Journal of APPLIED BIOMEDICINE
ISSN 1214-0287 (on-line)
ISSN 1214-021X (printed)
Volume 9 (2011), No 4, p 219-224
DOI 10.2478/v10136-011-0008-1
The nonlinear dependence between administered pro-oxidant doses and intensity of free-radical processes observed in rats
Sergey Ivanovich Krasikov, Alexey Alexeyevich Tinkov, Natalia Vasilievna Sharapova, Mikhail Anatolievich Bogatov
Address: Sergey Ivanovich Krasikov, PhD. Department of Chemistry and Pharmaceutical Chemistry, Orenburg State Medical Academy, Sovetskaya Street 6,
460000, Orenburg, Russia
krasikov.s.i@gmail.com
Received 24th March 2011.
Revised 19th May 2011.
Published online 4th July 2011.
Full text article (pdf)
Summary
Key words
Introduction
Materials and Methods
Results
Discussion
References
SUMMARY
The influence of iron, copper and nitrate ions on free-radical processes in rats and the dependence between dose and effect of pro-oxidants were
studied. Rats were divided into 14 groups and administered differing concentrations and combinations of chemicals with drinking water.
Concentrations of iron, copper and nitrate in the water were 1, 0.5 and 0.33 of maximum permissible concentrations (MPCs) for every chemical. The
action of the investigated pollutants on the intensity of free-radical processes was estimated by the determination of conjugated dienes in liver
homogenate and the intensity of Fe2+-induced chemiluminescence of the blood serum. It is estimated that chemicals entering the organism
in doses that do not exceed their MPC lead to an increase in free-radical oxidation in comparison to the controls. A maximal effect of iron on the
concentration of conjugated dienes was observed in a dose equal to 0.33 MPC, while copper and nitrate possess maximal activity in concentrations of
0.5 MPCs. Fast flash amplitude of chemiluminescence in serum was not dose-dependent in rats obtaining iron and copper, while nitrate had a reverse
dose-dependent effect. Total luminosity was maximal in doses of chemicals equal to 0.33 MPCs. The combined action of pollutants was more evident in
comparison to isolated chemicals in doses equal to 1 MPC.
KEY WORDS
iron; copper; nitrate; oxidative stress; rats
INTRODUCTION
It is well known that many pollutants possess a
significant pro-oxidant effect, but the intensity of this
effect during the administration of chemicals to the
organism is insufficiently studied. In particular, the
relation between the intensity of free-radical
oxidation and the administered pollutant dose remains
unknown. The investigation of this problem is not
only theoretically significant, but is also useful
practically, as the ability of some chemicals to induce
free-radical oxidation is the basis of their toxicity
(Valko et al. 2005, Flora et al. 2008). For this reason,
the purpose of our investigation was to study the
influence of iron, copper and nitrate ions on
free-radical processes in the organism and the
dose-dependent effect of pro-oxidants.
MATERIALS AND METHODS
The current research was approved by local Ethics
Committee. 105 female Wistar rats divided into
14 groups were used. The control group contained the
maximum number of rats in order to estimate the
basal level of free-radical processes in animals. The
animals were given free access to the drinking water
and a standard diet. The light and dark cycle in the
animal room was 12 hours. The animals were
acclimatized for one week to the laboratory
conditions before the study. The experiment lasted for
28 days. On the 29th day the animals were sacrificed
by decapitation.
All groups of animals except the control ones
received pollutants Fe2+, Cu2+ and NO3-.
Concentrations of chemicals used in the current study
were calculated on the base of maximum permissible
concentrations (MPC) for these pollutants accepted as
per Russian norms. MPCs for single chemicals Fe2+,
Cu2+ and NO3- are 0.3; 1.0 and 45.0 mg per litre of
drinking water respectively. For the adequate
dispensing of the pollutants, we recalculated the
MPCs for the salts used in the experiment. The
animals received the investigated chemicals in the
form of FeSO4·7H2O; CuSO4 and NaNO3. The
recalculated MPCs for these salts in the drinking
water were 1.5; 2.44 and 61.6 mg/l respectively.
