Effect of prenatal exposure of lindane on alterations in the expression of cerebral cytochrome P450s and neurotransmitter receptors in brain regions

Food and Chemical Toxicology 77 (2015) 74–81

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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x

Effect of prenatal exposure of lindane on alterations in the expression of cerebral cytochrome P450s and neurotransmitter receptors in brain regions Shikha Srivastava, Anshuman Singh, Rajendra K. Shukla, Vinay K. Khanna, Devendra Parmar * Developmental Toxicology Division, CSIR-Indian Institute of Toxicology Research, P.O. Box 80, M.G. Marg, Lucknow, U.P 226 001, India

A R T I C L E

I N F O

Article history: Received 18 June 2014 Accepted 13 December 2014 Available online 5 January 2015 Keywords: Lindane Rats Cytochrome P450s Neurotransmitter receptors Expression

A B S T R A C T

Prenatal exposure to low doses (0.0625- or 0.125- or 0.25 mg/kg b. wt., orally) of lindane, an organochlorine insecticide, from gestation day (GD) 5–21 was found to produce a dose-dependent increase in the mRNA expression of cytochrome P450s (CYPs) and associated transcription factors in frontal cortex, cerebellum and corpus striatum isolated from the offsprings. Though the increase in the expression persisted up to postnatal day 60, the increase was significant at postnatal days 21-, and 45- in the offsprings exposed prenatally to relatively higher doses (0.125- or 0.25 mg/kg) of lindane and even up to postnatal day 60 in the offsprings exposed prenatally to the highest dose of lindane. A similar increase in the expression of dopamine D2, 5HT2A and GABAA receptors and associated neurotransmitter receptor binding was observed in the brain regions of the exposed offsprings. Scatchard analysis also suggested an increase in the levels of these neurotransmitter receptors in offsprings prenatally exposed to lindane. The data indicating similarities in the alterations of neurotransmitter receptors and CYPs in brain regions in prenatally exposed offsprings have suggested that neurotransmission processes and CYPs are closely linked that will eventually help in understanding the developmental neurotoxicity of lindane. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Epidemiological studies have suggested that exposure of organochlorine pesticides during pregnancy is associated with poor cognitive, impaired motor functions and neurologic development and an increased risk of chronic diseases later in life (Siddiqui et al., 2003; Sagiv et al., 2007; Stillerman et al., 2008; Boucher et al., 2013). Likewise, exposure of polychlorinated biphenyls (PCBs), chlorinated hydrocarbons during pregnancy have been reported to influence birth weight, birth size of the infants and even reduce the gestation in humans (Kezios et al., 2012; Lignell et al., 2013; Lopez-Espinosa et al., 2011). Lindane (γ-hexachlorocyclohexane), an organochlorine pesticide, used in agriculture and public health, is known to persist in the environment (Alegria et al., 2006). It is thus likely that pregnant women and even children during early development could be exposed to lindane and other organochlorine pesticide. Lindane has been shown to accumulate in the fatty tissues and thus can be transferred through the placenta to the

* Corresponding author. Developmental Toxicology Division, CSIR-Indian Institute of Toxicology Research (IITR), P.O. Box 80, M. G. Marg, Lucknow, U.P 226001, India. Tel.: +91 522 2627586 Ext. 261; fax: +91 522 2628227, 2621547. E-mail address: [email protected] (D. Parmar). http://dx.doi.org/10.1016/j.fct.2014.12.010 0278-6915/© 2014 Elsevier Ltd. All rights reserved.

fetus and to the newborns through the mother’s milk (Albertson et al., 1985). Studies have suggested a link between exposure to lindane during pregnancy and fetal effects. Behavioral, neurochemical and even structural changes were reported in the brain isolated from developing rats treated with doses of lindane, which do not produce any effects in the adults (Rivera et al., 1998; Serrano et al., 1990). Lindane has been reported to induce the release of neurotransmitters by increasing the synaptosomal internal content of Ca+2 (Rivera et al., 1990). Though γ-amino butyric acid (GABA) receptor ionophore complex is the primary target of the pesticide, it has been shown that lindane causes neurobehavioral toxicity by several mechanisms, such as altering neurotransmitter levels and influencing the expression of cytochrome P450s (CYPs), involved in its metabolism and toxicity (Anand et al., 1998; Parmar et al., 2003a, 2003b; Rivera et al., 1998). Toxicokinetics of lindane play an important role in its neurobehavioral toxicity with DBA/2 mice, that lacks Ah receptor, reported to be more vulnerable to the convulsant effects of lindane (Liu and Morgan, 1986). The developing rats were found to be particularly sensitive to the neurotoxicity of the pesticide, with subconvulsant doses of lindane inducing an imbalance in the central monoaminergic systems (Rivera et al., 1998). As the CYPs are not fully developed in the fetus and developing animals (Moscovitz and Aleksunes, 2013), any exposure of lindane

