PEROXIDATIVE INDEX: A NEW MARKER IN KIDNEY TOXICITY

INDUCED BY MERCURY
Trilianty Lestarisa1*, Francisca Diana Alexandra2, Helena Jelita3, Eko Suhartono4
1

Department of Public Health, Faculty of Medicine, Palangkaraya University, Palangkaraya,

Central Kalimantan, Indonesia
2
Department of Pharmacology, Faculty of Medicine, Palangkaraya University, Palangkaraya,
Central Kalimantan, Indonesia
3
Department of Oral and Dental, Faculty of Medicine, Palangkaraya University,
Palangkaraya, Central Kalimantan, Indonesia
2
Department of Medical Chemistry/ Biochemistry, Faculty of Medicine, Lambung Mangkurat
University, Banjarmasin, South Kalimantan, Indonesia
Corresponding author: email: ekoantioxidant@gmail.com

ABSTRACT
Oxidative stress is an important molecular mechanism for kidney injury in mercury (Hg)
poisoning. Usually, oxidative stress was measured by measuring the levels reactive oxygen
species (ROS) and antioxidant enzymatic activity. In this present study, we proposed a new
marker for oxidative stress in kidney damage induced by Hg. The new marker is the
peroxidative index (PI). In this experiment, a kidney sample was taken from male rats (Rattus
novergicus). Samples then homogenized and divided into five groups with; T1 served as
control which contains kidney homogenate only; T2 which contains kidney homogenate+0.1
mg/l of mercury chloride (HgCl); T3 which contains kidney homogenate+1 mg/l of HgCl; T4
which contains kidney homogenate+10 mg/l of HgCl; and T5 which contains kidney
homogenate+100 mg/l of HgCl. After treatment, kidney catalase (CAT) and peroxidases
(Pox) activity, PI, hydrogen peroxide (H2O2) and PC level were estimated. The results
revealed that Hg level is strong negatively correlated with both CAT and Pox activities, and
strongly positively correlated with PI. Also, the results revealed that PI is strongly positively
correlated with PI with the presence of Hg in different concentration of Hg in kidney cells. In
conclusion, PI might be a useful marker for oxidative stress in kidney damage induced by
Hg. For our knowledge, the proposed mechanism according to our results is Hg inhibited
antioxidant enzymatic activity and increase ROS in the kidney. Thus, induced oxidative stress
which promotes a further reaction to damage protein and resulted in kidney damage.
Key Words: Kidney, Mercury, Oxidative Stress, Peroxidative Index
INTRODUCTION
Mercury (Hg) is ubiquitously distributed in the environment and is non-essential and toxic to
the human body.1 Hg is considered as one of the highest priority pollutants to humans. 2 Hg is
widely used in industry, agriculture, and medicine, and circulates in ecosystems, but is never
destroyed.1 Hg exists in two forms; as inorganic Hg or organometallic Hg compounds. 2 The
fate and behavior of mercury in the environment depend on these chemical form. 3 Although
organic Hg is the most toxic form, inorganic Hg is the most common form of mercury
released into the aquatic environment by industries, having a more significant effect on fish
tissue.4
High exposures to inorganic Hg may result in damage to the several of human organs, mainly
kidney.3,5 Inorganic Hg salts are taken up and accumulated in the proximal tubules of the
1