Animals of the first group (n=11) received
high-quality drinking water (general mineralization
< 250.0 mg/l) and served as a control group. Rats
from 2 (n=9), 3 (n=5), and 4 (n=5) groups received
drinking water containing FeSO4·7H2O in doses equal
to 1; 0.5 and 0.33 MPC respectively. The animals
from 5 (n=9), 6 (n=5), and 7 (n=5) groups obtained
CuSO4-containing drinking water in doses
corresponding to 1, 0.5 and 0.33 MPC respectively. In
groups 8 (n=9); 9 (n=5) and 10 (n=5) animals
received drinking water with NaNO3 in doses of 1;
0.5 and 0.33 MPC respectively. As for animals in 11
(n=9), 12 (n=9), and 13 (n=9) groups, they received
water containing the following combinations of
chemicals respectively: [Fe2++Cu2+]; [Fe2++NO3-] and
[Cu2++NO3-]. According to principles of hygiene the
sum of concentrations of all chemicals in water
should not exceed 1MPC:
C1/MPC1 + C2/MPC2 + … + Cn/MPCn 1
For that purpose the concentration of each
compound from the two-component combination was
equal to its 0.5 MPC. Finally, animals from the 14th
(n=10) group were given drinking water containing
the three-component combination of
[Fe2++Cu2++NO3-], where every chemical had a
concentration of 0.33 MPC and the total
concentration of the combination also did not exceed
1 MPC. It is important to mention that the groups of
rats given isolated chemicals in concentrations equal
to its 0.5 and 0.33 MPCs were also used as inner
controls for groups obtaining different mixtures.
All groups of rats were given the abovementioned
doses of chemicals daily. Water consumption was
measured by daily weighing of the water bottles. The
subsequent analysis has showed that there were no
significant differences in water consumption by rats
from the different groups.
The action of the investigated pollutants on the
intensity of free-radical processes was estimated in
liver homogenates and blood serum.
The level of free-radical oxidation in liver
homogenates was estimated by the concentration of
conjugated dienes. Liver homogenates were mixed
with heptane-isopropanol solution. The mixture was
centrifuged for 5 minutes at 5000 g. The supernatant
was further used for determination of optical density
at 233 nm. The final concentration of conjugated
dienes in liver homogenates was measured in units of
optic density per mg of protein (D/mg protein) (Placer
1968). Protein determination in liver homogenates
was carried out using the method of O. Lowry (Lowry
et al. 1951). Bovine serum albumin was used as a
standard.
The intensity of free-radical oxidation in the
serum was estimated on the base of the parameters of
iron-induced chemiluminescence by using
"Chemiluminomer-003" (Ufa, Russia). After the
addition of serum into the device and stabilization of
the signal, the inductor FeSO4·7H2O was entered into
the system. After the induction of free-radical
processes in the system fast flash amplitude and
general luminosity were recorded. Fast flash
amplitude characterizes the intensity of reactive
oxygen species generation as a response to Fe2+
addition and indirectly shows the concentration of
hydroperoxides in the serum. General luminosity
serves as an indicator of free radical oxidation of
biomolecules in the serum. The intensity of
chemiluminescence was expressed in conventional
units (c.u.) of luminosity (Lopukhin et al. 1983).
Data were expressed as mean values ± SEM and
evaluated using Mann-Whitney U-test at the
significance level 2alpha=0.05.
RESULTS
The data obtained are shown in Table 1 and indicate
that intake of any of the investigated pollutants led to
an increase in the concentration of conjugated dienes
in rats. After administration of iron in a dose equal to
its maximum permissible concentration, the level of
conjugated dienes was enhanced by 1/3 in comparison
with the controls. The intake of FeSO4 in
concentration of 1/2 MPC led to significantly increased levels of conjugated dienes; nearly 2-fold as
compared with controls. Finally, after administration
of water containing 1/3 MPC of iron, the
concentration of this product of lipid peroxidation
was elevated 2.3-fold (statistically significant).
Table 1. Concentration of conjugated dienes in liver homogenates (D/mg protein) and intensity of serum chemiluminescence
(c.u.) in rats obtaining different doses and combinations of investigated pollutants.
