exposure to environmental contaminants poses a significant threat to normal growth and differentiation of the developing brain .
it has also been reported that nervous system is susceptible to toxic chemicals and subsequent developmental perturbation may lead to long - term irreversible consequences that affect nervous system function in adult animals .
benzo[a]pyrene ( b[a]p ) , a polycyclic aromatic hydrocarbon ( pah ) , is known for its neurotoxic potential causing neurobehavioral alterations in animal models .
reports also suggest that exposure to b[a]p through early postnatal development leads to impairment in locomotor activity in adolescence .
a recent report revealed that subchronic oral administration of b[a]p in rats leads to spontaneous locomotor hyperactivity .
behavioral and motor dysfunction following exposure to environmental toxicants like b[a]p is well documented but the neurodegenerative potential of these compounds can not be ignored .
exposure to b[a]p produces neuromuscular , physiological , autonomic abnormalities and also shows decreased responsiveness to sensory stimuli [ 6 , 7 ] .
earlier reports suggest that microinjection of diesel exhaust fraction containing pah into rat hippocampus and striatum leads to neuronal lesions .
epidemiological study also revealed that parental occupational exposure to pah was associated with an increased risk of neuroectodermal tumors in children .
maternal exposure to airborne pahs during pregnancy was associated with a reduction in head circumference , lower iq , and reduced cognitive functioning in children .
investigation into pathophysiological manifestations following exposure to persistent anthropogenic neurotoxicants like benzo[a]pyrene might address new insight into their neurodegenerative potential .
studies have also addressed the effect of highly specific neurotoxin 6-hydroxydopamine ( 6-ohda ) on catecholaminergic neurons with an extensive and irreversible loss of dopaminergic neurons in the mesencephalon which is associated with behavioral deficits [ 1113 ] .
investigation of potential environmental neurotoxicants like b[a]p in animal model provides a new insight into serious human neurodegenerative diseases .
the purpose of the present study was to investigate the neurotoxic potential of b[a]p following intracisternal administration during early phase of postnatal development and its consequences in early adolescent period of rats .
we also investigated the neurotoxic effect of benzo[a]pyrene and its possible association with 6-hydroxydopamine induced neurobehavioral changes during early adolescence period .
the chemicals used in this experimentation were procured from sigma - aldrich chemicals ( st . louis , mo , usa ) , unless otherwise mentioned such as benzo[a]pyrene ( b[a]p , catalogue number b1760 ) , corn oil ( catalogue number c8267 ) , 6-hydroxydomine hydrochloride ( 6-ohda , catalogue number h4381 ) , and paraformaldehyde .
pregnant wistar rats ( rattus norvegicus ) were maintained in home cages in standard laboratory conditions and fed laboratory chow and filtered water ad libitum .
the animals were subjected to 12  hr light and 12  hr dark cycle in standard laboratory environment .
five - day - old male wistar pups were arbitrarily consigned to lactating dams and intracisternal administration of b[a]p and 6-ohda was carried out at postnatal day 5 .
the b[a]p solution was freshly prepared by dissolving 2  g / kg bw of b[a]p into 10  l of corn oil and was subsequently used for intracisternal administration while the control rats received only corn oil ( 10  l ) .
rats of 6-ohda group were injected with 25  g / kg , desipramine in 100  l ( i.p . ) , 30 minutes before 6-ohda injection .
then 150  g / kg bw of 6-ohda dissolved in 10  l of saline ( containing 0.2% ascorbic acid as antioxidant ) was injected into rats of 6-ohda group while rats of control group received 10  l saline solution , respectively .
five - day - old male wistar pups were designated into four groups ( 12 male pups / group ) , namely , control a ( corn oil ) ; b[a]p ( b[a]p dissolved in corn oil ) ; control b ( saline ) ; and 6-ohda ( 6-ohda dissolved in saline ) .
we aimed to study the neurodegenerative potential of b[a]p and its symptomatic similarities with 6-ohda induced neurobehavioral alteration , rather than address the confounding effect as a result of coadministration of these neurotoxicants .
sma of rats at pnd 30 was individually measured in a home cage with a supermex system ( muromachi kikai , ikeda , japan ) .
rats were maintained on a 12  h light : dark cycle during the recording and total distance covered for a period of 24  hr was recorded and the data was represented in 2  hr intervals as absolute time .
food and water were supplied at the beginning of recordings and rats were never disturbed in anyway .
open field activity was monitored in a circular arena ( 75  m diameter and 1.5  cm height ) wall with grey color paint .
the animals were placed individually in a specific side of the wall of the arena and the time spent in the central zone and total distance travelled were automatically monitored for 5  min using any maze software ( stoelting co. , usa ) .
briefly , elevated plus maze consisted of two opposite open arms ( 50  cm long  10  cm wide ) and two enclosed arms ( 50  10  40  cm ) that extended from a common central platform ( 10  10  cm ) , elevated 75  cm above the floor .
time spent in open arm and total distance travelled were recorded and considered as an indicator of general locomotor activity independent of anxiety . the video - tracking system ( any maze software , stoelting co. , usa )
the apparatus was cleaned with 70% ethanol and dried with paper towels before testing with another rodent .
