Download Di (n)-Butyl Phthalate Induced Neuronal Perturbations in Rat Brain

Mahaboob Basha P J..Biosci Tech,Vol 8(1),2017,794-800
ISSN: 0976-0172
Journal of Bioscience And Technology
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Di (n)-Butyl Phthalate Induced Neuronal Perturbations in Rat Brain Tissues:
A Multigenerational Assessment
Radha M. J. and Mahaboob Basha P*
Department of Zoology, Bangalore University, Bangalore 560056, INDIA
*Email: [email protected]
Abstract
Di-n-butyl phthalate (DBP) is a well-known plasticizer and cause a wide range of reproductive or endocrine
disruptive disorders. Exposure to DBP during gestation shown to disrupt testosterone synthesis and male sexual
development in the fetal rat, however, the underlying mechanisms and its impact on the cortical and cerebellar
neuronal development remain poorly understood. Here, we examine the effect of multigenerational exposure of
DBP on rat cortical as well as cerebellar neurons. In this study 1/16th LD50 (i.e., 500mg/kg BW) administered
through oral gavage during gestation day 6 to till parturition; exposure continued until they were weaned on
postnatal (PND) 30. All pups at PND30 were analysed for cytoarchitectural alternations in brain cortex and
cerebellum. Variations were observed in the rate of feed and water consumption, litter size, organ (brain) somatic
index and mortality. Significant degenerative changes in neuroglial cells of brain cortex of F1; focal aggregates
of inflammatory infiltration found invariably in the CC region of F2 and F3 rats while CB region showed a
remarkable degeneration in Purkinje cell layer in experimental rats. These findings reveal that direct neuronal
somatic injury as a consequence of oxidative stress origin, and developing brain tissues are vulnerable and
multigenerational exposure to DBP caused disturbance elicited neuro-pathological changes within the CNS,
generating heterogeneous injury and reactive alteration within both axons and neuronal somata in the same
domains. In brief, these perturbations of neuronal structure and function are likely to contribute to the
pathogenesis and affect the learning process.
Keywords: Di-n-butyl Phthalate toxicity, Generational effects, Cerebral cortex, Cerebellum, Histopathology
1. Introduction
Prevalence of phthalates in the environment found to be ubiquitous as they are widely used as a
plasticizer in polyvinyl chloride (PVC) products, food wraps and several medical devices [1]. As
phthalates are non-covalently bound to the plastic matrix, can migrate easily into surrounding
environment [2] and recognised as environmental contaminant owing to their association with an
array of health issues. Series of research findings suggest that in vitro and in vivo exposure to
phthalates are well known for their action as endocrine disruptor responsible for the increased
incidences of decline in sperm counts, hypospadias, undescended testes, shortened anogenital
distance (AGD), retained nipples, endometriosis, impaired, early pregnancy loss, precocious
puberty leading to defective reproductive phenotypes in animals and humans [3, 4, 5]. Recent
studies of Ma et al. (2015) [6] indicate ability of phthalates to induce toxicity even in nonreproductive tissues viz., hepatic and renal. Di-n-butyl phthalate (DBP) broadly used in
cosmetics and consumer products thereby exposing humans routinely, while daily exposure of
DBP in humans found to be 0.84-5.22µg/kg/day [7]. Experiments carried in gestational rats
indicated DBP was metabolised rapidly into their monoesters viz., monobutyl phthalate (MBP)
which profoundly induced developmental and reproductive toxic effects on male progeny [8];
finally eliminated out through milk (lactating mother) and urine. The elimination of metabolites
found in milk was higher than in urine according to Frederiksen et al. (2007) [9] studies wherein
infants are of a high risk than children and adults. Kim et al. (2009) [10] studies have shown
cause of hyperactivity and impulsivity in rodents, which was identical to attention
deficit/hyperactivity disorder (ADHD) occurred in children exposed to phthalates. In vitro
794 Mahaboob Basha P J..Biosci Tech,Vol 8(1),2017,794-800
ISSN: 0976-0172
Journal of Bioscience And Technology
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studies of Tetz et al. (2013) [11] indicated the human placental cell line treated with MEHP
resulted in oxidative stress leading to adverse pregnancy outcomes. This may be the result of
phthalate-induced increases in oxidative stress or inflammation in animal tissues; however,
Oxidative stress was interrelated with inflammation has been proposed to be part of the etiologic
pathway for DEHP-induced tumorigenesis [12]. Besides the potential effects of DBP on
reproductive functions, the endocrine disruptive actions of phthalates may alter neuronal
functions and bring histological alterations; however, the underlying mechanisms and its impact
on the cortical and cerebellar neuronal development remain poorly understood. Although few
findings documented neurotoxicity of DBP, data on mutigenerational effects is lacking thereby,
the study was conducted to explore the vulnerability of neuronal tissue by evaluating histological
aspects in cerebral cortex and cerebellum.