| No |
Chemicals |
Dose of each
chemical (MPC) |
Conjugated
dienes |
Fast flash
amplitude |
General
luminosity | | I |
Control group |
- |
0.24±0.02 |
0.32±0.05 |
1.47±0.17 | | II |
Fe2+ |
1 |
0.33±0.03 |
0.38±0.05 |
1.48±0.15 | | III |
Fe2+ |
0.5 |
0.43±0.05 a |
0.42±0.07 |
1.68±0.19 | | IV |
Fe2+ |
0.33 |
0.56±0.07 a, b |
0.39±0.11 |
1.72±0.20 | | V |
Cu2+ |
1 |
0.32±0.03 |
0.49±0.06 a |
1.67±0.15 | | VI |
Cu2+ |
0.5 |
0.47±0.05 a |
0.33±0.04 |
1.64±0.21 | | VII |
Cu2+ |
0.33 |
0.43±0.04 a |
0.53±0.05 e |
2.56±0.11 a, b | | VIII |
NO3- |
1 |
0.35±0.03 |
0.37±0.04 |
1.31±0.12 | | IX |
NO3- |
0.5 |
0.55±0.06 a, d |
0.41±0.02 a |
1.88±0.17 a, d | | X |
NO3- |
0.33 |
0.44±0.04 a |
0.52±0.10 a, d |
2.12±0.27 a, d | | XI |
Fe2+ + Cu2+ |
0.5 |
0.50±0.04 a, b, c |
0.35±0.05 |
1.68±0.18 | | XII |
Fe2+ + NO3- |
0.5 |
0.44±0.03 a |
0.38±0.05 |
1.67±0.18 | | XIII |
Cu2+ + NO3- |
0.5 |
0.47±0.04 a |
0.39±0.05 |
1.76±0.10 d | | XIV |
Fe2+ + Cu2+ + NO3- |
0.33 |
0.47±0.03 a, b, c, d |
0.54±0.08 a, d |
1.81±0.19 d, f |
a statistically significant versus control group
b statistically significant versus group receiving Fe2+ in 1MPC
c statistically significant versus group receiving Cu2+ in 1MPC
d statistically significant versus group receiving NO3- in 1MPC
e statistically significant versus group receiving Cu2+ in 0.5 MPC
f statistically significant versus group receiving Cu2+ in 0.33 MPC
Nonlinear dependence between the dose of
incoming pollutants and the intensity of lipid
peroxidation, estimated on the base of conjugated
dienes in liver homogenates, was observed in rats
receiving Cu2+ and NO3- with drinking water. The
maximal effect of single chemicals was observed in
concentrations equal to 0.5MPC (statistically
significant).
The data from the table 1 also show that the
effects of different pollutants acting in combinations
were not cumulative in relation to their pro-oxidant
action. At the same time the concentration of
conjugated dienes in the liver homogenates was
significantly higher than in the case of the isolated
action of pollutants in a dose of 1 MPC.
A similar dependence between the intensity of
free radical processes and concentration of
investigated pollutants was observed in the rats'
serum. After administration of iron and copper, the
concentration of lipid hydroperoxides as estimated by
the amplitude of fast flash did not depend on dose. In
groups of animals obtaining different concentrations
of nitrates, this dependence had an inverse character.
A significant increase of fast flash amplitude was
observed after administration of three-component
combination of pollutants.
It can also be seen from Table 1 that the intensity
of free-radical oxidation of biomolecules in serum as
estimated by general luminosity was maximal after
intake of pollutants in doses equal to 1/3 MPC. In the
case of combinations, no increase in pro-oxidant
action was observed, though the general luminosity in
these groups was higher than in the controls.
DISCUSSION
The data obtained allow us to make the following
conclusions. First of all, chemicals entering the
organism in doses that do not exceed their maximum
permissible concentrations are able to induce
oxidative stress. The observed pollutant-dependent
activation of free-radical processes is a consequence
of their pro-oxidant features. As redox metals, iron
and copper have the same mechanisms of pro-oxidant
action taking part in the generation of superoxide
anion (Haber and Weiss 1934), Fenton-reaction
(Fenton 1894) and lipid peroxidation (Valko et al.
2005). Moreover d-metals can also cause the
depression of antioxidant enzymes (Ribarov et al.
1982).
The activation of free-radical oxidation in rats
receiving NaNO3 with drinking water is possibly a
consequence of nitrate reduction to nitrite. It is known
that nitrite uptake by erythrocytes can cause oxidative
stress (May et al. 2000). It is possible that such an
effect may be connected with the further reduction of
nitrite to nitric oxide by different macromolecules
(Gladwin et al. 2005). This hypothesis is confirmed
by the data showing that inorganic nitrate can be a
source of nitric oxide (Lundberg and Govoni 2004).
NO·, except when serving as a signalling molecule,
can interact with superoxide and nitrite forming
peroxynitrite and N2O3 respectively (Wink and
Mitchell 1998).
Secondly, there is an inverse dependence between
pro-oxidants entering the organism and intensity of
free-radical oxidation. This thesis can be supported by
earlier data indicating that an increase in iron
concentration leads to depression of chemi-luminescence in vitro (Suslova et al. 1968). It was
supposed that high doses of iron in vitro enhance the
amount of lipid peroxidation chains, and their length
seems to be significantly reduced (Vladimirov and
Archakov 1972).