after completion of behavioral recording , rats of all the experimental groups were sacrificed after being deeply anesthetized with sodium pentobarbital and subsequently perfused intracardially with ice - cold 0.1  m phosphate buffer saline ( pbs ) followed by 4% paraformaldehyde .
brain samples were cryoprotected in 30% sucrose in pbs for 48  hr and then serial cryosectioning was carried out for histopathological studies . for biochemical and molecular study ,
rats were sacrificed by cervical dislocation and the requisite brain region was immediately dissected out at 4c in ice - cold 0.1  m pbs and then snap frozen in liquid nitrogen .
quantification of striatal dopamine level was carried out by high performance liquid chromatography - electrochemical detector ( hplc - ecd ) with minor modification .
briefly , 100  mg wet brain tissue ( striatum ) was homogenized by sonification in 0.5  ml of 0.2  m hclo4 ( perchloric acid ) containing isoproterenol as an internal standard substance .
then the supernatant was added with 1  m sodium acetate to adjust the ph 3.0 .
after that 10  l of sample solution was injected into a separation column ( eicompak sc-5ods , i d 3.0  100  mm ) in a hplc - ecd system for measurement of dopamine and the result was expressed as ng / mg of protein .
brains were postfixed in 4% paraformaldehyde for 24  hr and then washed for 24  h under running tap water and dehydrated in graded series of ethanol concentrations ( 50% , 70% , 90% , and 100% ) .
then they were cleared in xylene , embedded in paraffin wax , serially sectioned at 7  mm thickness , and stained with hematoxylin and eosin ( h&e ) .
the slides were observed under microscope and images of striatal region were taken with requisite magnification with the help of a digital camera ( olympus , bx43f , japan ) .
the area and perimeter of the neurons were measured with the help of ocular micrometer .
the mean and standard error of mean of each set , that is , control a , b[a]p , control b , and 6-ohda , were calculated .
the post hoc analysis was done by the newman - keuls test in all experimental groups by using one - way anova .
bonferroni posttest was conducted to study the effect of b[a]p and 6-ohda on sma by using two - way anova and difference below the probability level p < 0.001 was considered statistically significant .
b[a]p administration to rat neonates at pnd 5 led to significant increase in spontaneous motor activity during 24  hr of recording ( f(3,528 ) = 380.8 , p <
similarly , intracisternal administration of 6-ohda to rats pups significantly increased the spontaneous motor activity ( f(3,528 ) = 380.8 , p < 0.001 ) with respect to control b and b[a]p treated rats ( figure 1 ) .
open field test showed significant increase in the stay time at center in b[a]p group as compared to control a ( f(3,44 ) = 11.71 , p < 0.05 ) ( figure 2(a ) ) .
however the total distance travelled in oft was significantly augmented following 6-ohda administration ( f(3,44 ) = 14.69 , p < 0.05 ) as compared to control b group ( figure 2(b ) ) .
similarly , there was a significant increase in total distance travelled in b[a]p treated group when compared to control a ( f(3,44 ) = 14.69 , p < 0.05 ) .
the results showed significant increase in total distance travelled in oft following 6-ohda administration ( f(3,44 ) = 14.69 , p < 0.05 ) which showed similar effects following b[a]p administration .
the analysis of the stay time in open arm following b[a]p administration showed significant increase when compared to the control a group ( f(3,44 ) = 84.96 , p < 0.05 ) whereas no significant difference was observed between 6-ohda and control b
the b[a]p and 6-ohda administration also showed significant increase in total distance travelled in epm ( f(3,44 ) = 28.23 , p < 0.05 ) as compared to control a and control b , respectively ( figure 3(b ) ) .
dopamine ( da ) level in striatum significantly ( f(3,20 ) = 14.79 , p < 0.05 ) declined in b[a]p treated group as compared to control a ( figure 4 ) .
comparable depletion of da was detected in striatum following intracisternal administration of b[a]p as compared to 6-ohda group ( f(3,20 ) = 14.79 , p < 0.05 ) ( figure 4 ) .
the histopathological examination of striatal region following h&e staining showed significant increase in abnormal striatal neuron morphology in b[a]p and 6-ohda treated groups ( figure 5(a ) ) .
the mean area ( f(3,20 ) = 7.985 , p < 0.05 ) and perimeter ( f(3,20 ) = 8.603 , p < 0.05 ) of striatal neurons were significantly decreased in b[a]p treated group as compared to control a and similar alleviation of mean area ( f(3,20 ) = 7.985 , p < 0.05 ) and perimeter ( f(3,20 ) = 8.603 , p < 0.05 ) of striatal neurons was also observed in 6-ohda group as compared to control b ( figures 5(b ) and 5(c ) ) .
however , there was a significant increase in pyknotic cell counts in 6-ohda group when compared with b[a]p .
polycyclic aromatic hydrocarbons ( pahs ) are predominantly anthropogenic in source and are liberated as a consequence of partial combustion carbon containing organic compounds [ 21 , 22 ] .
benzo[a]pyrene ( b[a]p ) is extensively used as a prototype of pah which demonstrates significant genotoxicity .
