Introduction

A foreign body in the form of a chemical stress like insecticide or physical stress like cold or their interaction is sufficient enough to give a jolt either mild or severe in functioning potential of central nervous system (CNS). The CNS is especially sensitive to free radical oxidative damage as it contain post mitotic cells, high levels of iron, easily oxidizable fatty acids, low antioxidant defence system and use large amount of oxygen which renders the tissue susceptible to oxygen radicals [1]. Moreover, the heterogeneity of developing nervous system, with different cell types and function makes it vulnerable to environmental contaminants than the adult nervous system [2].

Indiscriminate use of pesticide for crop protection and for the control of vector borne disease(s) caused widespread harmful effects in human and other non-target biosystem. Chlorpyrifos (O,O1-diethyl O-3,5,6-trichloro-2-pyridinyl phosphorothioate, CPF) is a synthetic chlorinated OP insecticide utilized extensively in agriculture and for residential pest control throughout the world under the registered trademarks {LORSBAN Insecticide and DURSBAN Insecticide}. Despite the restrictions imposed on its use, CPF continues to be one of the most commonly used and extensively studied OP insecticides. Although, the mechanism of CPF toxicity involves acetylcholinesterase (AChE) inhibition, other mechanisms unrelated to AChE inhibition, including the induction of oxidative stress, have been implicated [3]. As a lipophilic molecule, CPF easily passes through the cells into the cytoplasm [4] and induce damage to the cellular molecules [5]. Oxidative modifications of proteins in vivo may affect a variety of cellular functions involving receptors, signal transduction mechanisms, transport systems and enzymes [6]. Reactive oxygen species (ROS = free radicals) can directly affect the conformation and/or activities of all sulfhydryl containing molecules, by oxidation of their thiol moiety [7]. The combined effect of these ROS-triggered cellular changes may eventually lead to cellular dysfunction and ultimate destruction.

Cold stress, a physical environmental stressor, cause detrimental effect(s) on organism by altering cellular homeostasis and plays a considerable role either in accelerating or modifying the toxic mechanisms. Exposure to extreme cold stress can lead to wind-chill, frostbite, and hypothermia [8] and many other harmful effects [9]. Cold stress can disrupt the balance in an oxidant/antioxidant system and cause oxidative damage to several tissues by altering antioxidant status, protein oxidation and lipid peroxidation [10]. Earlier studies of Maguire and Williams [11] reported that a cold stress enhances brain acetylcholinesterase (AChE) inhibition induced by chlorpyrifos in Northern bobwhites.

The effects of single stressor, either cold stress or CPF toxicity are well known. In this study attention has been drawn not only to assess the effects of CPF but also to its interaction with cold stress. Since co-exposure can result in antagonistic or synergistic effects, studies on these interactions will be of immense use to understand the realistic situation in the field especially on non-target organisms. Studies made in our laboratory have reported that increased lipid peroxidation and decreased antioxidant status causing architectural damage to the membranes of nervous system of rat in an age-related manner during co-exposure with CPF and cold stress [12]. Cold stress is an important contributor to enhance the neurotoxicity of CPF.

CPF is among the OP pesticides known to have adverse impact on the developing brain of children as well as laboratory animals [13]. Several other studies using different routes of CPF administration have reported greater sensitivity in younger rats [14, 15] and the mechanism(s) for the greater susceptibility in young rats is not well defined. Laboratory evidences, obtained largely through the use of rodents, suggest that acute or chronic exposure to CPF and/or its metabolic product(s) may overtly injure the central nervous system (CNS) or produce marked changes in neuronal function that persist even after exposure has ceased, particularly during the early postnatal period [16].

A major area of biomedical and societal concern regarding age-related sensitivity to the effects of pesticides led to the passage of the Food quality Protection Act in 1996, which mandated greater assurance of protection for the young. Understanding potential age-related sensitivity is especially important in light of data suggesting greater exposure of children to pesticides [17]. While the actions of OP pesticides have been widely studied for decades, almost all such studies have used only adult laboratory animals and there are few data concerning relative sensitivity in the young [18, 19]. The limited data available suggest some differential effects on developing as well as maturing animals on exposure to CPF [18]. In spite of the extensive use of CPF in crop protection and in the household, information related to its interaction with cold stress, and resulted effects on health with particular reference to neurotoxicity are scarcely available. Thus, the mechanism of CPF and cold stress interaction induced CNS dysfunction remains to be elucidated. Keeping in view the lacuna in the literature and the role of cold stress in aggravating quantum of toxicity, the present study was therefore initiated to determine an array of responses of interactive effects of CPF and cold stress in an age-related manner in discrete regions of rat CNS.