kidneys. Clinical findings are polyuria and proteinuria (especially low molecular proteinuria)
which are the main indicators of tubular damage in kidneys. 6 Hg may also cause nephrotic
syndrome, either because of membranous nephropathy or minimal change disease.7
It is widely accepted that Hg exerts toxic health effects by different mechanisms including,
disturbing or inhibition of enzymes and inducing oxidative stress.6 Recent reports suggest that
mercury toxicity involves the generation of ROS with marked alterations in the antioxidant
defense systems and oxidative damage induction such as lipid peroxidation leading to cell
death.4 Begam and Sagupta8 result study shows that Hg exposure could decrease the catalase
(CAT) activity in intestinal macrophages of fish. Agarwal and Behari9 also show that Hg
exposure could decrease both glutathione peroxidase and CAT activity.
Recently, oxidative stress state not only evaluated by analyzing the level of oxidants and
antioxidants, but also analyze through a proportion or ratio. Several proportion ratio is
known, such as oxidative stress index and peroxidative index (PI). 10-11 Kania et al. study10
suggested that PI is the ratio between lipid peroxidation to hydrogen peroxide levels. We use
a different approach in this present study. In this present study, PI is measured by the ratio
between hydrogen peroxide (H2O2) level and the sum of peroxidase (Pox) and CAT activity,
as which have been done in Suhartono and Setiawan research.12
Among a wide range of oxidative stress state modifications, biomolecule carbonylation is
known to be a major hallmark of oxidative stress.13 Among biomolecule carbonylation,
protein carbonylation (PC) is a common parameter that measured to assess biomolecule
carbonylation. PC can be formed via the -amidation pathway, oxidative cleavage of
glutamyl residues, formation of protein-protein cross-linked derivatives, and cell membrane
damage by lipid oxidation products give rise to reactive aldehydes and ketones. 14-16 PC
content in blood and tissues is a reliable indicator of protein oxidation.17
As mentioned above, in this present study we try to evaluate the kidney damage induced by
Hg through the measurement of PI. Since the PI is the ratio between H 2O2 level and the sum
of Pox and CAT activity, each of these parameters is measured one by one. To investigate the
mechanism, we correlated Pox and CAT activities, and PI, with different concentrations of
Hg and we, also correlated the PI with PC with the presence of Hg in different
concentrations.
MATERIAL AND METHODS
Animals and Homogenate Preparation
Male rats (Rattus novergicus) weighing 200250 gram with 2-3 months old were obtained
from the Abadi Jaya farm at Yogyakarta, Indonesia, in healthy condition. The experiment was
approved by the Ethical Committee of the Lambung Mangkurat University, South
Kalimantan, Indonesia. Animals were fed under standard conditions and acclimatized with a
12 hours light/dark cycle. The animals were sacrificed by surgical procedure and the kidney
was removed. Then, the organs homogenized in phosphate buffer saline (pH 7.0) and were
ready to use for in vitro experimental models.
Experimental Models
Kidney cells will be exposed to different concentration of HgCl. The HgCl concentrations
were 0 mg/l, 0.1 mg/l, 1 mg/l, 10 mg/l, and 100 mg/l. Each solution then incubated at 37C
for 3 hours. After incubation, the CAT and Pox activity, the PI, and the PC and H 2O2 level
were estimated.
2

CAT activity analysis

CAT activity estimated by using Aebis method. 18 The first step was to prepare the stock
solution by using 2 ml and 1 ml from phosphate buffer at pH 7 and H 2O2 (30 mM)
respectively. Then, 50 l of the lysate was added to the stock solution. The ability of catalase
to work a reducing factor was measured by determining the changes in absorbance at 240 nm.
Peroxidase acivity analysis
Determination of Pox activity was measured by the method of Pruitt et al. 19 The assay was
performed by mixing 1.0 ml phosphate buffer (pH 7.0), 1.0 ml guaiacol solution and 1.0 ml
of a sample. The reaction was started by adding 1.0 ml of H 2O2 stock solution. Absorbance at
470 nm (A) and time (T) data were monitored.
H2O2 level analysis
The H2O2 level was calculated by the FOX2 method with slight modification. 14 Solutions
measured spectrophotometrically at = 505 nm. Standard and test solutions consisted of 1 M
H2O2 200 L and 200 L serum, respectively, with the addition of 160 L PBS pH 7.4, 160
L FeCl3 (251.5 mg FeCl3 dissolved in 250 ml distilled water) and 160 L o-phenanthroline
(120 mg o-phenanthroline dissolved in 100 ml distilled water) for both solutions. The
composition of the blank solution was identical to that of the test solution, except for the
absence of FeCl3 in the blank. Subsequent to preparation, all solutions were incubated for 30
minutes at room temperature, then centrifuged at 12,000 rpm for 10 minutes, and the
absorbance of the standard (As), test (Au) and blank (Ab) solutions measured at =505 nm,
using the supernatant of each solution.22
Peroxidative Index (PI) analysis
PI was a ratio between H2O2 level and CAT plus Pox activity. PI was calculated following to
equation:12
PI

[ H 2 O2 ]level
(CAT POx)activity

PC level analysis
PC was calculated by measuring the total protein carbonyl content. The total protein carbonyl
content was determined by colorimetric method. The liver homogenate (0.5ml) was pipetted
into 1.5 ml centrifuge tube and 0.5 ml of 10 mM 2,4-dinitrophenylhydrazine in 2 M HCl was
added and allowed to stand at room temperature for 1 hour, with vortexing every 10-15
minutes. Then, 0.5ml of 20% Trichloroacetic acid was added followed by centrifugation. The
supernatant was discarded and the pellets were washed 3 times with 1 ml ethanol-ethyl
acetate (1:1) to remove free reagent. The obtained precipitated protein was redissolved in 0.6
ml guanidine solution. Carbonyl content was calculated from maximum absorbance (390nm).
Statistical analysis
The results were expressed as meanSE for six replicates. The data was analyzed between
each parameter level and cyanide concentration. For analyzing the data, Microsoft excel 2010
was used and was examined by simple correlation regression.
RESULTS AND DISCUSSION
The present study is aimed to investigate the kidney damage induced by Hg through the
measurement of PI. First, we measured the activity of CAT and Pox with the presence of Hg
3