further , humans and rodents differ with respect to their sensitivity to varying doses of b[a]p and previous findings suggested that humans were 10100 times more sensitive to toxic effects of b[a]p than rodents .
additionally , infants and children are exceptionally susceptible to environmental neurotoxicants at levels far below those known to harm adults . as the early phase of postnatal life
is involved with rapid phase of growth and development , it becomes a target of exposure to environment neurotoxicants , the rapid growth period in rat neonates straddling the first ( 1 - 2 ) weeks of life to acquire numerous novel motor and sensory skills during the early stage of development .
b[a]p induced motor dysfunction leading to spontaneous motor hyperactivity in rodents addresses possible consequences of serious neurological problems .
several studies have been conducted to show spontaneous motor dysfunction and its association with neurodegenerative symptoms following administration of potent neurotoxicants [ 26 , 27 ] .
this neurodegenerative predisposition is potentially related to alleviated dopamine level at striatum . during early stage of postnatal development striatal dopaminergic neurons
regulate the storage and release of dopamine . as the blood - brain barrier is not fully developed in rat neonates , 6-ohda , a potent neurotoxicant , might adversely affect the brain development during early adolescence period .
hence , the present study was conducted at pnd 5 in male wistar pups following single intracisternal administration of b[a]p that showed significant increase in spontaneous motor activity at pnd 30 .
further investigation showed that neonatal 6-ohda administration leads to spontaneous motor hyperactivity in adolescent rats and the findings are in agreement with previous reports .
our results demonstrated that 6-ohda administration leads to significant increase in total distance travelled in both oft and epm and similar behavioral change was also found after b[a]p administration .
the motor dysfunction following b[a]p administration was found to be positively associated with the neurobehavioral manifestation induced by potent neurotoxicant , that is , 6-ohda , and the above findings are in agreement with previous reports [ 32 , 33 ] .
the behavioral observations as stay time in oft and epm significantly increased in the group to which b[a]p was administered as compared to control .
however , no significant difference was observed after 6-ohda administration , from which it can be inferred that intracisternal b[a]p administration has anxiolytic potential and the findings are in agreement with the previous report .
we extended our findings to ascertain the possible relationship of neurobehavioral manifestation induced by intracisternal administration of b[a]p with that of dopamine level in striatum of adolescent rats .
our results showed significant alleviation of striatal dopamine level following b[a]p administration and the result finds support from previous report . similarly
, 6-ohda administration leads to significant decrease in dopamine level as compared to b[a]p and the above findings are in agreement with previous report [ 34 , 35 ] .
the possible effect of neonatal b[a]p administration on striatal dopamine level during adolescence was found to be linked with the effect of 6-ohda .
the findings of striatal dopamine alleviation by b[a]p administration were significantly associated with altered histopathological observation .
our results demonstrated a significant decrease in area and perimeter of striatal neurons following b[a]p administration .
the above findings are comparable with the neurodegenerative symptoms exhibited by 6-ohda and our results are supported by previous report .
the possible neurodegenerative consequences following b[a]p administration at pnd 5 and its neurobehavioral manifestation in adolescent rats may be associated with reduced striatal dopamine level .
the basic findings of the present study indicate that 6-ohda produces alterations in behavior that are similar to those produced by b[a]p .
in conclusion , the findings suggest that early postnatal life is highly susceptible to environmental neurotoxicants leading to neurobehavioral perturbation in adolescence .
early exposure to potent environmental neurotoxicants like b[a]p may lead to reduction in dopamine neurotransmitter level that can cause striatal neurodegeneration .
we addressed a pioneer effect of b[a]p exposure during early postnatal life leading to neurodegenerative processes and its symptomatic similarities with progressive neurodegenerative diseases .
 exposure to persistent genotoxicants like benzo[a]pyrene ( b[a]p ) during postnatal days causes neurobehavioral changes in animal models . however , neurotoxic potential of b[a]p and its association with 6-hydroxydopamine ( 6-ohda ) induced neurobehavioral changes are yet to be explored . 
 the growth of rat brain peaks at the first week of birth and continues up to one month with the attainment of adolescence . 
 hence , the present study was conducted on male wistar rats at postnatal day 5 ( pnd 5 ) following single intracisternal administration of b[a]p to compare with neurobehavioral and neurotransmitter changes induced by 6-ohda at pnd 30 . 
 spontaneous motor activity was significantly increased by 6-ohda showing similar trend following b[a]p administration . 
 total distance travelled in novel open field arena and elevated plus maze was significantly increased following b[a]p and 6-ohda administration . 
 neurotransmitter estimation showed significant alleviation of dopamine in striatum following b[a]p and 6-ohda administration . 
 histopathological studies of striatum by hematoxylin and eosin ( h&e ) staining revealed the neurodegenerative potential of b[a]p and 6-ohda . 
 our results indicate that b[a]p - induced spontaneous motor hyperactivity in rats showed symptomatic similarities with 6-ohda . in conclusion 
 , early postnatal exposure to b[a]p in rats causing neurobehavioral changes may lead to serious neurodegenerative consequences during adolescence . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion