CHAPTER II

Evaluation of the effect of antineoplastic

drug and herbicide on the HPA axis of mice

IIA: Effect of cyclophosphamide on the HPA axis of mice

Cyclophosphamide (CPA), a synthetic antineoplastic drug is used widely to

treat various types of cancer as well as an immunosuppressant in organ

transplantation, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis

and other benign diseases. It binds covalently to DNA and induces DNA damage

(Aguilar-Mahecha et al. 2005). CPA after metabolism produces two active

metabolites, phosphoramide mustard and acrolein and these metabolites are bi-

functional alkylating in nature. The cytotoxic effect of CPA is mediated by alkylation

of DNA at the N7 position of guanine and the formation of DNA-DNA cross-links,

DNA-protein crosslinks and single-strand breaks (Hemminki and Kallama, 1986;

Crook et al. 1986).

CPA treatment is associated with biochemical and histological alterations in

the testes and epididymis of rodents and humans. It also causes oligospermia,

azoospermia, reduced spermatogenesis in males (Aguilar-Mahecha et al. 2005) and

reduced no. of follicles in female (Montz et al. 1991). In addition to above effects, this

treatment is also reported to disrupt normal physiological functions, upsets body

metabolism, digestive functions and mood. It has been reported that chronic low dose

administration of CPA can decrease the weight of reproductive organs (Das et al.

2002), impair male fertility (Trasler et al. 1986), increase the post implantation loss

and fetal malformation (Higuchi et al. 1995) in rats. CPA induced germ cell toxicity is

mediated via generation of reactive oxygen species (Selvakumar et al. 2006; Sikka,

2001).

Inspite of wide use of CPA for the treatment of various pathological conditions

including cancer and as a immunosuppressant drug, its adverse or side effects are not
studied in detail except on the reproductive system. To understand its adversity on the

body physiology it was thought worthwhile to investigate whether CPA contributes to

stress responses in mice. Hence present experiment was designed to study the effect

of CPA on the HPA axis i.e. stress responses of mouse.

Experimental Design

Male laboratory mice weighing 252 grams were divided in 2 groups (n=12 in

each group). i) Control, received normal saline, ii) CPA, received cyclophosphamide

(Endoxan-N, Zydus Oncosciences, India) i.p.100 mg/kg body weight once a week for

5 weeks. At the end of treatment, mice of both the groups were weighed and

sacrificed by decapitation. Serum was collected for measuring the serum

corticosterone level. Brains of 4 mice in each group were dissected out; hypothalamus

was separated, processed for extraction of total RNA for reverse transcriptase-PCR of

AVP, CRH, nNOS, NF-B and -actin. The PCR products were processed for

electrophoresis on 2% agarose gel and the gels were photographed by a digital camera

(Olympus). The densitometric analysis of bands was done by ImageJ software. For

the RT PCR, band intensity of AVP, CRH, nNOS and NF-B was measured and

normalized with actin to be expressed as % relative expression.

Brains of other 4 mice were fixed in 4% PFA for immunohistochemistry/

immunofluorescence after the whole body perfusion and those of remaining 4 mice

were homogenised in chilled PBS (0.02 M, pH 7.4) for the biochemical estimation of

nitrate-nitrite (indicating nitric oxide) and SOD and catalase (antioxidant enzymes)

activity. The antioxidant enzyme activity, nitric oxide estimation, RT-PCR of various

genes (AVP, CRH, nNOS, NF-B and -actin), immunohistochemistry (AVP and

CRH), immunofluorescence (nNOS and Hsp70) and ELISA (serum corticosterone)

were performed using the methods described earlier in the General Materials and

Methods section. The photographs of brain IHC and immunofluorescent slides were

taken from Carl Zeiss Axioskop 2 Plus microscope.

The experiment was conducted in accordance with Institutional practice and

within the framework of the revised Animals (scientific procedures) Act of 2002 of

the Govt. of India on Animal Welfare.

Statistical analysis

All the data were analysed by Students t test for the comparison of treated

group from control. Difference was considered significant at the level of P<0.05.

Results

Effect of CPA on the antioxidant enzyme activity in the hypothalamus

Antioxidant enzyme superoxide dismutase (SOD) activity decreased

significantly (Fig. IIA.1A); however catalase activity increased (Fig. IIA.1B) in the

hypothalamus of CPA treated mice compared to their respective control.

Effect of CPA on the nitric oxide level in the hypothalamus

In the hypothalamus, NO was measured biochemically in the form of

nitrate/nitrite level which was not changed in the hypothalamus of CPA treated mice

as compared to control (Fig. IIA.1C).

Effect of CPA on the expression of hypothalamic AVP mRNA

RT-PCR results showed the increased expression of hypothalamic AVP mRNA

in CPA treated mice as compared to control (Fig. IIA.2).

Effect of CPA on the expression of immunoreactive AVP in the PVN and SON
 Immunohistochemical localization of ir-AVP showed decreased expression of

AVP in the PVN and SON of CPA treated mice as compared to control (Fig. IIA.3A

and IIA.3B).

Effect of CPA on the expression of hypothalamic CRH mRNA

RT-PCR results showed increased expression of hypothalamic CRH mRNA in

CPA treated mice as compared to control (Fig. IIA.4).

Effect of CPA on the expression of immunoreactive CRH

Immunohistochemistry of ir-CRH showed increased expression of CRH in the

PVN of CPA treated mice compared to control (Fig. IIA.5).

Effect of CPA on the expression of hypothalamic nNOS mRNA

The expression of hypothalamic nNOS mRNA remained unchanged after the

treatment of CPA compared to control shown by RT-PCR result (Fig IIA.6).

Effect of CPA on the expression of immunoreactive nNOS

Immunoreactivity of nNOS in CPA treated mice was unaltered compared to

control mice as shown by the immunofluorescent staining of nNOS in PVN region of

hypothalamus (Fig. IIA.7).

Effect of CPA on the expression of hypothalamic NF-B mRNA

After the treatment of CPA, expression of NF-B mRNA in the hypothalamus

increased significantly as compared to control mice (Fig. IIA.8).

Effect of CPA on the expression of immunoreactive Hsp70 in the PVN

The expression of ir-Hsp70 remained unchanged in the PVN of CPA treated

mice compared to control (Fig. IIA.9). Although no. of immunopositive neurons

appear to decrease but the decrease was not statistically significant.

Effect of CPA on serum corticosterone

CPA treatment caused significant increase in serum corticosterone level as

compared to control mice (Fig. IIA.10).

Discussion

Present study demonstrated that antineoplastic drug treatment with CPA

resulted in a significant decrease in SOD activity however, increase in catalase

activity. Hypothalamic AVP and CRH mRNA as well as ir-CRH peptide in the PVN

and serum corticosterone level increased significantly, although, ir-AVP decreased in

both PVN and the SON of CPA treated mice. On the other hand, NF-B mRNA was

upregulated after the CPA treatment but no change was observed in the expression of

hypothalamic nNOS and Hsp70 in CPA treated mice.

The PVN in the hypothalamus is an integrative site that regulates stress

induced neuroendocrine systems such as activation of the HPA axis (Sawchenko,

1991). Activation of the HPA axis is controlled by a set of parvocellular neurons

located in the PVN of the hypothalamus. Upon the stimulation by stress or circadian

drive, these neurons release neural factors, such as CRH and AVP, into the

hypophyseal portal circulation. These factors then travel to the anterior pituitary and

cause release of ACTH into the systemic circulation which in turn causes synthesis

and secretion of glucocorticoids by the adrenal cortex. Once released, glucocorticoids

are able to bind high-affinity mineralocorticoid receptors (MR) or lower-affinity

glucocorticoid receptors (GR), which function as ligand-gated transcription factors to

positively or negatively regulate gene expression (de Kloet et al. 1998).