S. Srivastava et al./Food and Chemical Toxicology 77 (2015) 74–81

occurring during gestation may not be detoxified readily resulting in the behavioral effects in the offsprings. Studies from our laboratory have shown that prenatal exposure to low doses of lindane alters the ontogeny of CYPs, involved in the neurobehavioral toxicity of lindane (Parmar et al., 2003a), in liver and brain of the offsprings at birth. The increase in the expression of CYPs in brain and liver of the offsprings was found to persist during postnatal development and were found to be associated with the neurobehavioral effects in the prenatally exposed offsprings (Johri et al., 2007, 2008a, 2008b). Interestingly, CYPs have been reported to be closely associated with the various endogenous functions in the brain (Miksys and Tyndale, 2004; Parmar et al., 2003a, 2003b). The dopamine transporters have been shown to exhibit overlapping substrate specificity with the CYP2E1 (Wójcikowski et al., 2008). The role of CYP2D6 isoenzymes has been demonstrated in the conversion of tyramine to dopamine (Bromek et al., 2011). Ligands that interact with the GABAA receptor are also reported to increase the activity of CYP1A1, 2B1/ 2B2 and 3A1 isoenzymes in liver (Parmar et al., 2003b; Roberge et al., 2004). As animal studies have shown high vulnerability of the fetus to the neurotoxic effects of pesticides, the present study was initiated to investigate the effect of prenatal exposure to low doses of lindane on the expression of CYPs in brain regions, that catalyze specific neurotransmission processes, during postnatal development. Attempts were also made to study the prenatal effect of these doses on the expression of neurotransmitter activity in offsprings during postnatal development and to correlate these alterations in the neurotransmitter receptors with the cerebral CYPs. 2. Materials and methods 2.1. Chemicals Lindane-technical grade was procured from Sigma-Aldrich, St. Louis, MO, U.S.A. Trizol reagent was obtained from Life Technologies, USA. The reagents for RT-PCR have been procured from ABI Biosystems U.S.A. All other chemicals used were of the highest purity commercially available and procured either from BDH (a subsidiary of E. Merck, India) or SISCO Research Laboratories Pvt. Ltd. (India). Phenobarbital sodium salt (PB) was a gift from Biodeal Laboratories (India). 2.2. Animals and treatment Adult male (~12 weeks old) and female (~10 weeks old) Wistar rats of proven fertility were obtained from the Animal House facility of CSIR-Indian Institute of Toxicology Research (IITR), Lucknow. All the animals were maintained on a commercial pellet diet and water ad libitum in a temperature controlled room with a 12/12-h light/dark cycle and cared for in accordance to the policy laid down by Animal Care Committee of IITR, Lucknow. The animal experimentation was approved by the Ethical Committee of the Institute. Fifty-seven (57) female rats were allowed to mate with 19 adult males (3:1). On day 0 of pregnancy (confirmed by a positive vaginal smear), the pregnant rats (54 numbers) were randomly divided into 3 batches (one batch each for studying effects on expression of CYPs, neurotransmitter receptors and receptor binding) of four groups each. Animals in groups 1, 2 and 3 in each batch received 0.0625- or 0.125- or 0.25 mg/kg b. wt. of lindane, orally from gestation day 5 (GD5) to GD21. Animals in group 4 served as control and received corn oil in an identical manner. On the day of parturition, the average litter size was adjusted to eight per dam in all the groups with equal number of males and females as far as possible. The male offspring born to the control and treated dams were sacrificed on postnatal age of 21, 45 and 60 days. Brains were immediately removed and dissected into regions (cerebellum, frontal cortex and corpus striatum) and processed for isolation of total RNA and membrane preparation for receptor binding. For isolating RNA, brain region tissues were snap frozen in liquid nitrogen and stored at −80 °C. The tissues were processed for isolation of RNA with Trizol (Life Technologies, USA) using the manufacturer’s protocol.

75

Table 1 Ligands and competitors used in receptor binding studies. Receptor

Brain regions

Radioligands

Competitors

Dopamine (DAD2)

Corpus striatum

3

Serotonin (5HT2A)

Frontal cortex

GABAA

Cerebellum

Haloperidol (1 × 10−6 M) Cinenserin (1 × 10−6 M) GABA (1 × 10−6 M)

H-Spiperone (1 × 10−9 M) 3H-Ketanserin (1 × 10−9 M) 3 H- Muscimol (1 × 10−9 M)

et al., 2006; Li et al., 2006; Ruddell et al., 2006). Likewise, RT-PCR for transcription factors such as aryl hydrocarbon receptor (AhR), aryl hydrocarbon nuclear transporter (Arnt), constitutive androstane receptor (CAR) and pregnane X-receptor (PXR) was carried out using primers and conditions as described earlier (Qin and Meng, 2006; Shirota et al., 2006; Vondracek et al., 2006; Wyde et al., 2005). 2.4. Receptor binding of dopamine-D2 (DAD2), serotonin-2A (5HT2A) and GABAA receptors in brain regions Assay of DAD2 receptors in corpus striatum, 5HT2A receptor in frontal cortex and GABAA receptor in cerebellum was carried out by the radioligand binding assays following the standard procedure (Khanna et al., 1994). Briefly, crude synaptic membrane was prepared by homogenizing the brain regions in 19 volumes of Tris–HCl buffer (5 mM, pH 7.4) following centrifugation at 40,000 × g for 15 min at 4 °C. The sedimented pellet was washed twice and recentrifuged and pellet suspended in Tris– HCl buffer (40 mM, pH 7.4) for the binding assays. Protein concentration in membrane preparations were measured following the method of Lowry et al. (1951) using bovine serum albumin (BSA) as a reference standard. For receptor binding assays the details of radioligands, competitors and their conditions for the assay of specific receptors are mentioned in Table 1. In brief, the reaction mixture containing Tris–HCl buffer (40 mM, pH 7.4), together with membrane protein (300–400 μg) and appropriate radioligand were incubated for 15 min at 37 °C in the presence or absence of competitor to assess the non-specific and total binding respectively. The contents of the binding tubes were immediately filtered on glass fiber discs (25 mm diameter, 0.3 μ pore size, Whatman GF/B). Filteration on the glass fiber discs was carried out using Manifold Filteration assembly (Millipore, U.S.A.) under vacuum, washed twice and filters were dried and transferred into vials containing scintillation fluid. The scintillation cocktail used for radioactive counting was the mixture of 1, 4-bis (5-Phenyloxazole-2-y) benzene (POPOP), 2,5-diphenyloxazole (PPO), napthalene, toulene, methanol and 1–4-dioxane. The radioactivity was counted on scintillation counter (Packard, USA) at an efficiency of 30–40% for 3H to determine the membrane bound radioactivity. Although the efficiency varies from instrument to instrument, 30–40% efficiency is appropriate to count the radioactivity. Specific binding was determined by subtracting the non-specific binding from the total binding and has been expressed as pmoles ligand bound/g protein. Scatchard analysis was carried out at varying concentrations of radioligands (generally 1/10 to 10 times of the affinity) to ascertain whether change in the binding is due to alteration in the affinity (Kd) or number of receptor binding sites (Bmax). Scatchard analysis was carried out at different concentrations of radioligands (normally 1/10 to 10 times of the affinity of radioligand). Linear regression analysis using GraphPad Prism ver 3.0 was carried out to determine the affinity (Kd) and maximum number of receptor binding sites (Bmax). R value was close to 0.9. 2.5. Statistical analysis All values are presented as mean ± SEM. Main effects of dose and duration of exposure of lindane on expression of individual CYP isoforms, their associated transcription factors and neurotransmitter receptors were ascertained using student’s t-test to calculate the statistical significance between control and treated groups. p < 0.05 was considered to be significant when compared with the controls.

3. Results 3.1. Effect of prenatal exposure of lindane on mRNA expression of CYP isoforms in the different brain regions

2.3. Real time PCR (RT-PCR) analysis qRT-PCR for the different CYP isoenzymes was carried out as described earlier (Shah et al., 2009). The sequences of primers used for CYP1A1, CYP1A2, CYP2B1, CYP2B2, CYP2E1, CYP3A1, CYP2D1 and GAPDH have been described earlier (Baldwin et al., 2006; Yamaguchi et al., 2005). The threshold cycle value (Ct Values) of each sample was normalized with Ct values of endogenous control (GAPDH). Fold Change was calculated from ΔΔCt value of each sample, which was derived from ΔCt of treated − ΔCt of control. Similarly, RT-PCR for neuroreceptors such as GABAA, DAD2 and 5HT2A was carried out using primers and conditions as described earlier (Andoh

Quantitative Real Time PCR (qRT-PCR) revealed that prenatal exposure of different doses of lindane increased the levels of CYP1A-, 2B-, 2D1, 2E1 and 3A1 isoenzymes and associated transcription factors in the brain regions of the offsprings during postnatal development (Figs. 1 and 2). Prior to relative quantification of CYPs, each sample was normalized with housekeeping gene (GAPDH), which served as an endogenous control. The expression of GAPDH

76

S. Srivastava et al./Food and Chemical Toxicology 77 (2015) 74–81

3

*

2.5

*

2 1.5

*

*

*

*

*

*

*

*

*

*

*

*

1

Fold change

Fold change

2.5

3

*

* 1.5

**

*

0

0

21 45 60 Days

21 45 60 Days

CYP1A 1 *

2.5

*

* *

* *

*

*

*

*

*

*

* *

*

1

Fold change

Fold change

*

*

* *

*

*

21 45 60 Days

21 45 60 Days

3

2 1.5

*

*

CYP1A2

3 2.5

*

1 0.5

21 45 60 Days

*

2

0.5

21 45 60 Days

*

*

* 1.5

*

*

*

*

* *

* *

*

* *

1 0.5

0

0

Cerebellum

*

2

0.5

21 45 60 Days

*

21 45 60 Days

21 45 60 Days

21 45 60 Days

21 45 60 Days

21 45 60 Days

Frontal cortex

Corpus striatum

Cerebellum

Frontal cortex

Corpus striatum

CYP2B1

CY P2B2

Fig. 1. Effect of prenatal exposure of different doses of lindane on mRNA expression of CYP1A- and 2B- isoenzymes in brain regions of rat offsprings at postnatal day 21- or 45- or 60. Fold change is calculated from ΔΔCt# value of each sample (#ΔΔCt = ΔCt of treated − ΔCt of control). *p < 0.05 when compared with the controls.

was found to be uniform in all the samples (control and treated) analyzed, confirming the integrity of RNA used in assays. Relative quantification data revealed a dose dependent increase in the levels of CYP1A-, 2B-, 2D1, 2E1 and 3A1 isoenzymes in cerebellum, frontal cortex and corpus striatum isolated from prenatally exposed offsprings at postnatal day 21 when compared to the controls. This increase was found to be statistically significant in the offsprings raised on mothers treated with higher doses of lindane (0.125- or 0.25 mg/kg). No significant increase was observed in the mRNA expression of any of the CYP isoenzymes in any of the brain regions isolated from offsprings exposed to the lowest dose (0.0625 mg/kg) of lindane (Figs. 1 and 2). Further, the increase in the expression of CYP isoenzymes was found to persist up to adulthood (60 days). Though the magnitude of increase declined with postnatal development, the increase in the mRNA expression of CYPs was found to be significant even at postnatal day 60 in the brain regions isolated from the offsprings prenatally exposed to the highest dose (0.25 mg/kg) of lindane (Figs. 1 and 2). Differences were also observed in the induction of CYP isoenzymes in different brain regions isolated from prenatally exposed offsprings. While CYP1A-, 2B- and 2D1 isoenzymes exhibited maximum increase in cerebellum followed by frontal cortex and

corpus striatum, CYP2E1 was increased maximally in frontal cortex followed by corpus striatum and cerebellum isolated from prenatally exposed offsprings during postnatal development. Likewise, CYP3A1 was found to be induced maximally in corpus striatum followed by cerebellum and frontal cortex isolated from prenatally exposed offsprings (Figs. 1 and 2). As observed with CYPs, differences were also observed in the mRNA expression of transcription factors (AhR, Arnt, CAR and PXR) in the different brain regions isolated from the prenatally exposed offsprings (Fig. 3). The increase in the mRNA expression of AhR, Arnt, CAR and PXR was found to be statistically significant in the offsprings raised on mothers exposed to the higher doses (0.125- or 0.25 mg/kg) of lindane, while no significant increase in the mRNA expression of AhR, Arnt, CAR and PXR was observed in the offsprings exposed prenatally to the lowest dose (0.25 mg/kg) of lindane (Fig. 3). The magnitude of increase declined with postnatal development, though the increase observed in the mRNA expression of AhR, Arnt, CAR and PXR was found to be statistically significant even at postnatal day 60 in the brain regions isolated from offsprings exposed prenatally to the highest dose (0.25 mg/kg) of lindane (Fig. 3). The data also revealed differences in the induction of mRNA of these transcription factors in different brain regions. While AhR,

S. Srivastava et al./Food and Chemical Toxicology 77 (2015) 74–81

2.5

* *

1.5

*

*

*

*

* *

*

*

*

*

*

1 0.5

Fold change

Fold change

2

*

0

21 45 60 Days Cerebellum

21 45 60 Days Frontal cortex

2 18 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

21 45 60 Days Corpus striatum

* *

77

* *

*

* *

*

21 45 60 Days Cerebellum

*

21 45 60 Days Frontal cortex

CY P 2 E 1

* *

21 45 60 Days Corpus striatum

C Y P3 A 1 3

Fold change

2.5

*

*

2

* 1.5

* *

*

*

* *

1

* *

* *

*

0.5 0

21 45 60 Days

Cerebellum

21 45 60 Days

Frontal cortex

21 45 60 Days

Corpus striatum

CYP2D1 Fig. 2. Effect of prenatal exposure of different doses of lindane on CYP2E1, 2D1 and 3A1 mRNA in brain regions of rat offsprings at postnatal day 21- or 45- or 60. Fold change is calculated from ΔΔCt# value of each sample (#ΔΔCt = ΔCt of treated − ΔCt of control). *p < 0.05 when compared with the controls.

Arnt and CAR exhibited maximum increase in the cerebellum followed by frontal cortex and corpus striatum isolated from the prenatally exposed offsprings, maximum increase in the PXR mRNA was observed in corpus striatum followed by cerebellum and frontal cortex (Fig. 3). 3.2. Effect of prenatal exposure of lindane on mRNA expression of neurotransmitter receptors in different brain regions qRT-PCR study in brain regions isolated from prenatally exposed offsprings revealed alterations in DAD2, 5HT2A and GABAA receptors in corpus striatum, frontal cortex and cerebellum respectively isolated from the offsprings during postnatal development. Prior to calculating the relative values of expression of neuroreceptors, each sample was normalized with GAPDH. The relative quantification data revealed a dose dependent increase in the mRNA expression of DAD2, 5HT2A and GABAA in corpus striatum, frontal cortex and cerebellum respectively isolated from prenatally exposed offsprings postnatally at 21 days when compared to the respective control. This increase in the mRNA expression of these neurotransmitter receptors was found to be statistically significant in the offsprings raised on mothers treated with relatively higher doses (0.125- or 0.25 mg/kg) of lindane (Table 2). As evident from the table, maximum

increase in the mRNA expression of DAD2, 5HT2A and GABAA receptors was observed postnatally at 21 days and the magnitude of increase then declined during postnatal development at 45 and 60 days in corpus striatum, frontal cortex and cerebellum respectively. Though the increase in mRNA expression of neurotransmitter receptors declined with development, the increase in the DAD2, 5HT2A and GABAA mRNA was found to be statistically significant postnatally at 45 days in the offsprings exposed prenatally to higher doses (0.125- or 0.25 mg/kg) of lindane. Even though the increase in mRNA expression of these neurotransmitter receptors persisted postnatally up to 60 days, the increase was not found to be statistically significant at this time point at any of the doses (Table 2). 3.3. Receptor binding analysis in prenatally exposed offsprings Exposure of pregnant rats to lindane (0.25 mg/kg/body weight) from GD5 to GD21 caused an increase in the binding of 3H-spiperone to corpus striatal membranes, 3H-ketanserine to frontocortical membrane and 3H-muscimol to cerebellar membranes isolated from the brain of the offsprings when compared to the membranes isolated from offsprings nursing on control mothers (Table 3). As evident from the table, maximum increase in the binding of 3H-spiperone (30.43%), 3 H-ketanserine (63.76%) and 3H-muscimol (38.12%) was observed

78

S. Srivastava et al./Food and Chemical Toxicology 77 (2015) 74–81

Fold change

1.4

1.6

* *

* *

*

* *

*

1.2

* *

*

1.4

**

*

1 0.8 0.6 0.4

Fold change

1.8 1.6

**

1.2

**

*

* *

*

*

0.8 0.6 0.4

0

21 45 60 Days

21 45 60 Days

21 45 60 Days

21 45 60 Days

AhR 1.6

*

21 45 60 Days

1.6

* *

*

*

**

* *

* * **

*

1.4

*

1 0.8 0.6 0.4

1.2

*

*

*

* *

*

*

0.4

0

F r o nt a l co r t e x

*

0.8

0.2

C er eb e ll u m

*

0.6

0

21 45 60 Days

* *

1

0.2

21 45 60 Days

21 45 60 Days

A rnt

Fold change

Fold change

*

1

0

1.2

*

0.2

0.2

1.4

**

21 45 60 Days

21 45 60 Days

Corpus striatum

Cerebellum

CAR

21 45 60 Days

21 45 60 Days

F ro nt al co r tex

Corpus striatum

PXR

Fig. 3. Effect of prenatal exposure of different doses of lindane on mRNA expression of transcription factors in brain regions of rat offsprings at postnatal day 21- or 45- or 60. Fold change is calculated from ΔΔCt# value of each sample (#ΔΔCt = ΔCt of treated − ΔCt of control). *p < 0.05 when compared with the controls.

Table 2 Relative quantification of neurotransmitter receptors in brain regions of prenatally exposed offsprings during postnatal development by qRT- PCR.

in the membranes prepared from brain regions of the exposed offsprings postnatally at 21 days. This increase in the binding of ligands specific for dopaminergic, serotonergic and GABAergic receptors was found to be statistically significant in the offsprings prenatally exposed to higher doses (0.125- or 0.25 mg/kg) of lindane, while

DAD2 Days

Control

0.0625 mg

0.125 mg

0.25 mg

21 45 60

1 ± 0.08 1 ± 0.06 1 ± 0.09

1.68 ± 0.07* 1.51 ± 0.06* 1.22 ± 0.04

1.82 ± 0.07* 1.61 ± 0.05* 1.24 ± 0.09

21 45 60

1 ± 0.06 1 ± 0.05 1 ± 0.05

1.65 ± 0.05* 1.25 ± 0.04* 1.16 ± 0.11

1.75 ± 0.06* 1.34 ± 0.05* 1.22 ± 0.04

21 45 60

1 ± 0.05 1 ± 0.06 1 ± 0.05

1.24 ± 0.05 1.12 ± 0.11 1.03 ± 0.09 5HT2A 1.25 ± 0.05* 1.09 ± 0.09 1.03 ± 0.08 GABAAα1 1.18 ± 0.11 1.10 ± 0.09 1.06 ± 0.09

1.26 ± 0.05* 1.19 ± 0.12 1.10 ± 0.10

1.31 ± 0.05* 1.25 ± 0.05* 1.19 ± 0.04

All the values are mean ± S.E. of 3 animals. The threshold cycle value (Ct values) of each sample was normalized with Ct values of endogenous control (GAPDH) [ΔCt]. Fold change is calculated from ΔΔCt# value of each sample (#ΔΔCt = ΔCt of treated − ΔCt of control). * p < 0.05 when compared with the controls.

Table 3 Receptor binding analysis in offsprings prenatally exposed to lindane (0.25 mg/kg) during postnatal development. Receptor

Days

Control

Treated

DAD2

21 45 60 21 45 60 21 45 60

318.8 ± 13.1 434.8 ± 27.5 450.3 ± 8.0 59.6 ± 8.8 68.9 ± 3.8 93.0 ± 6.5 130.1 ± 10.9 132 ± 15.1 129 ± 2.5

415.8 ± 15.4* 482.9 ± 5.2 496.9 ± 14.8 97.6 ± 10.3* 102.0 ± 13.3* 112.2 ± 13.1 179.7 ± 4.3* 148.4 ± 9.0 133 ± 6.0

5HT2A

GABAA

All the values are mean ± S.E. of 5 animals each. * p < 0.05 is considered as significant from control and treatment. All the values are expressed in pmoles ligand bound/g protein.

S. Srivastava et al./Food and Chemical Toxicology 77 (2015) 74–81

Table 4 Determination of binding characteristics by Scatchard analysis. Receptors Dopamine (DAD2) Serotonin (5HT2A) GABAA

Kd Bmax. Kd Bmax. Kd Bmax.

Control

Treated

2.0 ± 0.16 912.2 ± 56 2.99 ± 0.19 285.4 ± 20 2.02 ± 0.11 176.8 ± 14

1.21 ± 0.12* 1633 ± 134* 1.29 ± 0.16* 435.2 ± 27* 1.24 ± 0.19 203.9 ± 20

Values are mean ± SEM of three observations in each group. Kd – dissociation constant expressed in nM. Bmax – maximum number of binding sites expressed in pmoles 3H-spiperone (DAD2), 3H-ketanserin (5HT2A) and 3H-muscimol (GABA ) bound/g protein. A Data have been analyzed by one-way analysis of variance followed by t-test. * p < 0.05 when compared with the controls.

no significant change was observed in the offsprings prenatally exposed to the lowest dose (0.0625 mg/kg) of lindane. Though the increase in the binding of 3H-spiperone, 3H-ketanserine and 3Hmuscimol persisted up to adulthood, the magnitude of increase declined with postnatal development. The increase observed in the neurotransmitter receptor binding postnatally at 60 days was not found to be statistically significant at any of the doses (Table 3). Scatchard analysis revealed that the increase in binding of 3Hspiperone to striatal membrane, 3H-ketanserin to frontocortical membranes and 3H-muscimol to cerebellar membranes was due to increased number of receptor binding sites (Bmax). This increase in the number of receptor binding sites was found to be associated with a decrease in the Kd (Table 4). 4. Discussion Consistent with our previous reports (Johri et al., 2007, 2008a, 2008b), the present study has now provided evidence that prenatal exposure to low doses of lindane induces a dose dependent increase in the expression of xenobiotic metabolizing CYP1A-, 2B-, 2E1, 2D1, 3A1 isoenzymes in brain regions such as cerebellum, frontal cortex and corpus striatum, that catalyze specific neurotransmission pathways, during postnatal development. Dose dependent increase in mRNA expression of the transcription factors such as AhR, Arnt, CAR and PXR may account for an increase in the expression of CYPs isoenzymes observed in the whole brain reported earlier (Johri et al., 2007) or brain regions isolated from exposed offsprings during postnatal development in the present study. Though the prenatal exposure of these low doses of lindane used in the present study may result in very low levels of lindane being transferred through the placenta, the increase in the expression of CYPs in different brain regions may, nevertheless, help in explaining the distribution and accumulation of lindane and its metabolites in different brain regions. The present study indicating persistence in alterations in the expression of cerebral CYPs, particularly CYP1A, 2B and 2E1 isoenzymes in the prenatally exposed offspring during postnatal development, is of significance as these CYPs are involved in the metabolism of lindane and pesticide induced convulsions and mortality in rodents (Parmar et al., 2003a). Previous studies from our laboratory have attributed this persistence in increase in the expression of CYPs in brain and liver isolated from offsprings to the potential of lindane to imprint these CYPs because of its endocrine disrupting activity (Johri et al., 2007, 2008a, 2008b). Recent studies from our laboratory have shown that prenatal exposure of cypermethrin, a synthetic type II pyrethroid imprints the expression of CYPs in brain and liver of prenatally exposed rat offsprings (Singh et al., 2013). The persistence in alterations in the expression of CYPs in different brain regions is important in view of the endogenous role of CYPs in brain. It is reported that ligands that interact with GABAA receptors also induce the expression of CYP2B1/2B2 and CYP3A