2. Materials and Methods
2.1. Chemicals
Di-n-butyl phthalate, purchased from Sigma-Aldrich Ltd. Standards and other chemicals (AR
grade) procured from Merck Ltd.
2.2. Animals
Albino rats of Wistar strain were obtained from Sri Raghavendra Enterprises, Bangalore, and
they were acclimatised for a week and maintained at 25 ± 2 °C and relative humidity of 50%
under a controlled 12-h light-dark cycle before the commencement of the experiment. Animals
were fed with standard rodent diet (Amrit Feeds, India) and water ad libitum throughout the
experiment. Design and protocol of the study were approved by the Institutional Animal Ethics
Committee, Bangalore University, Bangalore (CPCSEA No. 402, File No. 25/525/2009 dated
23.03.2011).
2.3. Experimental design and procedure
Twenty-four adult Wistar rats (18 females and 6 males weighing 200 - 250g) were housed in
cages in the ratio of 3 females: 1 male (3F:1M) for a week and examined daily for the vaginal
plugs to confirm pregnancy. Out of 18, only 12 females were confirmed pregnant and they were
further divided into two groups. Group-I consisting control rats (n=6) were administered olive oil
(2ml/kg BW) and experimental group-II (n=6) received DBP at a dose of 500mg/kg body weight
(BW)/day dissolved in 0.1ml olive oil through oral gavage from gestation day (GD) 6-21.
Pregnant rats were considered parental generation (F0) dams were housed individually in cages;
the pups born to them were labelled as the F1 generation. Dams and litter remained together until
weaning. F0 dams of the experimental group were continued receiving DBP exposure throughout
the lactation and control group with olive oil. Offsprings (F1) belonging to experimental group
received the same amount of DBP (500mg/kg BW) and control group with olive oil until the
termination. Pups, sexed on PND-21, were isolated from each group and housed together with
the same sex. One-month-old male rats (n=6) from each generation were euthanized by spinal
dislocation under 1% pentobarbital sodium (0.4ml/100g BW) anaesthesia, brain tissue was
excised quickly to separate cerebral cortex (CC) and cerebellum (CB) for the histopathological
observations.
795 Mahaboob Basha P J..Biosci Tech,Vol 8(1),2017,794-800
ISSN: 0976-0172
Journal of Bioscience And Technology
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On attaining maturity, 18 females and 6 males (weighing 200 - 250g) of F1 generation were
selected to continue the multigenerational reproduction. They were then kept for breeding in a
similar way as their parental generation (F0). Care was taken to avoid breeding between siblings
of each generation. Out of 18, only 14 females were confirmed pregnant. The pups obtained
from the F1 rats were considered as the second generation (F2). Pups of F2 generation were made
use for various studies as described above and left over were allowed to mature while continuing
DBP exposure. On maturity, these F2 rats (18 females: 6 males) were kept for breeding in a
similar manner as described above and 14 females were confirmed pregnant. The pups obtained
from F2 were considered as the third generation (F3). Control animals (F1, F2, and F3) were also
reared along with experimental groups to match the age group of each generation.
2.4. Rationale for dose selection
Toxicological studies interpret the response manifested in test animals to establish a safe dose.