Variable stressor paradigm produces a syndrome consistent with chronic

stress, including hypersecretion of corticosterone, ACTH and prolactin, adrenal

hypertrophy and increased AVP and CRH mRNA expression in the PVN (Herman et

al. 1995). AVP coexists with CRH in approximately 50% of parvocellular CRH -

positive axons and terminalis in rats (Whitnall et al. 1985, 1987). AVP and CRH act

synergistically to induce the release of ACTH (Rivier and Vale, 1983). Osmotic stress

causes an increase in plasma AVP level as well as AVP mRNA in the PVN of rats and

water and food deprivation cause increase in plasma corticosterone level in rats (Kiss

et al. 1994). Zimmermann and colleagues (2004) reported an increase in peripheral

plasma AVP with psychosocial stress as well. Psychological stress activates the

parvocellular neurons to secrete AVP which induces ACTH release into the

hypophyseal portal vein system (Herman, 1995).

Secretion of AVP is stimulated by different types of stress and/or physiological

conditions such as pain (Kendler et al. 1978), sexual arousal (Murphy et al. 1987) and

surgery (Moran et al. 1964) etc. Previous reports suggest that AVP mRNA and

hnRNA increased in the PVN of the chronically stressed rats (Ma et al. 1997, 1999;

Makino et al. 1995; Pinnock and Herbert, 2001). Subcutaneous injection of formalin

in rats causes the increase in AVP hnRNA in the PVN and serum AVP level (Kurose et

al. 2001). Recently, Yadawa and Chaturvedi (2016) reported the activation of HPA

axis of mice including AVP expression following water and food deprivation of 2 and

4 days (For detail see chapter IA). In the present study also, weekly treatment of CPA

over a period of 5 weeks in mice, causes an increase in AVP mRNA expression in

hypothalamus but a decrease in the ir-AVP peptide in the PVN and SON. A concurrent

increase in CRH mRNA as well as ir-CRH peptide and serum corticosterone level

suggests the overall activation of HPA axis of mice after the treatment of CPA. The

observed decrease in ir-AVP in the PVN and SON could be explained by increased
demand of stress peptide AVP in the system to cope up with the CPA induced

metabolic stress. Similar findings have been noted in avian system where long term

water deprivation (4 days) although upregulates arginine vasotocin (AVT) gene

expression but ir-AVT peptide decreased (Chaturvedi et al. 1994). These authors

suggested that although gene expression of AVT is upregulated the gene product (AVT

peptide) is released immediately into the system on increased demand hence

immunosignaling is not evident resulting into decreased no. and amount of ir- AVT

peptide. Present finding (increased AVP mRNA but decreased ir-AVP) may be also

explained on the same ground. Further all other variables (CRH, corticosterone)

strongly support the activation of HPA axis and AVP is a major player of this

phenomenon in CPA treated mice. This differential response of AVP and CRH

(specially ir-AVP and CRH) may be also explained on the basis of the fact that

corticosterone differentially modulates AVP and CRH expression in rats (Pinnock and

Herbert, 2001).

Stress is a potent activator of CRH release from the hypothalamus and

extrahypothalamic sites as well (Habib et al. 2000). After CPA administration, CRH

mRNA level increased significantly in the central amygdala as well as the PVN of

mice (Nishii et al. 2007) and in the PVN of rats (Mineta et al. 2004). Being a regulator

for the activation of HPA axis (Pacak et al. 1995), increased expression of CRH

mRNA in the hypothalamus of CPA treated mice may contribute to activation of HPA

axis. CRH plays a major physiological role in regulating the HPA axis to stressors

(Aguilera et al. 2001; Sawchenko, 2000). The expression of CRH and of its receptors

in hypothalamus, amygdala and hippocampus is age-dependent and is modulated by

stress throughout life (Korosi and Baram, 2008). CRH involves in various stress-

related human pathologies, e.g. depression, Cushing's syndrome, alcoholism, anorexia

nervosa and Alzheimer's disease (Von Bardeleben and Holsboer, 1990). In our study

also we noted an increase in expression of CRH mRNA and CRH peptide which

denote the activation of HPA axis of CPA treated mice.

It has been reported that immobilization stress caused upregulation of

expression of nNOS gene in the PVN (Calza et al. 1993; Kishimoto et al. 1996; de

Oliveira et al. 2000) and an increase of NOS activity in the PVN, anterior pituitary

and adrenal gland of rats (Kishimoto et al. 1996; Turnbull and Rivier, 1996). Although

CPA treatment in the present study did not alter nNOS expression in mice, but Ota et

al. (1993) have reported that NO can act centrally to stimulate AVP activity.

Many exogenous and endogenous agents that represent a threat to the

organism are capable of inducing NF-B activity (reviewed in Baldwin, 1996) such as

viral infection, bacterial lipids, parasites, UV irradiation, shear stress,

chemotherapeutic agents, oxidative stress, proinflammatory cytokines and DNA

damage. Similar to these reports, CPA induces increased NF-B mRNA expression in

CPA treated mice and supports NF-B as a reliable marker of various types of stresses

and hence an indicator for the activation of HPA axis. Hsp70, another stress protein,

was not modulated by the CPA treatment in male mice.

Glucocorticoids are the final effectors of the HPA axis and participate in the

control of whole body homeostasis and the organisms response to stress.

Glucocorticoids modulate the stress response at the molecular level by altering the

gene expression, transcription and translation among other pathways (OConnor et al.

2000).

In general, present study indicates that the expression of AVP, CRH and NF-

B mRNA was upregulated in the hypothalamus of CPA treated mice. AVP peptide
was decreased while CRH peptide was increased in the PVN. Serum corticosterone

level was elevated in CPA treated mice. On the other hand, the expression of both

nNOS mRNA and its peptide did not change in CPA treated group compared to

control. From these findings we can conclude that antineoplastic drug

cyclophosphamide induces the activation of HPA axis via modulation of AVP and

CRH expression. This study also suggests that AVP and CRH act synergistically to

activate the HPA axis supporting the additional role of antidiuretic AVP as a potent

stress hormone not only during the environmental stress (see chapter I) but during

treatment with antineoplastic drug like CPA. The activation of HPA axis during drug

treatment supports and suggests the metabolic side effects of life saving

drugs/treatments.
IIB: Effect of paraquat on the HPA axis of mice

Paraquat (PQ, 1-1 dimethyl 4-4 bipyridylium dichloride), together with

diquat and difenzoquat, belongs to the group of bipyridylium herbicides. This

quaternary ammonium herbicide has been in use since 1960s and is among the most

commonly used herbicides worldwide. It is a quick-acting, nonselective, contact,

broad spectrum herbicide which is mainly used in railways and roadsides to prevent

growth of broad leaf weeds and grasses. It destroys tissues of green plants upon

contact with and by translocation within the plant (Saenz et al. 1997), but does not

harm mature bark. PQ can be rapidly absorbed by inhalation and through the intestine

after ingestion. Absorption after oral intake is about 10% (EC, 2003). Absorption

through intact skin is generally low, but is substantially increased if the skin is

damaged, and has also led to death in humans (UNEP, 2006). After oral intake, there

is high initial concentration in the liver and kidneys, which then reduces. Plasma

concentration is relatively stable for 30 hrs and concentration in the lungs increases. It

is actively concentrated in the lungs (UNEP, 2006). Low levels of paraquat may be
retained in muscle tissue after skin exposure and slowly released into the blood (Lee

et al. 2008).

It is a highly toxic compound for humans and animals (Suntres, 2002). Severe

PQ poisoning is characterized by multiple organ failure, involving mainly the lung,

kidney and liver. Short-term contact leads to irritation of the skin and delays in the

recovery of cuts and wounds on the skin. PQ induced oxidative stress impairs insulin-

dependent mTOR activation and this impairment probably causes inhibition of

insulin-dependent repression of IGFBP-1 expression (Kimura et al. 2010). Paraquat

can also cause endocrine disruption. It decreases testosterone in male rats, inhibits the

production of testosterone in the testis and 17-beta-oestradiol in the ovary of the frog

Rana esculenta (Quassinti et al. 2009). Haley (1979) reported that, when injected into

fertile hen eggs, paraquat caused pseudofeminization of male chick and quail

embryos. There is some evidence of its effects on the immune system and it may also

be implicated in type II diabetes (Watts, 2011, 2012).

Positively charged, PQ is actively transported across the blood-brain barrier by

the same neutral amino acid transporter used by L-valine and L-dopa (Manning-Bog

et al. 2003; Shimizu et al. 2001). Inside neurons, PQ induces oxidative stress by

producing superoxide anions through redox cycling (McCormack et al. 2002).

Paraquat causes extensive damage to the mitochondria of cells through the production

of free radicals and oxidative stress, resulting in the interruption of important

biochemical processes, cell death, and multi-organ failure (Suntres, 2002;

Mohammadi-Bardbori and Ghazi-Khansari, 2008; Cocheme and Murphy, 2009).

Effects have been measured in rats in the mitochondria of brain cells (Castello et al.

2007), in brain neurons (Yang and Tiffany, 2007; Zaidi et al. 2009) and in the
hippocampus of mouse brain (Chen et al. 2010), in blood, liver, lung and kidney cells

(Ray et al. 2007).

In spite of the reported adverse effect of PQ on the body system, effect of PQ

on stress responses is not yet known. Considering this lacuna, present study was

designed to investigate the effect of PQ on the HPA axis of mice.

MATERIALS AND METHODS

Male laboratory mice weighing 252 grams were divided in 2 groups (n=12 in

each group). i) Control received normal saline ii) paraquat (PQ) treated received10

mg/kg body weight PQ (Sigma Aldrich, Germany, Cat No. 36541) i.p. once a week

for 5 weeks. 24 hrs after the last injection, mice of both the groups were weighed and

sacrificed by decapitation. Serum was collected for measuring the serum

corticosterone level. Brains of mice in each group were dissected out. The

hypothalamus of 4 mice were separated and homogenised in chilled PBS (0.02 M, pH

7.4) for the biochemical estimation of nitric oxide and antioxidant enzyme activity.

Hypothalamus of other 4 mice were separated, processed for extraction of total RNA

for reverse transcriptase-PCR of AVP, CRH, nNOS, NF-B and -actin. RT-PCR

products were processed for electrophoresis on 2% agarose gel and gels were

photographed by a digital camera (Olympus) and the densitometric analysis of bands

was done by ImageJ software. For the RT PCR, band intensity of AVP, CRH, nNOS

and NF-B was measured and normalized by actin and expressed as % relative

expression. Brains of remaining 4 mice were fixed in 4% PFA for

immunohistochemistry or immunofluorescence after the whole body perfusion. The

photographs of brain IHC and immunofluorescent slides were taken from Carl Zeiss

Axioskop 2 Plus microscope.

 Biochemical estimation of antioxidant enzyme (SOD and catalase) activity

and nitric oxide level, RT-PCR of AVP, CRH, nNOS, NF-B and -actin,

immunohistochemistry of AVP and CRH, immunofluorescence of nNOS and Hsp70,

and ELISA of serum corticosterone were performed using the methods described

earlier in the General Materials and Methods section.

The experiment was conducted in accordance with Institutional practice and

within the framework of the revised Animals (scientific procedures) Act of 2002 of

the Govt. of India on Animal Welfare.

Statistical analysis

All the data were analysed by Students t test for the comparison of treated

group from control. Difference was considered significant at the level of P<0.05.

RESULTS

Effect of PQ on the antioxidant enzyme activity in the hypothalamus

Antioxidant enzyme - SOD activity was significantly decreased while catalase

activity was increased in the hypothalamus of PQ treated mice compared to control

(Fig. IIB.1A, B).

Effect of PQ on the nitric oxide level in the hypothalamus

Nitric oxide level measured in the form of nitrate-nitrite increased in the

hypothalamus of PQ treated mice compared to control (Fig. IIB.1C).

Effect of PQ on the expression of AVP mRNA in the hypothalamus

RT-PCR result shows that expression of AVP mRNA increased in the

hypothalamus of PQ treated mice compared to control (Fig. IIB.2).

Effect of PQ on the expression of ir-AVP in the PVN

Immunohistochemical localization of AVP shows that no. of immunopositive

neurons of AVP increased in the PVN of PQ treated mice. However, although

immunoreactivity was increased in few neurons, some exhibited depletion of

immunoreactivity compared to control (Fig. IIB.3).

Effect of PQ on the expression of CRH mRNA in the hypothalamus

The expression of CRH mRNA was upregulated in the hypothalamus of PQ

treated mice compared to control mice (Fig. IIB.4).