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isoenzymes (Roberge et al., 2004). Likewise, persistent increase in the expression of CYP2D1 and 3A1 during postnatal development in the different brain regions is of significance as these CYPs catalyze the metabolism of endogenous substrates such as neurotransmitters and neuroactive steroids (Miksys et al., 2000). A close association between dopaminergic system and CYP2E1 has been suggested with brain dopaminergic system acting as a physiological center that maybe involved in regulating the levels of CYPs in the liver (Konstandi et al., 2008; Wójcikowski et al., 2008). As CYP2E1 is located near or in the same compartment as stored DA in the substantia nigra, the persistence in increase in the expression of CYP2E1, involved in the generation of free radicals, may modulate dopaminergic neurotransmission by increasing the generation of ROS (Caro and Cederbaum, 2004; Gonzalez, 2007). Considering the high levels of polyunsaturated fatty acids (PUFA) and low levels of antioxidant enzymes in brain (Somani et al., 1996), significant increase in the CYP2E1 in different brain regions, particularly in corpus striatum, during postnatal development in prenatally exposed offspring further provides support to the previous studies indicating the sensitivity of brain regions of these offspring to oxidative stress and its possible role in lindane induced developmental neurotoxicity (Johri et al., 2007). That the alterations in the expression of CYPs and neurotransmitters receptors are closely associated events was demonstrated by a dose dependent increase in the mRNA expression of DAD2, 5HT2A and GABAA receptors in specific brain regions isolated from offsprings exposed prenatally to different doses of lindane at postnatal day 21 and persisting up to adulthood. A dose dependent increase in the binding as well as increase in the number of receptor binding sites (Bmax) and decrease in the Kd for ligands specific to these receptors have shown that the increase in their mRNA expression may lead to the increase in levels of these neurotransmitter receptors in offsprings prenatally exposed to lindane. Though the alterations in dopaminergic, serotonergic and GABAergic systems have been observed after exposure of lindane, these effects have been reported at doses, several fold higher than that used in the present study (Artigas et al., 1988; Rivera et al., 1991). At higher doses, lindane is reported to induce convulsions and seizures in rodents (Sunol et al., 1988, 1989). Previous studies have shown that lindane interacts with GABAergic receptors and block GABA-induced chloride channel influx in both insect and mammalian cells (Ogata et al., 1988; Vale et al., 2003; Wafford et al., 1989; Zhao et al., 2003). Similar inhibition of GABAA receptors has been reported after exposure of other organochlorine pesticides like dieldrin and dichlorodiphenyltrichloroethane (DDT) (Louis et al., 2006). Lindane has also been shown to induce differential effects on dopamine levels and augment the synthesis and release of 5HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), leading to the alterations in brain lipid peroxidation. These alterations have been further shown to be associated with the high anxiety states and convulsions reported in lindane treated rats (Bist and Bhatt, 2009; Rivera et al., 1991, 1998). As GABA can selectively regulate development of neurons of different neurotransmitter phenotypes, as well as developmental expression of its own receptors, in utero exposure to lindane could produce imbalances in monoaminergic versus GABAergic neurotransmission in the developing brain (Lauder et al., 1998). In conclusion, the results of the present study have shown that prenatal exposure to low doses of lindane induces significant dose dependent increase in the expression of xenobiotic metabolizing CYPs, involved in lindane-induced convulsions, in brain regions isolated from offsprings during postnatal development. Interestingly, these alterations and persistence in alterations in CYPs in brain regions were found to be associated with a similar increase in the expression of neurotransmitter receptors in the prenatally exposed offsprings during postnatal development. The persistence in these effects is of significance as the levels used in the study are often encountered in the environment and in view of the previous reports

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demonstrating birth defects, reduced gestation period in the offsprings born to mothers exposed to organochlorine during pregnancy (Lopez-Espinosa et al., 2011). Conflict of interest The authors declare that they do not have conflict of interests and, therefore, they have nothing to disclosure. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements The authors are grateful to the Director, CSIR-Indian Institute of Toxicology Research, Lucknow, for his keen interest and support in carrying out the study. Mr. AS is grateful to CSIR, New Delhi for providing Senior Research Fellowship. RKS is grateful to ICMR, N. Delhi for providing Senior Research Fellowship. The financial support of CSIR Network project entitled INDEPTH (BSC0111) in carrying out the above studies is gratefully acknowledged. The technical assistance of Mr. B. S. Pandey is also gratefully acknowledged. CSIRIITR communication number 3269. References Albertson, T.E., Joy, R.M., Stark, L.G., 1985. Facilitation of kindling in adult rats following neonatal exposure to lindane. Brain Res. 17, 263–266. Alegria, H., Bidleman, T.F., Figueroa, M.S., 2006. Organochlorine pesticides in the ambient air of Chiapas, Mexico. Environ. Pollut. 140 (3), 483–491. Anand, M., Agrawal, A.K., Rehmani, B.N., Gupta, G.S., Rana, M.D., Seth, P.K., 1998. Role of GABA receptor complex in low dose lindane (HCH) induced neurotoxicity: neurobehavioral, neurochemical and electrophysiological studies. Drug Chem. Toxicol. 21, 35–46. Andoh, H., Yoshikawa, M., Kawaguchi, M., Matsumoto, H., Yamazaki, K., Oka, T., 2006. The selective action of D2 dopamine receptor antisense oligodeoxynucleotide on the expression of the dopamine receptor subtype mRNA in rat striatum. Tokai J. Exp. Clin. Med. 31, 73–77. Artigas, F., Martinez, E., Camon, L., Rodriguez-Farre, E., 1988. Synthesis and utilization of neurotransmitters: actions of subconvulsant doses of hexachlorocyclohexane isomers on brain monoamines. Toxicology 49, 49–55. Baldwin, S.J., Bramhall, J.L., Ashby, C.A., Yue, L., Murdock, P.R., Hood, S.R., et al., 2006. Cytochrome P450 gene induction in rats ex vivo assessed by quantitative realtime reverse transcriptase-polymerase chain reaction (TaqMan). Drug Metab. Dispos. 34, 1063–1069. Bist, R., Bhatt, D.K., 2009. The evaluation of effect of alpha-lipoic acid and vitamin E on the lipid peroxidation, gamma-amino butyric acid and serotonin level in the brain of mice (Mus musculus) acutely intoxicated with lindane. J. Neurol. Sci. 276 (1–2), 99–102. Boucher, O., Simard, M.N., Muckle, G., Rouget, F., Kadhel, P., et al., 2013. Exposure to an organochlorine pesticide (chlordecone) and development of 18-month-old infants. Neurotoxicology 35, 162–168. Bromek, E., Haduch, A., Gołembiowska, K., Daniel, W.A., 2011. Cytochrome P450 mediates dopamine formation in the brain in vivo. J. Neurochem. 118 (5), 806–815. Caro, A.A., Cederbaum, A.I., 2004. Oxidative stress, toxicology, and pharmacology of CYP2E1. Annu. Rev. Pharmacol. Toxicol. 44, 27–42. Gonzalez, F.J., 2007. The 2006 Bernard B. Brodie Award Lecture. CYP2E1. Drug Metab. Dispos. 35, 1–8. Johri, A., Yadav, S., Dhawan, A., Parmar, D., 2007. Over expression of cerebral and hepatic cytochrome P450s alters behavioral activity of rat offspring following prenatal exposure to lindane. Toxicol. Appl. Pharmacol. 225 (3), 278–292. Johri, A., Dhawan, A., Singh, R.L., Parmar, D., 2008a. Persistence in alterations in the ontogeny of cerebral and hepatic cytochrome P450s following prenatal exposure to low doses of lindane. Toxicol. Sci. 101 (2), 331–340. Johri, A., Yadav, S., Dhawan, A., Parmar, D., 2008b. Responsiveness of cerebral and hepatic cytochrome P450s in rat offspring prenatally exposed to lindane. Toxicol. Appl. Pharmacol. 231, 10–16. Kezios, K.L., Liu, X., Cirillio, P.M., Kalantzi, O.I., Wang, Y., Petreas, M.X., et al., 2012. Prenatal polychlorinated biphenyl exposure is associated with decreased gestational length but not birth weight: archived samples from the Child Health and Development Studies pregnancy cohort. Environ. Health 11, 49. Khanna, V.K., Husain, R., Seth, P.K., 1994. Effect of protein malnutrition on the neurobehavioral toxicity of styrene in young rats. J. Appl. Toxicol. 14, 351–356.

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