TOXICOLOGICAL INVESTIGATIONS OF LEAD ACETATE AND ARSENIC TRIOXIDE FOLLOWING SIMULTANEOUS EXPOSURE IN ZEBRAFISH 3097
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Date
2020-09
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JAU, JUNAGADH
Abstract
Heavy metal contamination of aquatic environment is one of the most
common problem which affects the health of aquatic organisms including fish.
Assessment of heavy metals induced oxidative damage and antioxidant defenses in
fish can reflect the toxic effects of metals to the aquatic organisms and it can be
helpful to mitigate further human exposure. Hence, present study was carried out to
investigate the effects of lead acetate (PbAc, 20 µg/L) and arsenic trioxide (As2O3, 50
µg/L) alone as well as combined exposure for 21 days on anatomical confirmation,
behavioral and oxidative stress parameters, mRNA expression of antioxidant genes
and histopathological changes in adult female zebrafish.
In the present study, 224 adult female zebrafish were randomly divided into
four groups. The zebrafish of control group were maintained in normal condition
without any treatment (R.O water with standard range of temperature and pH). The
zebrafish of second group were maintained in R.O. water containing lead acetate at
the strength of 20 µg/L. The zebrafish of third group were maintained in R.O. water
containing arsenic trioxide at the strength of 50 µg/L of water. The zebrafish of fourth
group were exposed to both lead acetate and arsenic trioxide at above mentioned
strength.
Noticeable signs of toxicity were not observed except freezing and hiding
behavior in all toxicity groups. The exposure of lead acetate and arsenic trioxide alone
and in combination did not affect the physical parameters like body weight, body
width at middle and body length. Light-dark preference test was carried out at the day
7 and day 21 during the experiment. The mean values of time (sec.) spent in light
compartment by zebrafish of control, Pb, As and Pb + As groups at day 21 were
316.17 ± 6.32, 230.00 ± 17.08, 261.50 ± 6.63, 211.50 ± 13.91 sec., respectively and
time (sec.) spent in dark compartment were 283.83 ± 6.32, 370.00 ± 17.08, 338.50 ±
6.63 and 388.50 ± 13.91 sec., respectively. At 21st day, the zebrafish exposed to Pb,
As and Pb + As had spent significantly (p < 0.05) less time in light compartment and
more time in dark compartment as compared zebrafish of control group. At 21st day,
the zebrafish exposed to Pb, As and Pb + As had significantly (p < 0.05) decrease in
no. of entries in light compartment as compared to control group. Findings of present
study indicates that lead and arsenic exposure has induced anxiety in zebrafish. Novel
tank test was carried out at the day 7 and day 21 during the experiment. Novel tank
test findings revealed that at 21st day, the zebrafish exposed to Pb, As and Pb + As had significantly (p < 0.05) decrease in time spent in upper zone (sec.) and increase in
time spent in lower zone (sec.) of test tank as compared to zebrafish of control group.
In present study, SOD activity in brain tissue of zebrafish was significantly
(p < 0.05) decreased after 21 days of combined exposure of Pb + As compared control
group zebrafish. After 21 days of Pb, As and Pb + As combined exposure, CAT
activities in brain were non-significantly decreased, whereas levels of GSH in brain
were significantly (p < 0.05) reduced and MDA levels were significantly (p < 0.05)
increased as compared to control group zebrafish. In intestine, SOD activities were
significantly (p < 0.05) decreased in Pb, As and Pb + As treated groups as compared
to control group, whereas CAT activities and GSH levels were slightly decreased
(non-significantly) as compared to control group. The MDA levels of zebrafish
intestine were significantly (p < 0.05) increased in zebrafish of all treatment groups as
compared to control group. Alone Pb exposure for 21 days had shown to produce
significantly (p < 0.05) increased SOD activity in ovary of zebrafish as compared to
control group. The CAT activities and GSH levels were slightly decreased (non significantly) and MDA levels were significantly (p < 0.05) increased in ovary of
zebrafish exposed to Pb, As and Pb + As groups as compared to control group.
The SOD mRNA expression level in brain was significantly down regulated
by 12.96 fold (p < 0.05) in alone Pb exposure group as compared to control. After Pb,
As and Pb + As exposure, CAT mRNA expression levels in brain were non significantly down regulated by 12.42, 2.25 and 4.05 fold, respectively as compared
to control. Similarly, Nrf2 mRNA expression levels in brain after Pb alone and Pb +
As combination group were non-significantly down regulated by 19.04 and 1.04 fold,
respectively. Findings of mRNA expressions were concomitant with activities of
respective enzymes in brain of zebrafish. In intestine, after 21 days of Pb and As
alone exposure, SOD and CAT mRNA expressions were down regulated. Alone As
exposure showed up regulation of Nrf2 mRNA expression in intestine of zebrafish. In
ovary, SOD mRNA expression level was significantly (p < 0.05) up regulated by 0.03
fold in Pb + As exposure group. After Pb and As alone exposure, CAT mRNA
expression levels in ovary were non-significantly down regulated by 4.89 and 1.47
fold, respectively. The Nrf2 mRNA expression levels in ovary were non-significantly
up regulated by 0.63, 0.14 and 0.10 fold in Pb, As and Pb + As exposure group,
respectively as compared to control. In the present study, combined exposure of Pb
and As (Pb + As) had increased SOD and Nrf2 mRNA expressions in ovary of
zebrafish, but the extent of effect was much lower as compared to Pb and As alone
exposure.
After Pb, As and Pb + As exposure, microscopic examination revealed mild to
moderate degree of histopathological changes in brain, intestine, ovary, liver, kidney,
gill and retinal layer of eye in adult female zebrafish.
In conclusion, lead acetate and arsenic trioxide alone and in combination
exposure for 21 days had shown to produce anxiety like behavior and oxidative stress
mediated alterations in brain, intestine and ovary of zebrafish. Both lead acetate and
arsenic trioxide alone and in combination affected the SOD, CAT and Nrf2 mRNA
expressions in brain, intestine and ovary of zebrafish. The both metals alone and in
combination resulted in significant histopathological changes in brain, intestine,
ovary, liver, kidney, gills and retinal layer of eye in zebrafish. However, exposure of
lead acetate (20 µg/L) and arsenic trioxide (50 µg/L) for 21 days did not show the
synergistic toxicological effects upon simultaneous exposure in adult female
zebrafish.