Effect of PQ on the expression of ir-CRH in the PVN

Immunoreactivity of CRH as well as no. of ir-neurons increased significantly

in the PVN of PQ treated mice compared to control (Fig. IIB.5).

Effect of PQ on the expression of nNOS mRNA in the hypothalamus

The expression of nNOS mRNA was checked by RT-PCR and result shows

that expression of nNOS mRNA in the hypothalamus of PQ treated mice was not

different from the control (Fig. IIB.6).

Effect of PQ on the expression of ir-nNOS in the PVN

nNOS was immunolocalised in the PVN by immunofluorescent staining. Its

immunoreactivity as well as the no. of Immunopositive neurons increased in the PVN

of PQ treated mice compared to control (Fig. IIB.7).

Effect of PQ on the expression of NF-B mRNA in the hypothalamus

 The expression of NF-B mRNA was upregulated in the hypothalamus of PQ

treated mice compared to control (Fig. IIB.8).

Effect of PQ on the expression of ir-Hsp70 in the PVN

Immunofluorescent staining shows that immunostaining of Hsp70 as well as

the no. of Immunopositive neurons increased in the PVN of PQ treated mice

compared to control. (Fig. IIB.9).

Effect of PQ on the serum corticosterone level

Serum corticosterone level was elevated significantly in PQ treated mice

compared to control (Fig. IIB.10).

DISCUSSION

This experiment was undertaken to investigate the effect of paraquat, a widely

used herbicide, on the HPA axis of the adult male mice. Alteration in the antioxidant

enzyme activity indicates the induction of oxidative stress in PQ treated mice which is

supported by previous reports of other workers (Day and Crapo, 1996; Xi and Chen,

1997). Further this herbicide treatment is also reported to enhance nitrate-nitrite level

supporting the induction of nitrosative stress as well. Upregulation of hypothalamic

stress hormone (AVP, CRH) and NF-B transcripts as well the increased expression of

ir-AVP, CRH, nNOS and Hsp70 peptide in the PVN neurons of hypothalamus

supports the activation of normal components of HPA axis in PQ treated mice.

It is reported that PQ has toxic effects in the central nervous system.

Following systemic administration in rats, paraquat penetrates the bloodbrain barrier

causing neurotoxic effects and brain damage (Corasaniti et al., 1998; Shimizu et al.,

2001). Toxic damage to the brain has also been observed in humans who died from
paraquat poisoning (Grant et al. 1980; Hughes, 1988). These reported toxic and/or

fatal effects of PQ appear to be due to increased dose and/or its direct consumption.

PQ provokes other neuropathological and behavioural features such as

Parkinsons disease, nigrostriatal dopaminergic dysfunction coupled with a

neurobehavioural syndrome and neuroinflammatory/oxidative changes (Brooks et al.

1999; Chen et al. 2008; Litteljohn et al. 2011) which may be assigned to low intensity

of PQ toxicity. To the best of our knowledge, there is lack of study reporting the effect

of PQ on the HPA axis mainly in terms of AVP and CRH. There are few reports

suggesting the role of AVP in the stress (Tsigos and Chrousos, 1994; Phillips, 1987;

Holmes et al. 1986) and many reports on the modulation of CRH in stress but no

effect of PQ has been tested simultaneously on both AVP and/or CRH. The data of our

present study suggests that chronic PQ treatment may cause stress in the male mice

because in these mice in addition to AVP, CRH and corticosterone level i.e. HPA axis

has been activated. Increase in both the AVP peptide in the PVN and mRNA in the

hypothalamus of PQ treated mice may be caused by the oxidative stress which

provoked the PVN neurons to synthesise and release AVP hormone. CRH peptide as

well as mRNA was upregulated after the PQ exposure in this study. Increased

oxidative stress in PQ treated mice may be responsible for the increased expression of

CRH peptide as well as mRNA because CRH is reported to be involved in protection

of cell damage by oxidative stress (Lezoualch et al. 2000). The other reason of

enhanced CRH may be increased NO level in the hypothalamus which influences the

secretion of many endocrine hormones like CRH (Costa et al. 1993; Karanth et al.

1993). NO also mediates the neuroendocrine function in HPA axis (Nelson et al.

