Developmental Neurotoxicity of Nicotine in Neonatal Mice: Feiya Luo. Comparison Males and Females and Altered Susceptibility to Paraoxon at Adult Age

Transcription

1 Projektrapport från utbildningen i EKOTOXIKOLOGI Ekotoxikologiska avdelningen Nr 133 Developmental Neurotoxicity of Nicotine in Neonatal Mice: Comparison Males and Females and Altered Susceptibility to Paraoxon at Adult Age Feiya Luo

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3 Contents Preface /1 Abstract /3 Introduction /5 Neonatal exposure to toxic agent /5 Brain development /6 Critical period /7 Cholinergic system /8 Nicotine receptor /9 Muscarinic receptor /10 Nicotine /11 Organophosphorus Compounds /12 Comparison of male and female brain development /13 Aims /14 Materials and Methods /16 Chemicals and animals /16 Behaviour tests /17 Statistical analysis /18 Result /19 Effects of neonatal nicotine exposure on spontaneous behaviour and response to paraoxon in adult female mice /19 Comparison on developmental neurotoxicity of nicotine in males and females mice /28 Discussion /35 Concluding Remarks /39 Reference /40

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5 Preface This master s thesis was carried out in part fulfilment of the graduate studies program in Ecotoxicology at the Department of Environmental Toxicology, University of Uppsala, Sweden. The work has been financially supported by Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning. I wish to express my gratitude to my excellent adviser, Professor Per Eriksson, for his wholehearted and endless support, encouragement and never failing enthusiasm, to Professor Anders Fredriksson who conducted on behavioural testing, and statistical work. -1-

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7 Abstract Exposure to different xenobiotics, either persistent or nonpersistent, during the rapid development of the brain, can cause behavioural disturbances and changes in cholinergic receptor configuration in mice. Furthermore, exposure to low doses of toxicants during this neonatal period can make the animals more susceptible to adult exposure of different toxic agents. There are environmental chemicals that indicate gender differences for neurotoxic effects. This study was undertaken to investigate whether neonatal exposure of female mice to nicotine alter the susceptible of female mice to a known cholinergic agent, organophosphorous compounds. Furthermore, to study whether there are any differences between male and female mice in susceptible to a known cholinergic agent after neonatal exposure to nicotine. Neonatal, 10-day-old, female mice were exposed to nicotine-base (33 μg/kg or 66 μg/kg b.w.) or saline s.c. twice daily on five consecutive days. At 2 months of age the animals were exposed to paraoxon (0.3 mg/kg b.w.) or saline s.c. every second day for 7 days. Before the first injection, the animals were observed for spontaneous motor behaviour. Immediately after the spontaneous behaviour test, the animals received the first injection of paraoxon and were observed for acute effects of paraoxon on spontaneous motor behaviour. The spontaneous behaviour test did not reveal any differences between treatment groups. The acute response to paraoxon in the spontaneous motor behaviour test showed a decreased level of activity in mice neonatally exposed to nicotine 66 μg/kg b.w. Control animals showed no change in activity. Two months after the paraoxon treatment, the animals were again tested for spontaneous motor behaviour. Animals neonatally exposed to nicotine 66 μg/kg b.w. and exposed to paraoxon as adults showed a lack of habituation to a novel home environment and -3-

8 also a hyperactive condition in spontaneous motor behaviour. In comparison females and males it was seen that female mice, neonatally exposed to nicotine 66 μg/kg b.w., was less susceptible to paraoxon manifested as hypoactive response to paraoxon, while male mice showed the same hypoactive reaction when receiving nicotine 33 μg/kg b.w. neonatally. Key words: nicotine; paraoxon; development; female mice; cholinergic; behaviour; sexual differences -4-

