animal Experiments (Permission Number: 73/2021, Warsaw, Poland) according to Direc-
tive 2010/63/EU. This study conforms to the Guide for the Care and Use of Laboratory Animals
(NIH Publications No. 86–26, revised 2014), and was conducted in compliance with the replacement,
refinement, and reduction rule.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data supporting reported results are available on request.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Ognik, K.; Dworza´nski, W.; Sembratowicz, I.; Fotschki, B.; Cholewi´nska, E.; Listos, P.; Ju´skiewicz, J. The effect of the a high-fat
diet supplemented with various forms of chromium on rats’ body composition, liver metabolism and organ histology Cr in liver
metabolism and histology of selected organs. J. Trace Elem. Med. Biol. 2021,64, 126705. [CrossRef]
2.
Majewski, M.; Lis, B.; Ju´skiewicz, J.; Ognik, K.; Jedrejek, D.; Stochmal, A.; Olas, B. The composition and vascular/antioxidant
properties of Taraxacum officinale flower water syrup in a normal-fat diet using an obese rat model. J. Ethnopharmacol.
2021
,
265, 113393. [CrossRef]
3.
˙
Zary-Sikorska, E.; Fotschki, B.; Jurgo´nski, A.; Kosmala, M.; Milala, J.; Kołodziejczyk, K.; Majewski, M.; Ognik, K.; Ju´skiewicz, J.
Protective Effects of a Strawberry Ellagitannin-Rich Extract against Pro-Oxidative and Pro-Inflammatory Dysfunctions Induced
by a high-fat Diet in a Rat Model. Molecules 2020,25, 5874. [CrossRef] [PubMed]
4.
˙
Zary-Sikorska, E.; Fotschki, B.; Kołodziejczyk, K.; Jurgo´nski, A.; Kosmala, M.; Milala, J.; Majewski, M.; Ognik, K.; Ju´skiewicz, J.
Strawberry phenolic extracts effectively mitigated metabolic disturbances associated with a high-fat ingestion in rats depending
on the ellagitannin polymerization degree. Food Funct. 2021,12, 5779–5792. [CrossRef]
5.
Dworza´nski, W.; Cholewi´nska, E.; Fotschki, B.; Ju´skiewicz, J.; Listos, P.; Ognik, K. Assessment of DNA Methylation and Oxidative
Changes in the Heart and Brain of Rats Receiving a high-fat Diet Supplemented with Various Forms of Chromium. Animals
2020
,
10, 1470. [CrossRef]
6.
Majewski, M.; Jurgo´nski, A.; Fotschki, B.; Ju´skiewicz, J. The toxic effects of monosodium glutamate (MSG) - The involvement of
nitric oxide, prostanoids and potassium channels in the reactivity of thoracic arteries in MSG-obese rats. Toxicol. Appl. Pharmacol.
2018,359, 62–69. [CrossRef]
7.
Liu, B.; Zhan, M.; Zhang, Y.; Li, H.; Wu, X.; Zhuang, F.; Luo, W.; Zhou, Y. Increased role of E prostanoid receptor-3 in prostacyclin-
evoked contractile activity of spontaneously hypertensive rat mesenteric resistance arteries. Sci. Rep. 2017,7, 8927. [CrossRef]

Nutrients 2022,14, 5138 20 of 20
8.
Turck, D.; Bresson, J.L.; Burlingame, B.; Dean, T.; Fairweather-Tait, S.; Heinonen, M.; McArdle, H.J. EFSA NDA Panel (EFSA
Panel on Dietetic Products, Nutrition and Allergies), Scientific Opinion on Dietary Reference Values for chromium. EFSA J.
2014
,
12, 3845.
9.
St˛epniowska, A.; Tutaj, K.; Ju´skiewicz, J.; Ognik, K. Effect of a high-fat diet and chromium on hormones level and Cr retention in
rats. J. Endocrinol. Investig. 2022,45, 527–535. [CrossRef] [PubMed]
10.
Dworza´nski, W.; Cholewi´nska, E.; Fotschki, B.; Ju´skiewicz, J.; Ognik, K. Oxidative, epigenetic changes and fermentation processes
in the intestine of rats fed a high-fat diets supplemented with various chromium forms. Sci. Rep.
2022
,12, 9817. [CrossRef]
[PubMed]
11.
Dworza´nski, W.; Sembratowicz, I.; Cholewi´nska, E.; Tutaj, K.; Fotschki, B.; Ju´skiewicz, J.; Ognik, K. Effects of Different Chromium
Compounds on Hematology and Inflammatory Cytokines in Rats Fed A high-fat Diet. Front. Immunol.
2021
,12, 614000. [CrossRef]
[PubMed]
12.
Majewski, M.; Kasica, N.; Jakimiuk, A.; Podlasz, P. Toxicity and cardiac effects of acute exposure to tryptophan metabolites on the
kynurenine pathway in early developing zebrafish (Danio rerio) embryos. Toxicol. Appl. Pharmacol.
2018
,341, 16–29. [CrossRef]
[PubMed]
13.
Majewski, M.; Kozlowska, A.; Thoene, M.; Lepiarczyk, E.; Grzegorzewski, W.J. Overview of the role of vitamins and minerals on
the kynurenine pathway in health and disease. J. Physiol. Pharmacol. 2016,67, 3–19.
14.