At the same time the more expressed pro-oxidant
action of pollutants in relatively low doses can be
connected with the better gastrointestinal absorption
of these concentrations.
It is known that copper bioavailability decreases
with increasing amounts of dietary copper (Turnlund
et al. 1989). There is also data indicating that a
high-copper diet stimulates metal excretion (Turnlund
et al. 2005). Such effect can be a consequence of the
regulatory interaction of copper and transport proteins
during absorption. It is known that high doses of
copper lead to inhibition of apical copper transporters
DMT1 and Ctr1 (Tennant et al. 2002, Petris et al.
2003, Wu et al. 2009) and Menkes ATPase that acts
as a basolateral transporter (Monty et al. 2005).
A similar inhibitory action of high doses is
observed in iron. The only known apical transporter
of iron is DMT which can be inhibited by high doses
of iron acting by different mechanisms (Sharp et al.
2002, Zoller et al. 2002, Mena et al. 2008). Duodenal
reductase b (Dcytb), taking part in apical iron
absorption, is also down-regulated by iron (Frazer et
al. 2002). Iron transport through the basolateral
membrane by IREG1 also seems to depend on
incoming iron concentration (Zoller et al. 2002, Di
Domenico et al. 2007). There is some poor data on
reverse dependence between dose and bioavailability
of nitrates. It is supposed that nitrate is rather inert in
the organism and its active form is nitrite, which can
be formed by bacterial reduction of nitrates in the
gastrointestinal tract. In this connection data
indicating that the reverse dependence between
bacterial reduction and the dose of incoming nitrate
(Harada et al. 1975) can support our hypothesis.
Thirdly, the combined action of pollutants leads to
a slight inhibition of pro-oxidant features in
comparison with the isolated action of these
pollutants. According to our hypothesis this effect can
be a consequence of the interaction between
pollutants during absorption. There are a number of
works indicating a decrease in absorption of iron and
copper during combined administration (Arredondo et
al. 2003, 2006). It is believed that such an effect
occurs because of competition between these metals
for DMT1. Despite the absence of data on inhibition
of iron and copper absorption by nitrates there is a
theoretical basis for such action. It is shown that NO·
stimulates an NF-kappaB-mediated decrease in DMT1
transcription (Paradkar and Roth 2006). At the same
time inhibition of Fenton-mediated free-radical
oxidation by NO· was shown earlier (Gupta et al.
1997). These data can also explain the observed
inhibition of pro-oxidant activity of combinations
[Men++NO3-].
In conclusion it is important to note that the
observed activation of free-radical processes by low
doses of chemicals can play a part in the development
of a number of diseases known to be associated with
oxidative stress.
REFERENCES
Arredondo M, Munoz P, Mura CV, Nunez MT. DMT1, a physiologically relevant apical Cu1+ transporter of intestinal cells. Am J Physiol Cell Physiol. 284: 1525-1530, 2003.
[CrossRef]
Arredondo M, Martinez R, Nunez MT, Ruz M, Olivares M. Inhibition of iron and copper uptake by iron, copper and zinc. Biol Res. 39: 95-102, 2006.
Di Domenico I, Ward DM, Langelier C, Vaughn MB, Nemeth E, Sundquist WI, Ganz T, Musci G, Kaplan J. The molecular mechanism of hepcidin-mediated ferroportin down-regulation. Mol Biol Cell. 18: 2569-2578, 2007.
[CrossRef]
Fenton HJH. Oxidation of tartaric acid in the presence of iron. J Chem Soc. 65: 899-910, 1894.
Flora SJ, Mittal M, Mehta A. Heavy metal induced oxidative stress and its possible reversal by chelation therapy. Indian J Med Res. 128: 501-523, 2008.
Frazer DM, Wilkins SJ, Becker EM, Vulpe CD, McKie AT, Trinder D, Anderson GJ. Hepcidin expression inversely correlates with the expression of duodenal iron transporters and iron absorption in rats. Gastroenterology. 123: 835-844, 2002.
Gladwin MT, Schechter AN, Kim-Shapiro DB, Patel RP, Hogg N, Shiva S, Cannon RO 3rd, Kelm M, Wink DA, Espey MG, Oldfield EH, Pluta RM et al. The emerging biology of nitrite anion. Nat Chem Biol. 1: 308-314, 2005.
Gupta MP, Evanoff V, Hart CM. Nitric oxide attenuates hydrogen peroxide-mediated injury to porcine pulmonary artery endothelial cells. Am J Physiol. 272: 1133-1141, 1997.