9 Introduction Neonatal exposure to toxic agent There are a great number of hazardous contaminants in our environment. Most of them are man-made compounds used in various industrial processes and as pesticides, such as polychlorinated biphenyls (PCBs) and DDT. Many of these chemicals are persistent and accumulate in the food chain (Edwards 1970). Other possible environmental hazards, which can cause persistent toxic effect, are the short-acting substances, like nicotine and organophosphorus compounds (OPs) (Eriksson 1997). Each individual can be exposed to environmental toxic agents throughout his or her lifetime, start at the fertilization. At the gestational period, mammal s embryo/foetus can be exposed through the maternal intake of toxic compounds. After birth, the exposure continues via mother s milk, and direct exposure such as through the skin, ingestion and breathing. It has been shown in animal studies that both persistent compounds like PCB and DDT, and non-persistent, such as nicotine and organophosphorus compounds, can cause disruption of brain development when administrated during the critical period of neonatal development (Ankarberg et al. 2001; Eriksson 1997; Eriksson et al. 2000; Nordberg et al. 1991), and to induce persistent development neurotoxic effects (Eriksson 1997; Eriksson and Talts 2000; Talts et al. 1998). Another finding is that animals exposed to a low dose of a neurotoxic agent in neonatal life, can lead to an increased susceptibility in adults. This means, in adults, exposed to a xenobiotic, could result in additional behavioural disturbances and learning -5-

10 disabilities at doses not causing any effects in adults that not exposed as neonates (Eriksson 1997). For example, animals neonatally exposed to DDT were more susceptible to an adult exposure to low doses of paraoxon and pyrethroids, causing additional changes in spontaneous behaviour and muscarinic receptor density (Johansson et al. 1996). Brain development In mammals, the development of central nervous system (CNS) can roughly be divided into two major periods. The first part is known as the early embryonic development of the brain. It includes the structural development of brain when the neurones and precursors of glia multiply and the general shape of brain takes form. Exposure toxicants during this time can cause malformation of the brain. The second period is known as the Brain Growth Spurt (BGS) period (Davison and Dobbing 1986) which means a period of brain rapid growth and development. During this stage, there are series of rapid fundamental developmental changes taking place like axonal and dendritic outgrowth, establishment of neural connections, synaptogenesis, and multiplication of glia cells with accompanying myelinisation, and cell, axonal and dendritic death (Kolb and Whishaw 1989). During this period mice and rats acquire many motor and sensory faculties (Bolles and Woods 1964) and during this stage their spontaneous behaviour peaks (Campbell et al. 1969). In human the embryonic period constitutes 20% of the whole gestation period and the foetal period is 80%. The BGS period begins during the third trimester of pregnancy and continues during the first 2 years after birth. In research animals, such as mouse and rat, the embryonic period constitutes 80% and foetal -6-

11 period 20% of the gestational period, which is opposite to human. In mouse and rat the BGS is neonatal, spanning over the first 3-4 weeks of life and peak around postnatal day 10. Figure 1: Rate curves of brain growth in relation to birth in different species. Values are calculated at different time intervals for each species. (Data from Davidson and Dobbing, 1968, and Eriksson unpublished. Illustration by Ylva Stenlund) Critical period Earlier studies have shown that low-dose exposure to environmental agents during the BGS in neonatal mouse can lead to irreversible changes in adult brain function. The inductions of disturbances appear to be limited to a short period during neonatal development and are induced at doses that have no permanent effects when administered to the adult animal (Eriksson 1997). The studies also indicate that there is a defined critical -7-

12 period during neonatal mouse brain development when these persistent effects are induced (Ahlbom 1995; Ahlbom et al. 1994; Eriksson et al. 1992). Studies by Eriksson and co-workers (2000) have shown that mice exposed to nicotine on postnatal day 3-7, or 19-23, only animal exposed day displayed an altered response to nicotine at adult age and also a persistent change in nicotinic receptors. Cholinergic system The cholinergic system plays an important role in many behavioural phenomena, as one of the major transmitter systems in brain. This system is concerned with learning and memory, neurological syndromes, audition, vision, and aggression (Karczmar 1975). Acetylcholine (ACh) takes part in the cholinergic system as a transmitter of nerve signals and plays an important role in learning and memory (Frazier et al. 1998; Hasselmo et al. 1992; Lucas-Meunier et al. 2003). The life cycle of ACh conclude its synthesis, storage, release and metabolism and uptake. Briefly, ACh is synthesized through a reaction of choline and acetyl coenzyme-a by the enzyme choline acetyltransferase (ChAT). Then ACh is stored in vesicles and is released into the synaptic cleft when a nerve impulse reaches the terminal. In the synaptic cleft, the enzyme acetylcholine esterase (AChE) regulates the amount of ACh. AChE splits the ACh, via hydrolysis, into choline and acetate, which are then transported back into the presynapic neuron for reuse in the synthesis of ACh. In rodents, the rapid development of the cholinergic system -8-