Materials and Methods

Chemicals

A commercial grade OP compound having 20% w/v of CPF in organic solvent dimethyl sulphoxide (DMSO) marketed as Darsban was procured from Lupin Agrochemicals Pvt. Ltd., Bharuch, Gujarat (India) was used in this study. The commercial formulation was selected because it would reflect the realistic situation in the field and this product is extensively used for agricultural practices in India. The other chemicals were purchased from BDH and Sigma–Aldrich.

Animals

Male albino rats, Wistar strain (Rattus norvegicus albinus) of different age groups viz., neonatal (7-day old, 10–12 g wt), juvenile (21-day old, 35–40 g wt) and adult (90-day old, 160–180 g wt) were used throughout the experiment. Rats were procured from Sri Raghavendra enterprises, Bangalore and acclimatized to laboratory conditions (12 h dark/light cycle at 28 ± 1°C) for 1 week prior to commencement of the experiment. They were maintained on standard rodent pellet diet and tap water ad libitum; in accordance with the guidelines of National Institute of Nutrition, ICMR, Hyderabad, and experimental protocol was approved by the Institutional animal ethical committee, Bangalore University, Bangalore.

Experimental Protocol

Rats were divided into six groups: Group I—control animal group was kept at the laboratory room temperature (28 ± 1°C); Group II—exposed to CPF at the laboratory temperature, 28 ± 1°C; Group III—exposed to cold stress at 15°C; Group IV—exposed to cold stress at 20°C; Group V—exposed to CPF plus cold stress at 15°C; Group VI—exposed to CPF plus cold stress at 20°C. The number of animals in each group was six. The neonatal and juvenile animals were kept along with the dam and other littermates in order to avoid additional stress. We chose to administer CPF via subcutaneous injection in dimethyl sulfoxide (DMSO), a vehicle appropriate for water-insoluble agents and already known not to affect the corresponding measures of brain development [20]. The group II, V and VI animals were treated with sub lethal doses (1/3 of LD50) of CPF subcutaneously in a volume of 1 ml/kg body weight dissolved in DMSO. The group I, III and IV animals were injected with plain DMSO (vehicle controls). The 48 h LD50 of CPF (EC 20%) in rats by subcutaneous route was assessed by probit analysis method [21] and observed values were found to be 15, 242 and 510 mg per kg body weight for 7-day old, 21-day old and 90-day old age groups, respectively.

To induce cold stress, rats were housed in an acute cold stress apparatus (Colton BOD incubator) for 48 h on a 12-h light/12-h dark cycle with a built-in heater and cooler that could be controlled by self-timer. Rats were sacrificed by cervical dislocation 48 h after respective treatments and discrete regions like cerebral cortex (CC), cerebellum (CB), medulla oblongata (MO) and spinal cord (SC) were quickly separated and washed in ice cold 0.9% saline. The tissue homogenates were made by using appropriate buffer and supernatant was stored at a temperature of −20°C and used for biochemical assays.

Biochemical Methods

The biochemical estimations were performed spectrophotometrically using Jenway-6405(UV/VIS) Spectrophotometer by the following methods viz., Deoxy ribose nucleic acid (DNA) by Schneider [22], Ribose nucleic acid (RNA) by Munro [23], Proteins by Lowry et al. [24] Protein oxidation (Protein carbonyls) by Levine et al. [25], Protein thiols by Reznick and Packer [26], and Cellular damages were evaluated by measuring Alanine-aminotransferase (ALT) and Asparatate-aminotransferase (AST) activity levels by Reitman and Frankel [27].

Statistical Analysis

The results are expressed as mean ± standard deviation (SD) of six observations (n = 6) in each group. Differences between treatment groups of same age group were assessed by one-way analysis of variance (ANOVA) using the SPSS software package for windows version 15.0. Post hoc testing was performed for inter-group comparisons using Bonferroni test at probability (P) value <0.05 level of significance. To analyze the interactive effects, three-way ANOVA was carried out and tested by Duncan’s test for multiple comparisons to define the nature of the effect.

Results

The results of the present study are depicted in Figs. 1, 2, 3, 4 and Tables 1, 2.

