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1. Effect of o-phenylenediamine on biochemical parameters in
liver and brain of zebrafish
Journal: Toxicology Research
Manuscript ID: TX-ART-09-2014-000139
Article Type: Paper
Date Submitted by the Author: 20-Sep-2014
Complete List of Authors: Raman, Thiagarajan; SASTRA University,
K, Bhavana; SASTRA University,
S, Charupriya; SASTRA University,
R, Nitin Chandra; SASTRA University,
MKN, Sai Varsha; SASTRA University,
Toxicology Research
2. 1
Effect of o-phenylenediamine on biochemical parameters in liver and brain of zebrafish
Bhaavana, K. 1
, Charupriya, S. 1
, Nitin Chandra, R.1
, Sai Varsha, M.K.N. 1
, Thiagarajan, R1,2,
*
1
Department of Bioengineering, 2
Centre for Research on Infectious Diseases (CRID), School
of Chemical & Biotechnology, SASTRA University, Thanjavur 613401
*Correspondence to: raman@biotech.sastra.edu
Page 1 of 24 Toxicology Research
3. 2
Abstract:
o-phenylenediamine (oPD) is a commonly used industrial reagent. Though it is released into
water bodies, its environmental concentration and impact remains unexplored. This study
aimed to investigate the effect of oPD in an aquatic animal to highlight the toxicity
mechanism of this important toxicant. Zebrafish were exposed to 1ppm and 5ppm of oPD for
15 days and biochemical parameters in liver and brain were analysed. Biological Oxygen
Demand (BOD) of oPD treated water suggested moderate pollution at both the concentrations
tested. Further, exposure of zebrafish to oPD resulted in increase in liver oxidative stress in
both the concentrations of oPD. Interestingly activities of superoxide dismutase, and reduced
glutathione were found to be enhanced upon oPD exposure indicating protective response of
these two antioxidants against oPD-induced liver oxidative stress. Liver detoxification
enzymes, α- and β-carboxylesterase, were found to be altered upon oPD exposure. In the case
of α-carboxylesterase, the activity was enhanced at 1ppm exposure whereas inhibited at
5ppm, suggesting preferential formation of α-esters. However, inhibition at higher oPD
concentration suggests enzyme turnover during oPD metabolism. On the other hand β-
carboxylesterase was only slightly inhibited and that too at 5ppm of oPD. oPD exposure also
resulted in neuronal toxicity with significant decrease in brain monoamine oxidase activity in
both 1ppm and 5ppm of oPD. Taken together, our study shows that oPD exhibits its toxicity
at multiple levels by affecting the normal functioning of vital organs such as liver and brain,
thus producing toxicity and mortality in zebrafish.
Keywords: o-phenylenediamine; oxidative stress; toxicity; pollution; zebrafish
Page 2 of 24Toxicology Research
4. 3
Introduction
ortho-phenylenediamine (oPD), is commonly used in the synthesis of a variety of dyes,
pigments, fungicides, as a photographic developing agent and as a laboratory reagent1-3
.
It is also used in sensors4-7
,immunoassays8,9
, rechargeable cells10
, supercapacitors11
, corrosion
inhibitors12-14
and pharmaceuticals15,16
. Hence, there is a possibility that it may be released
into aquatic bodies through various waste water streams17
. Nevertheless, the concentration of
oPD in rivers remains unexplored even though it is a common industrial reagent17
. oPD has a
water solubility of 36000mg/L and an oxidative half life of 25-40 days18
, suggesting that it
has a good chance of interacting with biological systems. It is biodegradable and exhibited
33% removal after 5 days incubation (initial concentration: 25-30 ppm) in an aerobic
screening study using acclimated activated sludge inoculums19
. oPD absorbs light at a
wavelength of 290nm20
and above and has an overall photolysis half life of 0.26 days18
.