13 takes place during the first 3 to 4 weeks after birth. During this period, variables such as ChAT, AChE, sodium-dependent choline uptake and the cholinergic system receptors increase in various brain regions (Coyle and Yamamura, 1976; Kuhar et al., 1980; Falkeborn et al., 1983; Fiedler et al., 1987; Hohmann et al., 1995). There are two major classes of the cholinergic receptors: muscarinic and nicotinic (Dale 1914). Both of them are activated by acetylcholine, but belong to different gene families. Muscarine and nicotine serve as agonists for these two different receptors. Blockage of cholinergic transmission by nicotinic or muscarinic antagonists can produce learning and memory impairments in both humans and animals (Dale 1914). Nicotine receptor Neuronal nicotinic acetylcholine receptors (nachrs) belong to a gene super family of homologous receptors including GABA, glycine and 5-hydroxy tryptamine. The nachrs are believed to have a pentameric structure consisting of five membrane spanning regions around a central ion-channel (Karlin 2002). Various nicotinic ligands have been used in in vitro studies to characterize nicotinic receptors in tissue obtained from rodent and human post-mortem brains. There are at least three subtypes of nicotinic receptor binding sites have been described in human brain which are superhigh-, high-, and low-affinity sites (Nordberg et al. 1988). Neuronal nachrs various and consist of α2-α9 and β2-β4 subunits. So far, seven α (α2-α8) and three β (β2-β4) subunits have been cloned and show regional distributions in rodent brain (Arneric et al. 1995). The α7-9 subunits are known to form -9-

14 functional homo-oligomers consisting of a single a subunit subtype. Some receptors contain two distinct subunits, e.g. α4β2. It seems that α-bungarotoxin has a specific affinity to α7 receptor. The α4β2 subtype is suggested to correspond to nicotinic receptors presynaptically located and high-affinity binding sites (Paterson and Nordberg 2000). Study by Nordberg et al shows that during the first postnatal days, only high-affinity nicotinic receptors can be measured in mouse brain, while both high- and low-affinity sites are present in adult brain. Furthermore, neonatal exposure to low doses of nicotine (day 10-16) prevents the development of low-affinity nicotinic binding sites and can also change the behaviour in adult animals response to nicotine. Muscarinic receptor The muscarinic receptors belong to a superfamily of structurally related proteins which possess seven transmembrane spanning regions. They coupled to G-proteins and exhibit a slow response time. The G-proteins either act on ion channels directly or are linked to a variety of second messenger systems (Cooper, 1996). There were five cloned genes, m1 to m5 been characterised. These genes expressed five different types of receptor proteins called M1 to M5 (Lucas-Meunier et al., 2003). The muscarinic receptors subtypes were divided into two generally classes. The first one is M1-like receptors (Hammer et al., 1980) which are subtypes M1, M3, and M5. The M1-like receptors stimulate the phosphoinositol pathway. The other one is the M2-like receptors, subtypes M2 to M4, which act by inhibiting adenylate cyclise (Lucas-Meunier et al., 2003). The classic muscarinic antagonists, such as atropine and QNB, do not distinguish between the subtypes, but bind to all equally -10-

15 well (Cooper, 1996). Nicotine Nicotine is well known as a component in tobacco. It is one of the most commonly used dependence-producing substances (Henningfield and Woodson 1989). Nicotine will increase heart rate and cause vasoconstriction. It also can increase blood pressure in mother and reduce uterine blood flow, therefore decreasing a fetus s access to all nutrients. It is found that the most common effect of nicotine exposure is low birth weight (Ellard et al. 1996; Lambers and Clark 1996). Low birth weight is independently associated with cognitive deficits (Chaudhari et al. 2004; Corbett and Drewett 2004; Viggedal et al. 2004), regardless of the causative agent. Nicotine is also used as an insecticide. It causes unorthodox upregulation of nicotinic receptors which means it causes over stimulation and blockade of synapses associated with motor nerves. Nicotine activates reward mechanisms in the CNS, which is presumed to be the reason why people smoke (James and Nordberg 1995). It can stimulate the nicotinic receptors directly, and then increase release of acetylcholine, and other neurotransmitters such as dopamine, serotonin and norepinephrine (Wonnacott et al. 1989). Earlier studies by Nordberg et al. 1991, show that mice treated with nicotine (66 μg(-)nicotine base/kg b.w. s.c. twice daily days 10-16) displayed a hypoactive condition, while mice treated with saline displayed a hyperactive condition in response to an acute nicotine injection at adult age. Furthermore, it was found that mice treated with nicotine neonatally lacked the lowaffinity binding site after analysed the nicotinic receptors in the cerebral cortex by displacement studies. -11-

16 Organophosphorus Compounds The first synthesized anticholinergic organophosphate appeared in It was tetraethyl pyrophosphate (TEPP), and the researcher miraculously survived to describe its taste! Then organophosphorus compounds (OPs) were developed in the 1930s. German scientists synthesized several compounds designed for agricultural use. But unfortunately some of them developed into chemical warfare agents during the World War II era, e.g. Sarin Tabun, and Soman. The use of OP-pesticides greatly increased following the ban of DDT in many countries (Nimmo and MacEwen, 1994). OPs bind to the acetylcholine esterase, and turning it into a phosphorylated inactive complex. Therefore acetylcholine accumulates in the synaptic clefts and neuromuscular junctions, and then causes instant stimulation of the synaptic receptors (Eriksson 2000). The symptoms of OP insecticide poisoning were resulted from the overstimulation of: (a) muscarinic receptors in the parasympathetic autonomic postganglionic nerve fibres causing e.g. salivation, lacrimation, vomiting, cramps, diarrhea, bradycardia; (b) nicotinic receptors in parasympathetic and sympathetic autonomic fibres, causing e.g. tachycardia and pallor; (c) nicotinic receptors in somatic motor nerve fibres, causing e.g. cramps and muscle weakness; and (d) both muscarinic and nicotinic receptors in the CNS, causing drowsiness, fatigue, confusions, coma and depression of respiratory centres (cause of death) (Fukoto, 1990). Another distinct manifestation of exposure to OP insecticides is the intermediate syndrome with an onset 24 to 96 hours after the acute cholinergic crisis. The major effect of this is muscle weakness, primarily in muscles innervated by the cranial nerves (Johnson, 1982; Johnson, 1990). -12-

17 The OP used in this study is paraoxon (diethyl p-nitrophenyl phosphate), an active metabolite of the OP compound parathion, which is an insecticide first synthesized during World War II, and still extensively used in agriculture. Parathion is converted through oxidative desulfuration to paraoxon by a group of enzymes called the mixed function oxidases (MFO s). This is a reaction catalysed, in both insects and mammals. Earlier studies have shown that neonatal exposure to paraoxon affects behaviour and nicotine receptors in adult mice (Ahlbom 1995). Ankarberg et al showed that male mice neonatally exposed to nicotine and exposed to paraoxon at adult age showed a deranged spontaneous motor behaviour, including hyperactivity and lack of habituation. Comparison of male and female brain development Sexual dimorphism of the mammalian central nervous system has been widely documented. Morphological sex differences in brain areas underlie sex differences in function. Sex differences have been reported for virtually all central cholinergic markers. In Rhodes and Rubin (1999), the sex difference in the size of the cortex is assumed to be due to a difference in the size of dendritic trees. Ultimately, sex differences leading to different physiological functions that can be manifested by the manner in which the animal interacts with its environment. Some studies show that basal ACh concentrations, high-affinity choline receptor activity and choline acetyltransferase (ChAT) activity appear to be more sensitive to stimulation or blockade and less stable with age in female animals. In males, acetylcholinesterase (AChE) activity appears to be more sensitive to antagonism but exhibits more stability with age compared to females (Rhodes and Rubin 1999). In adult male mice, brain -13-

18 AChE activity did not return to control levels after receiving a single exposure to dose of OP, whereas in female mice, control levels was regained, albeit 20 days after exposure (Smolen et al. 1987). Smolen et al suggested that there was an increased susceptibility in males to neurotoxicity and longterm behavioural changes following OP poisoning. One possibility may have been due to sex-related pharmacokinetic differences, leading to metabolites with sustained activity in the male mice. Multiple Chemical Sensitivity (MCS) is a clinical phenomenon in which individuals, after acute or intermittent exposure to one or more chemicals, usually irreversible cholinesterase inhibitors such as OP pesticides or nerve gases, report hypersensitivity to a wide variety of chemically unrelated compounds. In some studies, there have been more female than male MCS patients reported, reaching a ratio of 4:1 (Overstreet et al. 1996; Miller et al. 1995). Some neurotoxic compounds are known to affect the two sexes in different ways and also affect different strains of the same species in different ways. There are numerous studies involving environmental chemicals that indicate gender differences for neurotoxic effects. But the contradicted results may be caused by differences in study design, endpoints, or simply the possibility that there are no gender differences (Viberg 2004). Aims The aims were to study the development neurotoxic effects of nicotine in both female and male mice. More specific: To study effects of neonatal exposure to nicotine and adult susceptibility to paraoxon in female mice. -14-

19 To compare the altered spontaneous behaviour of female mice and male mice regarding neonatal exposure to nicotine and later adult exposure to paraoxon or nicotine. -15-

20 Materials and Methods Chemicals and animals (-)nicotine-bi-(+)-tartrate and paraoxon (diethyl ρ-nitrophenyl phosphate) were obtained from Sigma, U.S.A. Pregnant NMRI mice were obtained from B&K, Sollentuna, Sweden. The animals were kept in plastic cages in a room with an ambient room temperature of 22 C and a 12/12 hours light/dark cycle and supplied with standardised pellet food (Lactamin, Stockholm, Sweden) and tap water ad libitum. Each litter was kept with its respective dam. Litter size was adjusted to 8-12 animals within 48 hours by killing excess pups. Female NMRI mice at the age of 10 days start to be given either 33 μg or 66 μg (-)nicotine-bi-(+)-tartrate per kg b.w. s.c. twice daily at 10 a.m. and 4 p.m. for five consecutive days. The dose 66 μg/kg b.w. is known to effect, and reduce the development of LA(low affinity)-binding sites in neonatal male mice (Eriksson et al 2000; Nordberg et al 1991). Furthermore, this dose equals the dose absorbed when smoking approximate ten cigarettes per day (Russel 1990). Nicotine was dissolved in saline. The ph of the (-)nicotine-bi-(+)-tartrate solution was adjusted to 7.0 to avoid necrosis in the mice necks. Control animals were given 10 ml/kg body weight s.c. of saline vehicle in the same manner. Each treatment group consisted of mice from 6 litters. At the age of 4 weeks, pups were weaned and only the females were left and raised in groups of 4-7 under the same conditions as above. At the age of 2 months, the animals were exposed to paraoxon (0.3 mg/kg b.w.), dissolved in saline, or saline vehicle s.c. every second day for 7 days (a total of four injections/mouse). -16-

21 The doses of paraoxon were selected to avoid clinical toxic symptoms in the animals and to be similar to an earlier study regarding increased susceptibility to paraoxon in adult male mice (Johansson et al. 1996; Ankarberg 2004). Behaviour tests Spontaneous behaviour is especially meaningful in studying behaviour in animals because it reflects the animals ability to integrate the sensory input into a motory output. The animals habituation to a novel home environment can also be viewed from the spontaneous behaviour. The habituation index in this activity test chambers situations, over repeated test periods maybe assumed to provide a simple, non-associative instance of learning. Spontaneous behaviour Spontaneous behaviour testing was done on female mice at the age of 2 and 4 months. The animals were tested once only between 8 and 12 in the morning under the same ambient light and temperature as the housing. Eight mice from each treatment group were randomly taken from 4 to 6 litters and tested. Motor activity was measured for 60 minutes, divided into three 20 minutes periods, in an automated device consisting of cages ( cm) placed within two series of infrared beams (low-level and high-level) (Rat-O-Matic, ADEA Elektronik AB, Uppsala, Sweden) (Fredriksson 1994). Locomotion: Registered when the mouse moved horizontally through the low-level grid of infrared beams. Rearing: Registered whenever and as long as the mouse moved vertical and crossed a high-level grid, at a rate of four counts per second (i.e. the number of counts obtained was proportional to the time spent rearing). -17-

22 Total activity: A pick-up (mounted on a lever with counter weight) registered all types of vibrations within the test cage, i.e. those caused by mouse movements, shaking (tremors) and grooming. Response to paraoxon Immediately after the spontaneous motor behaviour test in 2-month-old female mice, the animals received the first injection of paraoxon (0.3 mg/kg b.w.) or saline, and were then observed for another 60 minutes period ( min). Two months after the adult injection, the animals (aged 4 months) were again observed for spontaneous motor behaviour (0-60 min). Statistical analysis The data were evaluated in a split-plot ANOVA (analysis of variance) and pairwise testing between treated groups and their corresponding control groups was performed with Tukey HSD (honestly significant difference) test (α = 0.05) (Kirk 1968). -18-

23 Result Effects of neonatal nicotine exposure on spontaneous behaviour and response to paraoxon in adult female mice This section shows the results on spontaneous behaviour in female mice neonatally exposed to nicotine and as adults to paraoxon. The spontaneous behaviour tests were recorded and taken from: Ι spontaneous behaviour in 2-month-old female mice, II spontaneous behaviour in 2-month-old female mice in acute response to paraoxon, III spontaneous behaviour in 2-month-old female mice after exposure to paraoxon for one week, IV spontaneous behaviour in 4-month-old female mice. There were no clinical signs of toxic symptoms in the treated mice throughout the experimental period. Nor were there any differences in body weight gain between the different treatment groups during the whole experimental period. Ι Spontaneous Behaviour in 2-month-old female mice Results from locomotion, rearing and total activity variables in 2-month-old mice treated with either 33 or 66 μg nicotine-base/ kg b.w. and controls receiving 10 ml/kg b.w. of saline, twice daily s.c., between postnatal day 10 and 14 are shown in Figure 2. Saline treated animals (controls) displayed a decrease in activity over the 60 minutes period as the novelty of the test cage diminished, which is a normal behaviour (Ankarberg 2003; Viberg 2003). Animals neonatally exposed to either 33 or 66 μg nicotine-base/kg b.w. also displayed a decrease in activity over time, as the control group. No significant group period interactions were observed (F 10, 132 =0.64, F 10, 132 =1.33, F 10, 132 =0.85), for locomotion, rearing and total activity, respectively. -19-

24 a) Locomotion b) Rearing -20-

25 c) Total Activity Mean Figure 2: Spontaneous behaviour. a) Locomotion, b) Rearing and c) Total Activity Mean, variables in 2-month-old female mice after exposure to either 33 or 66 μg nicotine-base/kg b.w. s.c. twice daily or to 10 ml/kg b.w. s.c. of saline vehicle as control twice daily between postnatal day 10 and 14. For statistical evaluation, an ANOVA with split-plot design was used. No significant group period interactions were observed (F =0.64, F =1.33, F 10, =0.85), for locomotion, rearing and total activity , , 132 respectively. II Spontaneous Behaviour in 2-month-old female mice - acute response to paraoxon The results in female mice treated with saline or nicotine neonatally and response to paraoxon or saline at adult age are shown in Figure 3 below. Immediately after the 60 min observational period for spontaneous behaviour the mice received a single injection of saline or paraoxon (0.3 mg/kg b.w.) s.c. and were observed for an additional 60 min period. There were significant group period interactions (F 10, 132 =10.71, F 10, 132 =11.78, F 10, 132 =5.86), for the locomotion, rearing and total activity variables, respectively. -21-

26 Mice treated neonatally with 66 μg/kg nicotine-base/kg b.w. s.c. showed a significant decrease in activity when exposed to paraoxon (0.3 mg/kg b.w.) compared to nicotine 66μg-saline treatment group and saline-paraoxon treatment group during the fourth period (60-80 min, p 0.01). In mice neonatally exposed to 33 μg nicotine there were no significant change in the variables, locomotion or rearing, but in the total activity variable there was a significant (p 0.05) decrease. a) Locomotion b) Rearing -22-

27 c) Total Activity Mean Figure 3: Spontaneous behaviour. a) Locomotion, b) Rearing and c) Total Activity Mean, variables in 2-month-old female mice after exposure to either 33 or 66 μg nicotine-base/kg b.w. s.c. twice daily or to 10 ml/kg b.w. s.c. of saline vehicle as control twice daily between postnatal day 10 and 14, and challenged with a single injection of either paraoxon (0.3 mg/kg b.w.) or saline, s.c. For statistical evaluation, an ANOVA with split-plot design was used. A significant group period interactions were observed (F 10, 132 =10.71, F 10, 132 =11.78, F 10, 132 =5.86), for locomotion, rearing and total activity respectively. A= significantly different from its respective control, B= significant different from saline-paraoxon treatment. Capital letter indicates p-value 0.01; small letter indicates p-value III Spontaneous Behaviour in 2-month-old female mice after exposed to paraoxon for one week The mice were observed for spontaneous behaviour 24 hours after the last injection of paraoxon. The adult paraoxon exposure consisted of injections of paraoxon (0.3 mg/kg b.w.) every second day for one week (a total of 4 injections). The results are shown in Figure 4 below. -23-