Fig. 1
figure 1

Effects of CPF, cold stress and their co-exposure on protein oxidation parameters in the cerebral cortex of different age group rats: a DNA content; b RNA content; c Total protein; d Protein carbonyl content; e Total protein thiol; f ALT activity; g AST activity. Results are presented as mean ± SD, n = 6. Significantly different from control: *P < 0.05; significantly different from CPF: & P < 0.05; significantly different from neonatal in corresponding treatment groups: †P < 0.05; significantly different from juvenile in corresponding treatment groups: ¥ P < 0.05

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Fig. 2
figure 2

Effects of CPF, cold stress and their co-exposure on protein oxidation parameters in the cerebellum of different age group rats: a DNA content; b RNA content; c Total protein; d Protein carbonyl content; e Total protein thiol; f ALT activity; g AST activity. Results are presented as mean ± SD, n = 6. Significantly different from control: *P < 0.05; significantly different from CPF: & P < 0.05; significantly different from neonatal in corresponding treatment groups: †P < 0.05; significantly different from juvenile in corresponding treatment groups: ¥ P < 0.05

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Fig. 3
figure 3

Effects of CPF, cold stress and their co-exposure on protein oxidation parameters in the medulla oblongata of different age group rats: a DNA content; b RNA content; c Total protein; d Protein carbonyl content; e Total protein thiol; f ALT activity; g AST activity. Results are presented as mean ± SD, n = 6. Significantly different from control: *P < 0.05; significantly different from CPF: & P < 0.05; significantly different from neonatal in corresponding treatment groups: †P < 0.05; significantly different from juvenile in corresponding treatment groups: ¥ P < 0.05

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Fig. 4
figure 4

Effects of CPF, cold stress and their co-exposure on protein oxidation parameters in the spinal cord of different age group rats: a DNA content; b RNA content; c Total protein; d Protein carbonyl content; e Total protein thiol; f ALT activity; g AST activity. Results are presented as mean ± SD, n = 6. Significantly different from control: *P < 0.05; significantly different from CPF: & P < 0.05; significantly different from neonatal in corresponding treatment groups: †P < 0.05; significantly different from juvenile in corresponding treatment groups: ¥ P < 0.05

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Table 1 Comparison of mean body weight, mean absolute weight of the neurosomatic index in control and experimental groups at 48 h of exposure
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Table 2 Effect of CPF, cold stress and their co-exposure on protein/DNA and RNA/DNA ratio in the (a) cerebral cortex, (b) cerebellum, (c) medulla oblongata, (d) spinal cord of different age group of rats at 48 h of exposure
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Neuro-Somatic Index

Individual and co-exposures of CPF and cold stress resulted decline in body weights followed by brain and spinal cord weights indicating reduction in the neuro-somatic index in all age groups of rats studied (Table 1). Similarly CPF and cold stress exposure individually and concurrently resulted in a lower RNA/DNA ratio in all age groups of rat CNS regions while protein/DNA ratio found to be more in juveniles followed by neonates than young adults (Table 2), further results indicate that DNA concentration per gm of tissue decreased with age, from neonatal to adult.

Cerebral Cortex

Figure 1 presents data on individual and interactive effect of CPF and cold stress in rat cerebral cortex of different age group rats. The levels of DNA, RNA, total protein and protein thiols were significantly decreased, and protein carbonyl content and activity level of AST and ALT were markedly elevated. Moreover, interaction of CPF and cold exposure at 15°C exhibited pronounced change when compared to CPF and cold stress alone and together at 20°C. Comparatively, neonatal and juvenile animals showed higher sensitivity (P < 0.05) than adult animals (P < 0.05).

Cerebellum

In comparison with control values, the levels of DNA, RNA, total protein and protein thiols were statistically decreased, while the levels of protein carbonyl, AST and ALT were dramatically increased in different age group rats exposed to CPF or cold stress. However, interactive effects were more pronounced in neonatal and juvenile age groups exposed at 15°C over the adult. Comparatively, this region appears to be most vulnerable in neonatal and juvenile animals than young adults (data presented in Fig. 2).

Medulla Oblongata

The effect of CPF and cold exposure alone and together in medulla oblongata are shown in Fig. 3. The three-way ANOVA indicated a remarkable significant decrease in the levels of DNA, RNA, total protein and protein thiol, and significant elevations in the level of protein carbonyl, AST and ALT in neonatal, juvenile and young adult rats. Synergistic interaction of CPF and cold stress at 15°C showed marked macromolecular perturbations in comparison with CPF and cold stress alone and together at 20°C. Comparatively, this region appears to be more vulnerable in young animals.

Spinal Cord

Interactive changes in spinal cord are shown in Fig. 4. In comparison with control values, the levels of nucleic acids, total protein and protein thiols were drastically decreased, while the levels of protein carbonyl, AST and ALT were statistically increased in different age group rats exposed to CPF and cold stress. However, interactive effects were more pronounced in neonatal and juvenile age groups exposed at 15°C over the adult.

Discussion

Pesticides are known to alter protein and nucleic acid metabolism [28] and OP insecticides have an effect in addition to their specific inhibition of cholinesterase enzyme. Our previous study demonstrated that developing animals were more susceptible to CPF, cold stress; their interaction as a result of oxidative stress showed to be one of the decisive pathologic mediating factor(s) in CPF toxicity [12]. The generated data in the present study was concentrated on changes in indices of protein oxidation, a major event of oxidative stress, changes in transaminases and nucleic acid macromolecular alterations.

Protein Carbonyls

Oxidative damage to proteins by ROS can result in cleavage of the polypeptide backbone, their cross-linking, and thereby modify the side chains of amino acids that may be a more critical to cause damage to lipids, because enzyme inactivation can have rapid and suprastoichiometric effects [29]. Protein carbonyl content (PCC) [aldehyde or ketone] is a widely used marker to assess the presence of oxidative stress in physiological and pathological conditions [30].Our results clearly show that toxic insults induced a significant increase in protein carbonyl level in discrete CNS areas and oxidized protein accumulates in neuronal tissues and eventually resulting in age-associated brain dysfunction. An increase observed in protein carbonyl levels in the discrete regions of CNS is in accordance with earlier reported works where augmented carbonyl level formation observed upon several pollutant exposures [31].

Protein Thiols

Protein thiols are frequent sites for post translational modification mediated by oxidative stress [32] observed in a wide variety of disorders including aging, and pesticide neurotoxicity [33]. In line with the previous studies we found decrease in protein thiol in the discrete regions of CNS studied due to CPF administration, which may be due to increased degradation of protein or increased consumption of antioxidant in stress environment which confirm the role of OPs in disruption of body’s total antioxidant capacity [34, 35]. These findings indicate the efficiency of S-thiolation as a mechanism of antioxidant defence with pathological state that creates an increased risk of irreversible oxidation of—SH groups of proteins [36].

Transaminases

AST is present in rat cerebral homogenate at about the same concentrations as in liver suggests its importance in the brain amino acid pool homeostasis [37, 38]. Though ALT is indicated to be distributed in both mitochondrial and soluble fractions of the brain, unlike GOT, it is less active in brain, however, Matthews et al. [39] reported that both the enzymes degrade glutamate and GPT is able to reduce toxic (500 μM) levels of glutamate into the physiologic (<20 μM) range. The metabolism of amino acids is indicated by activities of specific transaminases, which were altered in response to CPF exposure. Further, increased aminotransferase activities may alter the levels of amino acid neurotransmitters in the nervous system. The transaminases are implicated to have a role in protein synthesis, are seriously affected by CPF. In the present study, a significant increase in the AST/ALT activities was observed. This result go hand-to-hand with Abdul Naveed et al. [40] who reported increased levels of brain AST and ALT enzymes in the fish treated with OP pesticide. Similar results showed the same trend in the activity level of AST/ALT caused by OP pesticides had been also reported in rabbit [41] and rat [42]. Indeed, it is believed that CPF causes damage to the nervous tissue probably through the induction of oxidative stress [12]. Although transaminase reactions are reversible, the equilibrium of the AST and ALT reactions favours the formation of aspartate and alanine respectively, leading to excitotoxicity [43]. In this study the regional variation of ALT/AST activities was prominent on CPF exposure but the response to cold stress and CPF toxicated insult showed exacerbation in the AST/ALT levels. Three factor ANOVA with replication showed that CPF exposure contributed significantly on the changes of the ALT activity in all the CNS regions, but the interactions of impact of CPF and Cold exposure were significant in all age groups at 15°C. These observations support region-wise specific sensitivity to toxic insults as indicated in transaminases. Thus, it may be suggested that co-exposure of CPF and cold stress cause region specific alteration in glutamate metabolism. Hence, altered amino acid distribution pattern within the CNS regions might be expected in response to exposures of CPF and cold stress.

Nucleic Acids

Nucleic acids are the most sensitive indices to access the extent of cellular damage upon the impact of pesticide because of the relative constancy of nucleic acid per somatic cell nucleus [44]. The reduction observed in the level of RNA, DNA and protein concentrations may be due to inhibition of their syntheses and/or enhancement of degradation in the cells and such reductions are in accordance with earlier report on OP pesticide [28]. It may be suggested that CPF affects the turnover of RNA and protein thereby changing the levels of macromolecular constituents in the neuronal tissues of the rat. CPF exposure resulted in a lower RNA/DNA ratio in all age groups of rat CNS regions. Further results indicate that DNA concentration per gm of tissue decreases with age, from neonatal to adult. The data indicate that cold stress also results in less protein and DNA. RNA/DNA ratio used as stress indicator and generated data (Table 2) suggests that neuronal tissues of younger age group animals affected more than young adult. Protein/DNA ratio used as an index of cell size and in comparison, the cell size was found to be more in juveniles followed by neonates than young adult indicating higher macromolecular content in developing rats. Decline observed in nucleic acids and proteins may also be due to DNA damage caused by the free radicals and inhibition of RNA by direct interaction of ROS. Oxygen radicals can attack proteins, nucleic acids and lipid membranes, thereby disrupting cellular functions and integrity. Therefore, the oxidative changes appear to be related to cellular alterations.

Cold Stress

Brain is the organ most vulnerable to thermal fluctuations, since most of the physiological acclimation responses are initiated by the CNS [45]. Cold shock cause severe pathologies in the mammalian brain [46]. The present finding that activity levels of transaminases, protein carbonyl levels, protein thiols, nucleic acids and protein content were reduced in cold stress exposed group seems to be correlated with brain tissue damage induced by free radicals likely formed by acute cold stress [12]. These findings run in agreement with previous reports of increased levels of protein carbonyls [47, 48] and decreased level of RNA content [49] in tissues of animals exposed to cold stress. The increased protein oxidation may be a result of the decreased GSH concentration, which would otherwise protect proteins against the oxidation of the sulfhydryl groups [50].

Influence of Age

The present results reveal that young animals are markedly more sensitive and vulnerable than young adults. Age-related differences in susceptibility have been attributed in general to the following differences in the young immature nervous system, poor development of metabolizing/detoxifying enzymes, weak kinetic ability, differences in permeability of membranes, distribution of fat and overall excretory functions in the very young when compared with adults [51]. The brain is heterogeneous both metabolically as well as structurally. This heterogeneity not only allow various parts to perform the specific functions, but also permits the same metabolite to be used for different functions [52]. It is inevitable that metabolic variations that occur are due in part to preferential uptake or forced accumulation of CPF into particular cells or their processes. Further the same metabolites are proportionately different in their relative distribution among cellular structure. In addition, the mitochondrial population of brain is apparently heterogeneous in size as well as enzyme compliment [52]. The mechanism behind regional alterations could lead to degeneration or loss of neurons in vulnerable area of anatomical resolution which are supposed to carry designated function. In this study perhaps young rat, CNS as a whole appears to be susceptible to oxidative damage; the medulla oblongata and cerebellum is observed to be more prone to oxidative macromolecular alterations which may be due to pronounced oxidative stress as indicated from our recent study [12]. It is known that the differential sensitivity to CPF neurotoxicity in different brain regions is due to preferential CPF metabolites accumulation, also due to alteration of biochemical or cellular process that are uniquely associated with or greatly enhanced in a particular region.

Impact of Cold Stress on CPF Toxicity

OP pesticides pose the threat of hypothermia, because they amplify body temperature decline that accompanies cold exposure [53]. In the present study, when different age group animals exposed to CPF toxicity at the temperatures of 15 and 20°C, their interaction exacerbated resulting in a synergistic action and substantially modified the toxicity in discrete CNS regions indicating potential effect of CPF and the quantum of synergistic interaction was most apparent at 15°C. In line with our findings, earlier studies [11] have reported that concomitant exposure of animals to CPF and cold stress exaggerates the neurotoxicity by provoking the AChE inhibition. The co-exposure of CPF and cold stress induced severe hypothermia and may retard the detoxification mechanisms of CPF resulting in its longstanding effect. Further, toxic insult of CPF and cold stress either alone or concurrently induced AChE inhibition might have triggered pronounced lipid peroxidation because of their interaction and the production of ROS in the tissues exceeds the ability of the antioxidant system to eliminate them, oxidative stress ensues [12]. Because of the high reactivity of ROS, most cellular components are likely to be targets of oxidative damage; lipid peroxidation, protein oxidation, GSH depletion, DNA single strand breaks, are all initiated by ROS in excess. As a whole, these events ultimately lead to cellular dysfunction and injury [54].

In conclusion, CPF and cold stress exposure either individually or in combination substantially increased the activity/levels of protein carbonyls, AST, ALT and decreased protein thiols, DNA, RNA and total proteins in discrete regions of CNS. Overall, the effects of co-exposure were appreciably different from either of the exposures. However, synergistic-action of CPF and cold stress at 15°C showed higher dyshomeostasis in comparison with CPF and cold stress alone and together at 20°C indicating the extent of oxidative macromolecular damage in discrete regions of brain and spinal cord. Furthermore, the present study demonstrates that macromolecular oxidative damage is highly pronounced in neonates and juveniles than the young adults. Indeed, this is an area of research whose results certainly have great potential to be translated in important advances in public health.