The para isomer of phenylenediamines has received a lot of attention in relation to its
toxicity. Para-phenylenediamine (pPD) has been shown to cause haemolytic anaemia and
rhabdomyolysis in rats subsequently leading to acute renal failure21
. The myotoxicity of pPD
and its derivatives is thought to be due to their oxidation by mitochondria, disruption of
respiratory control and ADP:O ratios causing muscle necrosis22,23
. pPD has also been
reported to cause testicular toxicity in male albino rats by inducing oxidative stress and
apoptosis of germ cells24
. Nephrotoxicity of pPD was shown to be associated with caspase-3
activation in human HK-2 proximal tubular epithelial cells25
. In guinea pigs, dermal
application of pPD induces an increase in skin enzymes, lipid peroxidation and histamine
levels, decrease in glutathione levels, degenerative changes in the liver and hyperkeratosis
with infiltration of cells in the dermis26-28
. Single stranded DNA breaks were observed when
human lymphocytes were exposed to pPD and its derivatives29
. When the ortho, para and
meta isomers were tested in rats and mice by administering 4-chloro-oPD, 4-chloro-mPD and
4-chloro-pPD in their diets, the order of carcinogenicity was found to be ortho > meta > para
with para isomer showing no significant neoplasia30
. Histopathological examination of spleen
from treated rats exhibit red pulp congestion, expansion of the germinal centre, hyperplasia of
the membrane capsule and extensive accumulation of hemosiderin pigments in the red pulp
of the spleen31
. pPD is thought to induce genotoxic carcinogenesis in SV-40 immortalized
human uroepithelial cells by accumulating mutant p53 and cyclooxygenase-2 proteins32
. In
Page 3 of 24 Toxicology Research
5. 4
Mardin-Darby canine kidney cells, it has been demonstrated that p-PD induces apoptosis via
oxidative stress causing the activation of caspases-8, 9, 3/733
and p5334
. In humans, acute
pPD intoxication manifests as cervicofacial and laryngeal edema, rhabdomyolysis, impaired
renal function, increase in liver transaminases, hyperkalemia and ventricular arrhythmia35
. It
has been demonstrated in rats, human hepatocytes and epidermis that pPD is converted to N-
monoacylated pPD and/or N,N-diacylated pPD which are biologically less reactive36-38
.
Effect of pPD in fishes has also been studied (U.S. EPA). Behavioural and mortality tests on
common carp were carried out and the mean toxic dose was determined to be 140 mg/kg.
Three mortality tests were performed for different exposure periods of <72 hours, 5.79 days
and <19 hours. The minimum and the maximum doses were determined to be 125 and 145
mg/kg for <72 hours and 110 and 133 mg/kg for 5.79 days. A similar study on goldfish was
done to determine the mortality and the mean toxic dose was determined to be 5740 µg/L.
Mortality test in Coho Salmon were carried out on fish for a period of 24 hours and the toxic
doses were reported to be 8000, 10000 and 12000 µg/L. Rainbow trout were exposed for 24
hours and the mean toxic dose was determined to be 8000, 10000 and 120000 µg/L. Northern
Squawfish ( 5-10cm long) was also subjected to a similar exposure for 24hours and the mean
toxic dose was determined to be 8000, 10000 and 120000 µg/L.
The effect of oPD has also been studied in many model organisms, primarily rodents. o-
phenylenediamine dihydrochloride (oPD.2HCl) was found to be carcinogenic in rats and
mice of both sexes, when administered with drinking water for two years39
. Mouse, Chinese
hamster and guinea pig were exposed to 4 derivatives of oPD, namely 4-methyl-, 4-nitro-,
4,5-dimethyl- and 4,5-dichloro- oPD, and evaluated for cytogenetic effects by micronucleus
test. Out of the 4 derivatives only 4-methyl-oPD caused chromosomal damage40
. In another
study, when 4-chloro-oPD, 4-nitro-oPD and pPD.2HCl were administered to mice
intraperitoneally, in vivo mouse bone marrow micronucleus assay revealed a significant dose
related increase in micronucleated polychromatic erythrocyte only for the 4-chloro
derivative30
. However, both 4-chloro-oPD and 4-nitro-oPD were shown to be carcinogenic in
big blue transgenic mice contradicting the previous study41
. It was demonstrated that oPD and
4-chloro-oPD cause oxidative DNA damage by undergoing SOD-enhanced Cu(II)-mediated
auto-oxidation42
. Effect of oPD has also been studied on different fishes (U.S. EPA).