Haber F, Weiss JJ. The catalytic decomposition of hydrogen peroxide by iron salts. Proc Roy Soc London Ser A. 147: 332-351, 1934.
Harada M, Ishiwata H, Nakamura Y, Tanimura A, Ishidate M. Studies on in vivo formation of nitroso compounds (I). Changes of nitrite and nitrate concentrations in human saliva after ingestion of Salted Chinese cabbage. J Food Hyg Soc Japan. 16: 11-18, 1975.
Lopukhin YM, Vladimirov YA, Molodenkov MN. Registration of serum constutients in presence of iron (In Russian). Bull Exp Biol Med. 95: 61-63, 1983.
Lowry OH, Rosenbourgh NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 193: 265-275, 1951.
Lundberg JO, Govoni M. Inorganic nitrate is a possible source for systemic generation of nitric oxide. Free Radic Biol Med. 37: 395-400, 2004.
[CrossRef]
May JM, Qu Z-C, Xia L, Cobb CE. Nitrite uptake and metabolism and oxidant stress in human erythrocytes. Am J Physiol Cell Physiol. 279: 1946-1954, 2000.
Mena NP, Esparza A, Tapia V, Valdes P, Nunez MT. Hepcidin inhibits apical iron uptake in intestinal cells. Am J Physiol Gastrointest Liver Physiol. 294: 192-198, 2008.
[CrossRef]
Monty JF, Llanos RM, Mercer JF, Kramer DR. Copper exposure induces trafficking of the menkes protein in intestinal epithelium of ATP7A transgenic mice. J Nutr. 135: 2762-2766, 2005.
Paradkar PN, Roth JA. Nitric oxide transcriptionally down-regulates specific isoforms of divalent metal transporter (DMT1) via NF-kappaB. J Neurochem. 96: 1768-1777, 2006.
[CrossRef]
Petris MJ, Smith K, Lee J, Thiele DJ. Copper-stimulated endocytosis and degradation of the human copper transporter, hCtr1. J Biol Chem. 278: 9639-9646, 2003.
[CrossRef]
Placer Z. Lipoperoxydationssysteme im biologischen Material 2. Mitt. Bestimmung der Lipoperoxydation im Saugetierorganismus. Food / Nahrung. 12: 679-684, 1968.
Ribarov S, Benov L, Benchev I, Monovich O, Markova V. Hemolysis and peroxidation in heavy metal-treated erythrocytes; GSH content and activities of some protecting enzymes. Experientia. 38: 1354-1355, 1982.
Sharp P, Tandy S, Yamaji S, Tennant J, Williams M, Singh Srai SK. Rapid regulation of divalent metal transporter (DMT1) protein but not mRNA expression by non-haem iron in human intestinal Caco-2 cells. FEBS Lett. 510: 71-76, 2002.
Suslova TB, Olenev VI, Vladimirov YA. Role of iron ions in chemiluminescence of lipids (in Russian). Biofizika. 13: 723-726, 1968.
Tennant J, Stansfield M, Yamaji S, Srai SK, Sharp P. Effects of copper on the expression of metal transporters in human intestinal Caco-2 cells. FEBS Lett. 527: 239-244, 2002.
Turnlund JR, Keyes WR, Anderson HL, Acord LL. Copper absorption and retention in young men at three levels of dietary copper by use of the stable isotope 65Cu. Am J Clin Nutr. 49: 870-878, 1989.
Turnlund JR, Keyes WR, Kim SK, Domek JM. Long-term high copper intake: effects on copper absorption, retention, and homeostasis in men. Am J Clin Nutr. 81: 822-828, 2005.
Valko M, Morris H, Kronin MTD. Metals, toxicity and oxidative stress. Curr Med Chem. 12: 1161-1208, 2005.
Vladimirov YA, Archakov AI. Lipid Peroxidation in Biological Membranes (in Russian). Nauka, Moscow 1972, pp. 56-87.
Wink DA, Mitchell JB. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med. 25: 434-456, 1998.
Wu X, Sinani D, Kim H, Lee J. Copper Transport Activity of Yeast Ctr1 Is Down-regulated via Its C Terminus in Response to Excess Copper. J Biol Chem. 284: 4112-4122, 2009.
[CrossRef]
Zoller H, Theurl I, Koch R, Kaser A, Weiss G. Mechanisms of iron mediated regulation of the duodenal iron transporters divalent metal transporter 1 and ferroportin 1. Blood Cells Mol Dis. 29: 488-497, 2002.
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