28 There were significant group period interactions (F 10, 132 =6.17, F 10, 132 =5.29, F 10, 132 =26.46), for locomotion, rearing and total activity respectively. Mice treated neonatally with 66 μg/kg nicotine-base/kg b.w. s.c. and exposed to paraoxon (0.3 mg/kg b.w.) s.c. at adult age show a significant decrease in activity both compared with its respective control and with saline-paraoxon treatment group during the first period (0-20 min, p 0.01). a) Locomotion b) Rearing -24-

29 c) Total Activity Mean Figure 4: Spontaneous behaviour. a) Locomotion, b) Rearing and c) Total Activity Mean, variables in 2-month female mice after neonatal exposure to either 33 or 66 μg nicotine-base/kg b.w. s.c. twice daily or to 10 ml/kg b.w. s.c. of saline vehicle as control twice daily between postnatal day 10 and 14, and exposed to saline or OP (paraoxon, 0.3 mg/kg b.w.) s.c. every second day for 7 days (4 injections in total). For statistical evaluation, an ANOVA with split-plot design was used. A significant group period interactions were observed (F 10, 132 =6.17, F 10, 132 =5.29, F 10, =26.46), for locomotion, rearing and total activity respectively. A= 132 significantly different from its respective control, B= significant different from saline-op treatment. Capital letter indicates p-value 0.01; small letter indicates p-value IV Spontaneous Behaviour in 4-month-old female mice The results from spontaneous behaviour in 4-month-old female mice treated with saline or nicotine neonatally and exposed to paraoxon at 2 months of age are shown in Figure 5 below. A significant group period interactions were observed (F =12.65, F =7.04, F 10, =20.65), for locomotion, rearing , , 132 and total activity respectively. Mice treated neonatally with

30 μg/kg nicotine-base/kg b.w. s.c. and exposed with paraoxon (0.3 mg/kg b.w.) s.c. showed a significant decrease in activity when compared with its respective control and with saline-paraoxon treatment group during the first period (0-20 min). But during the third 20-min period (40-60 min) they were significantly more active than the controls. a) Locomotion b) Rearing -26-

31 c) Total Activity Mean Figure 5: Spontaneous behaviour. Locomotion, rearing and total activity variables in 4-month-old female mice after exposure to either 33 or 66 μg nicotine-base/kg b.w. s.c. twice daily or to 10 ml/kg b.w. s.c. of saline vehicle as control twice daily between postnatal day 10 and 14, and exposed to saline or Paraoxon (0.3 mg/kg b.w.) s.c. every second day for 7 days (4 injections in total) at 2-month-old. For statistical evaluation, an ANOVA with split-plot design was used. A significant group period interactions were observed (F 10, 132 =12.65, F 10, 132 =7.04, F 10, 132 =20.65), for locomotion, rearing and total activity respectively. A= significantly different from its respective control, B= significant different from saline- OP treatment. Capital letter indicates p-value 0.01; small letter indicates p-value

32 Comparison on developmental neurotoxicity of nicotine in males and females mice There are several studies conducted on development neurotoxicity of nicotine in neonatal male mice. These studies concern adult response to nicotine or OP after neonatal exposure to nicotine. This section comparison results on males and females mice from five separately developmental neurotoxicity studies: Study 1 (Eriksson et al. 2000), study 2 (Ankarberg et al. 2001), study 3 (Ankarberg 2003), study 4 (Söderberg 2001, unpublished) and study 5 (the present study). The data constitute results from 5 different experiments taken from three different studies. The spontaneous behaviour tests were done in the same manner as in the present study but with male mice instead of females. The only variable used in this comparison is locomotion and data used were from 0-60 min and min period. All these data are shown in Table 1-4. Comparison Ι: Comparison between male and female mice on altered susceptibility to nicotine at adult age (study 1, 3 and 4) In study 1 and 3, the activity in the first period (Table 1), second period (Table 2) and third period (Table 3), there were no significant differences between male mice neonatally exposed to saline or nicotine 33 μg or 66 μg, in each respective study. In study 4, a similar activity in female mice were seen in period 1, period 2 and period 3, as male mice. This shows that in the spontaneous behaviour test, the variable showed a normal treatment time effect and indicates a normal habituation to a novel home environment. In comparing the reaction to nicotine, period 4 (60-80 min), -28-