Behavioural and mortality tests on Coho salmon were conducted and the toxic dose was
determined to be 1000 µg/mL. A similar study performed on Chinook salmon also showed
the toxic dose to be 1000 µg/mL. A mortality test on common carp was carried out and the
minimum and the maximum toxic dose were determined to be 67 and 194 mg/kg,
Page 4 of 24Toxicology Research
6. 5
respectively. Mortality test on rainbow trout and northern squawfish revealed the toxic dose
for both to be 1000 µg/mL. Acute toxicity dosage of oPD in zebrafish has also been reported
by Verschueren in the Handbook of Environmental Data on Organic Chemicals, 200143
. The
dosages at which 50% (LD50) and 100% (LD100) of the exposed fish die, were found to be
24mg/L and 33mg/L for 96 hrs, respectively. However the mechanism of toxicity of oPD in
aquatic animals, especially fish, has not received much attention and this area remains
unexplored.
Thus, in our study we aim to understand the chronic effects of oPD exposure on zebrafish and
decipher the possible mechanism involved. Zebrafish is one of the most commonly used
organisms for toxicity analyses due its small body size and ease of handling44, 45
. For this, we
tested oPD toxicity in zebrafish at two different concentrations, 1ppm and 5ppm, for 15 days
using static renewal assay. We used low concentrations of oPD and chronic exposure in order
to understand the biochemical changes produced by oPD.
Results
1. BOD Estimation:
Biological oxygen demand is the amount of dissolved oxygen required for the biological
decomposition of dissolved organic matter to occur at a standardized time and temperature (5
days, 20°C). The BOD of control, 1ppm oPD and 5ppm oPD solutions were found to be
2.10526mg/L, 6.31578mg/L and 8.0808mg/L, respectively. According to Taiwan
Environmental Protection Administration database, the given water sample is non-polluted if
BOD ≤ 3.0, lightly polluted if 3.0 ≤ BOD ≤ 4.9, moderately polluted if 5.0 ≤ BOD ≤ 15.0 and
severely polluted if BOD >15.0. Using this classification scheme, our control water is non-
polluted while 1ppm oPD and 5ppm oPD are both moderately polluted.
2. Toxicity evaluation
In the present study we have evaluated the toxicity of oPD against zebrafish for a period of
15 days. We have selected two concentrations of oPD for toxicity analysis-1ppm and 5ppm.
It is to be noted that oPD has been well studied in rodents and but studies in aquatic
organisms are very few and even these have concentrated on behavioural responses and
mortality. These studies have used very high doses of oPD, 1 mg/ml, and moreover, the
actual environmental concentration of oPD remains unclear. oPD toxicity for zebrafish has
been shown to be 24 mg/L (LD50) and 33mg/L (LD100) for 96hrs 43
. In the present study, both
1ppm and 5ppm were used since these two concentrations did not produce mortality up to 15
Page 5 of 24 Toxicology Research
7. 6
days of exposure. Earlier we also tested 10 ppm, however, at this concentration we observed
mortality of 3 to 4 fishes (out of 10) at the end of 15 days.
3. Effect of oPD on liver oxidative stress parameters
oPD toxicity in the case of rodents has been shown to involve oxidative stress. In the present
study we analysed whether exposure to oPD could enhance oxidative stress parameters in
zebrafish too. As can be seen from Figure 1a ,exposure to 1ppm and 5ppm of oPD resulted in
a significant enhancement (p<0.05) in nitric oxide generation (1ppm = 1.821±0.004 and
5ppm = 2.11±0.005) in the liver of zebrafish when compared to control fish (1.375±0.005).
These results show that oPD induces free radical generation and this could be one mechanism
of its toxicity. Enhanced generation of free radicals can be counteracted by endogenous
antioxidants. As shown in Figure 1b, exposure of zebrafish to oPD resulted in a significant
enhancement (p<0.05) in liver SOD activity (1ppm = 1.261±0.26 and 5ppm = 1.406±0.25)
when compared to control (0.892±0.062). Similar to increase in SOD activity, liver GSH
activity (Figure 1c) was found to be significantly enhanced only in zebrafish exposed to
5ppm of oPD (0.468±0.03, p<0.05) and not at the lower dose (1ppm= 0.301±0.06; NS),
which was comparable to the control value (0.321±0.03). On the other hand, liver catalase
activity (Figure 1d) was found to be significantly inhibited (p<0.05) in zebrafish that were
exposed to both 1ppm (0.018±0.003) and 5ppm (0.016±0.002) of oPD, when compared to
control (0.021 ± 0.006). Taken together, these results show that exposure to oPD results in
enhancement in liver oxidative stress. However, increase in the activities of SOD and GSH
due to oPD exposure suggests that this could be a protective response to oPD-induced
oxidative stress.