1997) and also drive the upregulation of AVP in PQ treated mice supported by reports

that NO influences the secretion of AVP (Ota et al. 1993; Kadowaki et al. 1994).
 CRH regulates both basal and stress induced release of pituitary corticotropin

(ACTH) and was considered as the major constituent of the HPA system (Vale et al.

1981) until the role of AVP was also established as hypothalamic hormone which also

stimulated pituitary corticotrophs. CRH is also required for adrenal response to

different stressors (Jacobson et al. 2000) including fasting (Jeong et al. 2004; Yadawa

and Chaturvedi, 2016) although such role of AVP is not investigated in detail.

Considering the role of CRH and AVP as well in the stress responses, from the data of

present study it might be concluded that PQ treatment leads to stress in mice

involving both the hypothalamic AVP and CRH component of HPA axis.

Corticosterone is the final effector hormone of the HPA axis. In this experiment serum

corticosterone level was significantly increased in PQ treated mice. A previous study

in dogs also reported the elevation in the circulating cortisol after PQ treatment (Giri

et al. 1983) which supports our finding. In the rodents, corticosterone involves in the

regulation of energy, immune reactions and stress responses (Charmandari et al. 2005;

Sapolsky et al. 2000) including regulatory role in stress-induced HPA axis activity in

rodents (Osterlund and Spencer, 2011).

Nitric oxide (NO) generation has been previously described following

paraquat (PQ) exposure both in vivo and in vitro (Ahmad et al. 2008; Djukic et al.

2007). In this experiment ir-nNOS expression in the PVN and NO level in the

hypothalamus increased following PQ treatment in the mice. Moran et al. (2010)

reported that after 250 M PQ exposure, N27 cells show constitutive transcription of

nNOS and PQ did not affect the level of nNOS peptide and mRNA over 24 h period

but dose and experimental models were different in this and present study. The

production of NO and superoxide has been also implicated in neuronal injury after

ischemia, trauma, and numerous neurodegenerative disorders, such as Parkinsons

disease (Ebadi and Sharma 2003) and later disorder is also reported to be associated

with PQ (Litteljohn et al. 2011).

Viral infection, bacterial lipids, parasites, UV irradiation, shear stress,

chemotherapeutic agents, oxidative stress and pro-inflammatory cytokines are some

agents which are capable of inducing NF-B (reviewed in Mercurio and Manning,

1999). Since the expression of NF-B mRNA, NO and ir-nNOS increased

significantly in PQ treated mice along with the modulation of antioxidant enzymes, it

is suggested that oxidative and nitrosative stress might be responsible for the

upregulation of NF-B mRNA in the hypothalamus. It is reported that PQ toxicity

regulates NF-B (Gupta et al. 2010) which appears to have potent effects upon CNS

processes important for neuronal survival and plasticity. The transcription factor may

have a neuroprotective role through the induction of antiapoptotic proteins, such as

Bcl-2 and the antioxidant enzyme, manganese superoxide dismutase (Mattson, 2005).

The over-expression or induction of Hsps has also been shown to confer

resistance towards a variety of insults including oxidative stress (Donati et al. 1990).

Heat shock proteins such as Hsp27 and Hsp70 in particular, have been shown to

protect cells against such environmental and physiological stressors as oxidants

(Gorman et al. 1999). In the present study also Hsp70 expression enhanced in the

PVN of PQ treated mice. It is suggested that PQ treatment provoked oxidative stress

and this oxidative load might be responsible for the induced expression of Hsp70 (a

marker of stress/ toxicity) in the PVN of PQ treated mice. It is also quite possible that

Hsp70 might be involved in the protection of brain cells against the toxicity caused by

chronic PQ treatment.
 The data obtained from the present study i.e. increased expression/ level of

AVP, CRH and serum corticosterone suggests the activation of HPA axis of mice

following chronic treatment with PQ. It is concluded that increased oxidative and

nitrosative stress in these mice might be the driver which lead to the activation of

HPA axis. It seems that stress induced reactive species (ROS, RNS) might be also

responsible for the induced expression of NF-B and Hsp70 (the reliable markers of

certain stresses) in PQ treated mice with special reference to neurohormones/

hypothalamic peptides, the important components of HPA axis.