The pilot study assessment was made to evaluate LD50 of DBP for Wistar rats and was found to
be 8012 mg/kg BW; thereby, a value of 1/16 LD50 (i.e., 500 mg/kg BW) was chosen for this
study, which was in accordance with the previous reports [13] and the chosen dose adversely
affected the reproductive development without maternal or systemic toxicity [14].
2.5. General observations
After birth, live and dead pups were counted to measure mortality rate; litter size of live pups
was also recorded. During the weaning period, pre-measured feed and water ad libitum to rats
and body weight was noted daily.
2.6. Organosomatic index of brain
Brain weight of control and intoxicated rats of all three generations on PND30 was recorded and
brain-somatic index was calculated using the formula,
OSI (brain) = (Weight of brain/Body weight)*100
3. Histopathology
Cerebral cortex and cerebellum were isolated immediately from the brain, fixed in 10% formalin,
later processed were done by fixation, dehydration, embedding, sectioning and finally stained
with hematoxylin and eosin. Images of stained tissue sections were captured with the aid of
Olympus microscope (CX-61) and Olympus camera (E-330) with the motorised stage at a
magnification of 40X.
4. Statistical analysis
The data was analyzed by one-way analysis of variance (ANOVA) followed by posthoc
Dunnett’s multiple comparison tests by using SPSS software (version 20.0). All the values were
expressed as means ± SE of six animals. A P value <0.05 was considered as statistically
significant.
796 Mahaboob Basha P J..Biosci Tech,Vol 8(1),2017,794-800
ISSN: 0976-0172
Journal of Bioscience And Technology
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Table 1:
Changes in the rate of feed and water consumption and body weight indices as a functional
consequence of DBP exposure in rats for three generations
Groups
Feed (g)
C
F1
14.3±0.2
F2
13.9±0.5
F3
14.2±0.2
E
11.5±0.1*
(-19.6)
10.2±0.3*
(-26.6)
9.3±0.2*
(-34.5)
Water (ml)
C
24.7±0.3
23.7±0.4
23.5±0.2
E
18.8±0.2*
(-23.9)
18.2±0.3*
(-23.2)
17.8±0.1*
(-24.3)
Body weight (g)
C
E
59.7±0.2*
62.5±0.4
(-4.5)
56.3±0.2*
61.7±0.8
(-8.8)
54.3±0.6*
63.6±0.5
(-14.6)
Values are represented as mean ± SE of six animals assessed for feed and water consumption on PND 30. Symbol
‘*’ indicates significantly different from the control, P < 0.05 as determined by one-way ANOVA followed by
posthoc Dunnett’s multiple comparison tests. Values in parenthesis represent the percent change and ‛-’ sign
indicates decrease over the control group. F1, F2 and F3 represents first, second and third generation. C-Control, EExperimental.
Figure 1
Changes in litter size at postnatal day (PND) 0 (a), mortality at PND 0 (b), and organosomatic index (OSI) of the
brain at PND 30 (c), in male Wistar rats upon DBP exposure for three generations. Values are mean ± SE of six
animals; symbol ‘*’ indicates significantly different from their corresponding control, P<0.05 as determined by oneway ANOVA followed by Dunnett’s multiple comparison test. F1, F2 and F3 represents first, second and third
generation. Litter size and OSI of brain was calculated using the formula,
Litter size at PND 0 = [Total no. of pups delivered (live and stillborn) / No. of dams delivered];
Mortality (%) = [(Total no. of dead pups)/ No. of live pups born] X100
OSI (brain) = (Weight of brain/Body weight)*100
797 Mahaboob Basha P J..Biosci Tech,Vol 8(1),2017,794-800
ISSN: 0976-0172
Journal of Bioscience And Technology
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Figure 2
Coronal sections of brain regions showing extent of cytoarchitectural alterations in cerebral cortex and cerebellum in
Wistar rats upon DBP exposure for three generations. a,g- F1 control, c,i- F1 intoxicated, e,k- F2 intoxicated and g,hF3 intoxicated. Impression (arrow): b - neuroglial cells with degenerative changes, d - focal aggregates of
inflammatory infiltration with decreased neurofibrillary tissue, f - focal aggregates of inflammatory infiltration, h, j,
l – degeneration of Purkinje cells (H& E staining, Scale bar - 100 µm).