4. Effect of oPD on liver carboxylesterases
Liver being the primary detoxification organ, we analysed the effect of oPD exposure on liver
carboxylesterases. As can be seen from the Figure 2a, exposure to 1ppm oPD led to a
significant stimulation of liver α-carboxylesterase activity (360.28±10.42, p<0.001); while
5ppm produced a significant inhibition of liver α-carboxylesterase activity (109.43±12.56),
when compared to control (199.65±9.87). On the other hand liver β-carboxylesterase activity
(Figure 2b) was found to be inhibited upon exposure to 5ppm oPD (1057.78±35.62) and no
change was observed at 1ppm (1222.65±28.26; NS). These results suggest that α-
carboxylesterase activities are primarily increased for the metabolism of oPD at 1ppm.
Page 6 of 24Toxicology Research
8. 7
However, at higher concentration, there is inhibition of even α-carboxylesterases, indicating
toxicity of oPD to liver enzymes that could affect liver functions.
5. Effect of oPD on neuronal functions
To understand the toxicity of oPD on neuronal tissues, we tested brain MAO activity after
exposure of fish to oPD for 15 days. As can be seen from the Figure 3, exposure of zebrafish
to oPD (1 ppm and 5 ppm) resulted in a significant inhibition of brain MAO activity. This
suggests that apart from promoting liver toxicity, oPD can also affect the physiological
functions of the organism by producing neuronal toxicity in zebrafish.
Discussion
In this study we present data showing the probable mechanism of toxicity of oPD in an
aquatic animal. More specifically we show that exposure of zebrafish to sub-lethal
concentrations (1 & 5ppm) of oPD for 15 days results in alterations in both liver and brain
parameters. oPD according to ‘Hazardous Substances Data Bank’ is a known contaminant in
waste water from where it can reach other aquatic habitats. It has good water solubility18
and
is biodegradable by almost 33% when the initial concentration was kept at 25-30ppm19
.
However, concentration of oPD in rivers and other aquatic bodies remains unexplored (US
EPA). In the present study the BOD values of 1ppm and 5ppm oPD solutions suggest
moderate pollution46
. If high concentrations of oPD are discharged into aquatic bodies, the
BOD value may be higher. Higher the BOD, greater is the microbial growth in the water,
causing depletion of dissolved oxygen levels which may prove lethal to aquatic life47
. This
increase in BOD could also have contributed to the toxicity of oPD to zebrafish as observed
in this study.
From literature it is clear that most oPD toxicity evaluation has been done using rodent
models30,39,40-42
. oPD toxicity in aquatic animals has also been studied, but these studies have
primarily looked at the levels required for toxicity. As per US EPA, oPD has been tested at
concentrations ranging from 10,000µg/L to 194mg/kg body weight against fish such as
salmon, carps, trout and squawfish. These studies have predominantly looked at mortality due
to oPD exposure. One study employing zebrafish has shown LD50 (24 mg/L) and LD100 (33
mg/L) for oPD after 96hrs43
. However, to the best of our knowledge, oPD toxicity in aquatic
animals has not been properly characterised. Moreover, as per US EPA, the actual
environmental concentration of oPD also remains to be analysed and information on this and
Page 7 of 24 Toxicology Research
9. 8
the mechanism of oPD toxicity in aquatic animals remains unexplored. In this study we have
tested two different concentrations of oPD (1 and 5 ppm) against zebrafish for 15 days. This
concentration is well below sub-lethal levels of oPD as per other studies. We selected these
two concentrations since we wanted to understand the basic mechanism of oPD toxicity in
both liver and brain of zebrafish, without producing mortality.