Majewski, M.; Ju´skiewicz, J.; Krajewska-Włodarczyk, M.; Gromadzi´nski, L.; Socha, K.; Cholewi ´nska, E.; Ognik, K. The Role of 20-
HETE, COX, Thromboxane Receptors, and Blood Plasma Antioxidant Status in Vascular Relaxation of Copper-Nanoparticle-Fed
WKY Rats. Nutrients 2021,13, 3793. [CrossRef] [PubMed]
15.
Cholewi´nska, E.; Ju´skiewicz, J.; Majewski, M.; Smagieł, R.; Listos, P.; Fotschki, B.; Godycka-Kłos, I.; Ognik, K. Effect of Copper
Nanoparticles in the Diet of WKY and SHR Rats on the Redox Profile and Histology of the Heart, Liver, Kidney, and Small
Intestine. Antioxidants 2022,11, 910. [CrossRef]
16.
Majewski, M.; Lis, B.; Olas, B.; Ognik, K.; Ju´skiewicz, J. Dietary supplementation with copper nanoparticles influences the
markers of oxidative stress and modulates vasodilation of thoracic arteries in young Wistar rats. PLoS ONE
2020
,15, e0229282.
[CrossRef] [PubMed]
17.
Majewski, M.; Ognik, K.; Ju´skiewicz, J. Copper nanoparticles modify the blood plasma antioxidant status and modulate the
vascular mechanisms with nitric oxide and prostanoids involved in Wistar rats. Pharmacol. Rep. 2019,71, 509–516. [CrossRef]
18.
Majewski, M.; Ognik, K.; Ju´skiewicz, J. Copper nanoparticles enhance vascular contraction induced by prostaglandin F2-alpha
and decrease the blood plasma cu-zn ratio in wistar rats. J. Elem. 2019,24, 911–922. [CrossRef]
19.
Majewski, M.; Ognik, K.; Zdunczyk, P.; Juskiewicz, J. Effect of dietary copper nanoparticles versus one copper (II) salt: Analysis
of vasoreactivity in a rat model. Pharmacol. Rep. 2017,69, 1282–1288. [CrossRef]
20.
Majewski, M.; Klett-Mingo, M.; Verdasco-Martín, C.M.; Otero, C.; Ferrer, M. Spirulina extract improves age-induced vascular
dysfunction. Pharm. Biol. 2022,60, 627–637. [CrossRef]
21.
Majewski, M.; Jurgo´nski, A. The Effect of Hemp (Cannabis sativa L.) Seeds and Hemp Seed Oil on Vascular Dysfunction in Obese
Male Zucker Rats. Nutrients 2021,3, 2575. [CrossRef] [PubMed]
22.
Abebe, W.; Liu, J.Y.; Wimborne, H.; Mozaffari, M.S. Effects of chromium picolinate on vascular reactivity and cardiac ischemia-
reperfusion injury in spontaneously hypertensive rats. Pharmacol. Rep. 2010,62, 674–682. [CrossRef] [PubMed]
23.
Kopilas, M.A.; Dang, L.N.; Anderson, H.D. Effect of dietary chromium on resistance artery function and nitric oxide signaling in
the sucrose-fed spontaneously hypertensive rat. J. Vasc. Res. 2007,44, 110–118. [CrossRef] [PubMed]
24.
Gluais, P.; Lonchampt, M.; Morrow, J.D.; Vanhoutte, P.M.; Feletou, M. Acetylcholine-induced endothelium-dependent contractions
in the SHR aorta: The Janus face of prostacyclin. Br. J. Pharmacol. 2005,146, 834–845. [CrossRef] [PubMed]
25.
Martorell, A.; Blanco-Rivero, J.; Aras-López, R.; Sagredo, A.; Balfagón, G.; Ferrer, M. Orchidectomy increases the formation of
prostanoids and modulates their role in the acetylcholine-induced relaxation in the rat aorta. Cardiovasc. Res.
2008
,77, 590–599.
[CrossRef]
26.
Blanco-Rivero, J.; Aller, M.A.; Arias, J.; Ferrer, M.; Balfagón, G. Long-term portal hypertension increases the vasodilator response
to acetylcholine in rat aorta: Role of prostaglandin I2. Clin. Sci. 2009,117, 365–374. [CrossRef]
27.
Noeman, S.A.; Hamooda, H.E.; Baalash, A.A. Biochemical study of oxidative stress markers in the liver, kidney and heart of high
fat diet induced obesity in rats. Diabetol. Metab. Syndr. 2011,3, 17. [CrossRef]
28.
Llévenes, P.; Rodrigues-Díez, R.; Cros-Brunsó, L.; Prieto, M.I.; Casaní, L.; Balfagón, G.; Blanco-Rivero, J. Beneficial Effect of a
Multistrain Synbiotic Prodefen
®
Plus on the Systemic and Vascular Alterations Associated with Metabolic Syndrome in Rats: The
Role of the Neuronal Nitric Oxide Synthase and Protein Kinase A. Nutrients 2020,12, 117. [CrossRef]
29.
Chen, W.Y.; Chen, C.J.; Liu, C.H.; Mao, F.C. Chromium attenuates a high-fat diet-induced nonalcoholic fatty liver disease in
KK/HlJ mice. Biochem. Biophys. Res. Commun. 2010,397, 459–464. [CrossRef]