5. Results
A considerable decrease in litter size was observed in DBP-treated groups (F1 –F3) (Figure 1a)
while the mortality rate (%) increased in experimental rats (Figure 1b) compared to their control
groups. Besides, the relative weight of brain (organosomatic index of the brain) showed a
gradual decrement over the generations which were exposed to DBP (Figure 1c). A significant
decrease in the feed and water intake (P<0.05) along with the decreased body weight (P<0.05)
was observed in DBP-treated groups (Table 1). Figure 2 indicates the histopathological
observations of CC with mild to severe focal aggregates of inflammatory infiltration in DBP
induced rats (F1- F3). Control rats showed cerebral cortex with intact architecture of parenchyma
while F1 rats showed degenerative changes in neuroglial cells; further, decreased neurofibrillary
tissue and focal aggregates of inflammatory infiltration was evident in F2 and severe focal
aggregates of inflammatory infiltration in F3 rats. While in CB region, remarkable degenerating
Purkinje cells were observed in DBP-treated rats.
798 Mahaboob Basha P J..Biosci Tech,Vol 8(1),2017,794-800
ISSN: 0976-0172
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6. Discussion
DBP being lipophilic in nature, gains entry through the placenta [15], and breast milk in
mammals over the generations. While DBP found to be environmental endocrine disruptor
(EED) acts as estrogenic or anti-androgenic which impedes the generation of gonadal hormones
found essential for the development of CNS during the critical period of development [16].
Markey et al. (2003) [17] findings specified EED target the developing nervous system. In this
study DBP exposure for three generations caused an impact on the prime tissues of brain by
showing alterations in the cytoarchitecture of CC and CB. Brain cortex was characterised by the
severity of focal aggregates of inflammatory infiltration upon DBP exposure while F1 rats
showed neuroglial cells with degenerative changes. While F2 and F3 rats had a focal aggregate of
inflammatory infiltration compared to the control rats with an intact architecture of brain
parenchyma. Furthermore, observations of Ma et al. (2015) [6] on mouse brain showed damage
to the pyramidal cells in the CA1 region characterised by loose and disordered arrangements of
cells, swelling deformations in cell shape, shortening or even disappearance of apical dendrites
and Nissl substance loss exposed to di-isononyl phthalate (DINP). Recent findings of Park et al.
(2015) [18] suggested that DEHP or DBP metabolites induced alterations in the neuronal tissue
subsequently leading to attention deficit disorders in children and observed negatively correlated
with the cortical thickness in the right middle and superior temporal gyri.
In the present investigation, a considerable decrease in body weight of rats upon exposure to
DBP attributed to decrease in feed and water consumption. Likewise, David et al. (2000) [19]
observed a significant decrease in body weight along with feed consumption upon DEHP
exposure at the high dose. Contrarily, findings of Salazar et al. (2004) [20] noticed no alterations
in litter size or body weight upon exposure of DBP at 50mg/kg BW, however mortality rate was
high in the third generation compared to the other two generations and control group. The
relative weight of brain and litter size also reduced profoundly in the third generation. Our
findings indicated a decrease in body weight, brain-somatic index, and litter size in F1, F2 and F3
rats primarily predict the plausibility of DBP in masking the metabolic activity thereby affected
the growth. It can be postulated from overall findings that DBP exposure affects the developing
neuronal tissues and cytoarchitectural alterations witnessed in cerebral cortex and cerebellum
found to be of oxidative stress origin and multigenerational exposures resulted higher extent
pathological changes within the CNS, causing heterogeneous injury and reactive alteration
within both axons and neuronal somata in some domains. Critical concern related to
neurotoxicity during the sensitive period of development has high impact in rats, also amortized
when extrapolated to the human population.
Acknowledgements
The authors thank University Grants Commission, New Delhi, India, for research grants
(F.No.41-31 / 2012 (SR) dated 10-07-2012).
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