Exposure of zebrafish to 1ppm and 5ppm of oPD resulted in significant enhancement in nitric
oxide generation in liver when compared to control fishes. Studies in Mardin-Darby canine
kidney cells have indeed shown 4-chloro-oPD induced enhancement in free radical
generation48
leading to the induction of apoptosis in these cells. Increase in oxidative stress is
counteracted by endogenous antioxidants that play a vital role in not only xenobiotic
metabolism but also protect biological tissues from xenobiotic-induced oxidative stress. In
the present study it was observed that exposure of zebrafish to 5ppm oPD alone resulted in
the enhancement in liver GSH activity when compared to both control and 1ppm oPD. This is
interesting since in a study by Onn et al48
the authors had demonstrated GSH depletion due to
oPD-enhanced free radical generation. This difference could be due to the nature of oPD and
dosage used. Similar to GSH, liver SOD activity was found to be significantly enhanced in
zebrafish exposed to both 1ppm and 5ppm oPD. However, liver catalase activity was found to
be inhibited by oPD at both 1ppm and 5ppm. These results suggest that GSH and SOD are
preferentially activated as a response to oPD-induced oxidative stress in liver of zebrafish.
However, reduction in catalase activity suggests that catalase is susceptible to oPD49
.
Interestingly, Murata et al42
have shown that oPD produces carcinogenicity by oxidative
DNA damage. Furthermore, they also showed that presence of SOD enhanced DNA damage
produced by oPD through Cu(II)-mediated auto-oxidation of oPD. These results suggest that
oPD exposure could have resulted in oxidative stress leading to increase in liver SOD activity
that we speculate is a host protective response to oPD toxicity. However prolonged exposure
to oPD can result in SOD-mediated auto-oxidation of oPD leading to DNA damage, which
can increase the toxicity of oPD to the host.
Carboxylesterases belong to α/β-hydrolase family that catalyze the cleavage of esters and are
important components of the biological xenobiotic detoxification system. In the present study
liver α-carboxylesterase activity was found to be enhanced when zebrafish were exposed to
1ppm oPD, while at 5ppm the activity was inhibited. Similarly, β-carboxylesterase activity
was also inhibited upon exposure to 5ppm oPD. This suggests that at 1ppm α-
carboxylesterase activity is at optimum level but is overwhelmed at 5ppm. However, decrease
in both α- and β-carboxylesterase in liver of zebrafish exposed to 5ppm oPD suggests the
Page 8 of 24Toxicology Research
10. 9
formation of α-esters due to oPD metabolism and enzyme turnover. Apart from this it is also
plausible that liver oxidative stress could result in inhibition of carboxylesterase activity and
moreover release of enzyme from tissues into soluble extra cellular compartments due to cell
injury, could also contribute to low tissue carboxylesterase activities.
Monoamine oxidases are amine oxidases and have important role in the central nervous
system. They are responsible for catalysing the oxidative deamination of a wide variety of
xenobiotic and endobiotic primary, secondary and tertiary amines. In the central nervous
system they are concerned with the deactivation of monoamine neurotransmitters such as
serotonin and dopamine and hence their proper functioning is critical to aminergic
transmission in neural tissues. In this study, exposure of zebrafish to oPD resulted in a
significant inhibition of brain monoamine oxidase activity. This leads us to speculate on two
possibilities; 1) Brain MAO activity is lowered due to oxidative stress-induced enzyme
deactivation and 2) MAO activity could also be responsible for deactivation of oPD, during
which there is rapid turnover of the enzyme. It is quite plausible that both these scenarios are
responsible for the observed toxicity of oPD in zebrafish brain. Thus, oPD exposure results in
inhibition of brain MAO that could adversely affect the neural physiology in zebrafish.
Taken together, our study shows the probable mechanism of toxicity of oPD in an aquatic
animal, namely zebrafish. oPD exposure resulted in enhanced oxidative stress in the liver and
deactivation of liver detoxification enzymes. Apart from this oPD also exhibited neuronal
toxicity by inhibiting brain MAO activity. Thus, oPD produces adverse biochemical changes
in an aquatic animal that can lead to disruption of physiological functions ultimately leading
to mortality.
Experimental
Animals
Adult zebrafish (Danio rerio) of length 4±1cm and weight 300±10mg were procured from a
local aquarium irrespective of sex. The fish were maintained in 10L tanks in filtered tap water
at RT and fed at regular intervals. The tanks were cleaned, sterilized and water replaced
periodically. Fish were allowed to acclimatize for a week before initiation of oPD exposure.
Chemicals
oPD (CAS registry no. 95-54-5) was purchased from LOBA Chemie. Naphthyl
ethylenediamine, sulphanilamide, α-naphthyl acetate, β-naphthyl acetate, Fast Blue B,
benzylamine, epinephrine, 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB) and diethyl ether
Page 9 of 24 Toxicology Research
11. 10
were purchased from Sigma-Aldrich. All other chemicals and reagents were of the highest
analytical grade and commercially available.
oPD purification
O-phenylenediamine was purified by recrystallization from water-ethanol mixture in
presence of charcoal followed by sublimation. Stock solution of OPDA (10-2
M) was
prepared freshly for each experiment50
.
Estimation of biological oxygen demand (BOD)
The BOD of control, 1ppm and 5ppm oPD solutions (prepared using filtered tap water) were
estimated according to Indian Standards:3025 (Part 44)-Reaffirmed 2003.
oPD exposure
Fish were exposed to two different concentrations of oPD (1 ppm and 5 ppm). oPD was
prepared by dissolving in filtered tap water. Exposure was performed in 4L tanks containing
20 fishes. Static renewal assay was performed and exposure was done for a period of 15 days.
There was no mortality in both the concentrations of oPD till 15 days of exposure. The water
and oPD were renewed every day.
Tissue preparation
At the end of the exposure period, fish were sacrificed, dissected and the liver removed. Liver
from two fishes of the same treatment group were pooled for all assays. Liver tissue was
homogenised in ice-cold buffer (as per the assay). The homogenate was centrifuged (10,000 x
g, 10 min, 4°C) and supernatant used for all analyses. Brain tissue was homogenised for
MAO assay alone.
Estimation of free radicals
Nitric Oxide assay: Nitric oxide was estimated according to Manikandan et al51
. 100µL of
the liver homogenate was mixed with 150µL of Tris HCl (pH 7.4) followed by the addition of
50µL of 0.1% naphthyl ethylenediamine dihydrochloride and 50µL of 1% sulphanilamide.
The mixture was incubated in dark at room temperature for 10min and then centrifuged at
12000 x g for 15min at 4°C. The absorbance of the clear supernatant was measured at 540nm
using a UV spectrophotometer. Values were presented as µM nitrite.
Page 10 of 24Toxicology Research
12. 11
Estimation of antioxidant enzymes
Superoxide dismutase (SOD) assay: SOD activity was estimated according to Manikandan
et al52
. 100µL of liver homogenate was mixed in 750µL of ethanol followed by the addition
of 150µL of ice cold chloroform. This mixture was centrifuged at 4000 x g for 5 min at 4˚C.
To 0.5mL of the supernatant, 0.5mL of 0.6mM EDTA was added followed by the addition of
1mL of bicarbonate buffer (0.1M, pH 10.2)and 500µL of 1.3mM epinephrine. The
absorbance was measured at 480nm using a UV spectrophotometer. The enzyme activity was
expressed as unit/mg protein.
Reduced glutathione (GSH) assay: GSH was quantified as per Manikandan et al52
. To
750µL of liver homogenate, 500µL of 10% trichloroacetic acid was added and the mixture
was centrifuged at 11,000 x g for 15min at 4˚C. 250µL of supernatant was taken and to it
1mL of DTNB was added. The volume was made up to 1.5mL using potassium phosphate
buffer (0.2mM, pH 8.0) and the absorbance measured at 412nm using a UV
spectrophotometer. The results were expressed as µmol/g wet weight.
Catalase assay: Catalase activity was measured according to Manikandan et al52
. To 100µL
of liver homogenate, 1.2mL of phosphate buffer (50mM, pH 7.0) and 1mL of 30mM
hydrogen peroxide solution were added. The absorbance was measured at 240 nm for 3 min
at 15 second interval using UV spectrophotometer. The values were expressed in units/ml
using the molar extinction coefficient of H2O2.
Estimation of metabolic enzymes
Monoamine oxidase (MAO) assay: MAO activity was measured as per Tabor et al53
. 900µL
of brain homogenate was mixed with 300µL of sodium phosphate buffer (1M, pH 7.2). To
this, 300µL of 50mM benzylamine and 1.5mL of double distilled water were added. The
absorbance was measured at 292nm every 5 min over a period of 30min.
Carboxylesterase assay: Carboxylesterases were measured according to Joseph et al54
.