in different concentrations in kidney homogenate and correlated. The results show in figure 1
and 2. From figure 1 and 2, we can see that Hg level is strong negatively correlated with both
CAT and Pox activities. It means, with the presence of Hg, both CAT Pox activities decrease.
Similarly, Ibrahim20 found that CAT and glutathione peroxidase (GPx) activities were
decreased in the kidney of Clarias gariepinus exposed to Hg, and Ogunrinola et al. 21 also
found the depletion of CAT activity in the kidney of rats exposed to Hg.

Figure 1. The correlation between Hg level and CAT activity in kidney cells homogenate. Hg:
mercury; CAT: catalase

Figure 2. The correlation between Hg level and Pox activity in kidney cells homogenate. Hg:
mercury; Pox: peroxidase
It is widely accepted that Hg exposure could lower the defense system of antioxidants. These
effects is caused two basic mechanisms according to Karantika et al. 22, i.e (1) heavy metals
including Hg can make a bond formation with the sulfhydryl groups (-SH) of cysteine, thus
inhibited the activity of enzymes; and (2) Hg can replace a metal ion in the bodys of several
enzymes, leading to inactivation of the enzymes. Furthermore, the inactivation of CAT and
Pox can cause the accumulation of ROS, such as H2O2, which in turn causes oxidative
damage in the cells.23-25
To prove the oxidative damage in kidney cells by Hg, in this present study, we measure the
correlation between Hg exposure and PI. To best of our knowledge, PI is a new parameter

that we try in this study to assess oxidative damage from Hg exposure. The result shows in
figure 3.

Figure 3. The correlation between Hg level and PI in kidney cells homogenate. Hg: mercury;
PI: peroxidative index
From figure 3 we can see that the PI has increased with the increasing of Hg concentrations
in kidney cells homogenate. However, as far as we know, there have been no investigations of
the association between Hg exposure and PI in kidney cells. This is the first study to examine
the effects of Hg exposure on PI in kidney cells. These results reinforce previous results in
figure 1 and 2 that Hg exposure can cause oxidative stress. The exact mechanism is still
unknown. Several studies indicated that Hg reacts with thiol groups (-SH), thus depleting
intracellular thiol, especially glutathione and causing cellular oxidative stress or predisposing
cells to it and forming ROS.26 In other hand, results of this study indicated that Hg could
interact with antioxidant enzymatic, thus also increase the level ROS resulted in oxidative
stress.

Figure 4. The correlation between PI and PC level in kidney cells homogenate. PI:
peroxidative index; PC: protein carbonylation
It is widely accepted that oxidative stress situations can cause damage of biomolecules. 27
Among biomolecules, proteins are the principal target of ROS because they are present in
high concentrations in biological systems and remove 50-75% of the generated ROS.28 The
5

ROS could oxidize the protein and change the structure of proteins. This process is known as
PC. PC has attracted much attention in the last four decades and recognized as a universal
marker of oxidative stress induced protein modification.13
To evaluate the link between oxidative stress and PC during Hg exposure, we correlated the
PI and PC level with the presence of Hg in different concentrations in kidney cells
homogenate. The results show in figure 4. From figure 4, we can see that PI is strong
positively correlated with PC level. It means, if the PI is increased, the PC level in kidney
cells also increase with the presence of Hg. Several study and review indicated that PC
appear through side-chain oxidation of proline, arginine, and lysine. They can also result
from backbone cleavage through the -amidation pathway or -scission. Alternatively, they
can be introduced into proteins through Michael addition of unsaturated aldehydes produced
by peroxidation of lipids.29
In conclusion, the present study indicated that the Hg exposure induced kidney cell damaged
through the oxidative stress condition, which indicated by the strong correlation between Hg
level and PI. PI might be a useful marker for oxidative stress-induced kidney damage by Hg
exposure. However, further longitudinal studies are needed to validate PI as a marker of
oxidative stress induced kidney damage in Hg exposure. In addition, this results studies also
suggested that oxidative stress by Hg might be through the depletion of antioxidant
enzymatic such as CAT and Pox, which promote a further reaction to promote PC.
CONFLICT OF INTEREST
We declare that we have no conflict of interest.
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