500µL of liver homogenate was mixed with 2.5mL of α–naphthyl acetate (250µM) or β –
naphthyl acetate (250µM) solution and incubated at room temperature for 30min. At the end
of incubation, 250µL of 0.3% Fast Blue B solution was added to stop the reaction and the
solution was left for 30min to develop a black colour for α–naphthyl acetate and red colour
Page 11 of 24 Toxicology Research
13. 12
for β–naphthyl acetate. The absorbance was measured at 430nm and 588nm using a UV
spectrophotometer for α–naphthyl acetate and β–naphthyl acetate, respectively. The amount
of α and β-carboxylesterase was calculated using standard graphs and expressed as µM of α-
or β-napthol released/min.
Protein estimation
Protein was estimated by the method of Lowry et al55
.
Statistical analyses
All assays were performed in duplicates. Results were expressed as mean±SD. One way
analysis of variance (p < 0.05) was followed by Tukey Post-hoc test to evaluate the
significance between different treatment groups.
Conclusion
Thus this study demonstrates the possible mechanism of toxicity of oPD in aquatic animals.
oPD toxicity is poorly understood and previous studies on fish have only looked at mortality.
oPD was shown to trigger oxidative stress and in the process inactivate enzymes concerned
with important physiological responses. The results of this study will open up further research
into aspects of oPD toxicity, an area which has been neglected so far.
Acknowledgements: TR would like to thank the Vice Chancellor, SASTRA University, for
support through TRR Fund.
Conflict of interest: The authors declare no conflict of interest
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Figure legends:
Figure 1a: Effect of oPD on nitric oxide generation by the liver of zebrafish. Each bar
represents mean±SD of 10 determinations using liver samples pooled from two fishes from
each treatment group. Assays were run in duplicates. Nitric oxide was estimated as nitrite and
quantified using nitrite in a standard graph. Asterisks indicate the increase in nitric oxide
generation in oPD (1ppm & 5 ppm) exposed fishes are statistically significant at p<0.05,
when compared to control fish.
Figure 1b: Effect of oPD on liver SOD activity. Each bar represents mean±SD of 7
determinations using liver samples pooled from two fishes from each treatment group.
Assays were run in duplicates. Asterisks indicate the increase in SOD activity in oPD (1 ppm
& 5 ppm) exposed fish are statistically significant at p<0.05, when compared to control fish.
Figure 1c: Effect of oPD on liver GSH activity. Each bar represents mean±SD of 7
determinations using liver samples pooled from two fishes from each treatment group.
Assays were run in duplicates. Asterisks indicate that the increase observed in the case of
oPD (5 ppm) exposed fish is statistically significant at p<0.05, when compared to control
fish.
Figure 1d: Effect of oPD on liver catalase activity. Each bar represents mean±SD of 7
determinations using liver samples pooled from two fishes from each treatment group.
Assays were run in duplicates. Catalase activity was determined using molar extinction
coefficient of hydrogen peroxide (0.0436cm2
/µmol). Asterisks indicate the decrease observed
in the case of oPD (1 ppm & 5 ppm) exposed fish are statistically significant at p<0.05, when
compared to control fish.
Figure 2a: Effect of oPD on liver α-carboxylesterase activity. Each bar represents mean±SD
of 10 determinations using liver samples pooled from two fishes from each treatment group.
Assays were run in duplicates. The activity was quantified by extrapolating the absorbance
Page 16 of 24Toxicology Research
18. 17
values in a standard graph of α-naphthol. Asterisks indicate the increase observed in the case
fish exposed to 1 ppm of oPD and the decrease in fish exposed to 5 ppm of oPD are
statistically significant at p<0.05, when compared to control.
Figure 2b: Effect of oPD on liver β-carboxylesterase activity. Each bar represents mean±SD
of 10 determinations using liver samples pooled from two fishes from each treatment group.
Assays were run in duplicates. The activity was quantified by extrapolating the absorbance
values in a standard graph of β-naphthol. Asterisks indicate the decrease observed in the case
of fish exposed to 5 ppm of oPD is statistically significant at p<0.05, when compared to
control.
Figure 3: Effect of oPD on MAO activity in the brain. Each bar represents mean±SD of 10
determinations using brain samples from each treatment group. Assays were run in
duplicates. Asterisks indicate the decrease observed in the case of fish exposed to oPD (1
ppm & 5 ppm) are statistically significant at p<0.05, when compared to control.
Page 17 of 24 Toxicology Research