33 there are some differences between the studies and also in the response between males and females. In all three studies mice given saline neonatally responded with increasing activity with increasing dose of nicotine (40 μg and 80 μg nicotine). This increase was seen in both male and female mice. However, in study 1, the response to nicotine in adult male mice (4 months old) neonatally receiving 33 μg or 66 μg nicotine responded to 80 μg nicotine with a decrease in activity. In study 4, female mice (2 months old) only showed this response when neonatally exposed to higher dose of nicotine (66 μg). In female mice neonatally receiving to 33 μg nicotine, an increase in activity, similar to neonatally saline treated mice, were observed. This indicates that male mice appeared to be more susceptible to developmental neurotoxic effects of nicotine compared to females. But it should be pointed out that the study conducted in female mice were 2 months younger than the male mice. Comparison II: Comparison between male and female mice on altered susceptibility to paraoxon at adult age (study 2 and 5) Study 2, no significant difference in activity in 5 months old male mice neonatally exposed to either saline or nicotine 33 μg were seen. These mice showed a normal decrease in activity during the 60 min period. A similar decrease in activity, and no difference between saline, nicotine 33 or nicotine 66 μg, was also seen in female mice, study 5. However, in female mice neonatally given 66μg nicotine and given paraoxon (0.3 mg/kg b.w.) for one week at adult age showed a significant decrease in activity 24 hours after the last paraoxon injection. Data on the spontaneous behaviour in male mice is lacking, and no comparison between males and females can be done. -29-

34 Two months after the adult exposure to paraoxon, male mice (7 months old) showed a significant hypoactive condition, after neonatal exposure to nicotine 33 μg. A similar hypoactive condition was only seen in female mice neonatally exposed to higher dose of nicotine (66 μg). Furthermore, in male mice a hyperactive condition was seen in period 3 (Table 3) when neonatally exposed to nicotine 33 μg and adult exposed to paraoxon (either 0.17 mg/kg b.w. or 0.3 mg/kg b.w.). The female mice, in study 5, showed the same hyperactive condition when neonatally exposed to nicotine 66 μg and exposed to paraoxon (0.3 mg/kg b.w.) at adult age. No alternation in activity was shown in neonatal nicotine 33 μg treated female mice. From study 2, in period 4 (Table 4), male mice which neonatally were exposed to nicotine 33 μg and responded to paraoxon (either 0.17 mg/kg b.w. or 0.3 mg/kg b.w.) with a significant decrease in activity compared to its control groups at 5-month-old. The female mice, in study 5, showed the similar tendency of activity in neonatally exposure of nicotine 66 μg and response to paraoxon (0.3 mg/kg b.w.), but this tend is not significant. Taken together these data indicate that male mice can be more susceptible to paraoxon at adult age after neonatally exposed to nicotine compared to female mice. However, it should also be said that males were tested 2 months older than female mice. -30-

35 Period month,M 2. 5-month,M 2. 7-month,M 3. 4-month,M 4. 2-month,F 5. 2-month,F 5. 2-month+1-week,F 5. 4-month,F Saline-Saline 245.6± ± ± ± ± ± ± ±108.8 Saline-OP1 (0.17mg/kg b.w.) 654.5± ±47.0 Saline-OP2 (0.3 mg/kg b.w.) 613.1± ± ± ± ±171.4 Saline-Nicotine 40μg 254.4± ± ±114.4 Saline-Nicotine 80μg 230.9± ± ±114.4 Nicotine 33μg-Saline 231.5± ± ± ± ± ± ± ±145.1 Nicotine 33μg-OP ± ±71.0 * Nicotine 33μg-OP ± ±48.2 * 549.9± ± ±130.0 Nicotine 33μg-Nicotine 40μg 277.3± ± ±151.8 Nicotine 33μg-Nicotine 80μg 247.9± ± ±151.8 Nicotine 66μg-Saline 228.6± ± ± ± ± ±191.9 Nicotine 66μg-OP ± ±54.9 * 256.5±49.2 * Nicotine 66μg-Nicotine 40μg 244.0± ± ±92.8 Nicotine 66μg-Nicotine 80μg 227.1± ± ±92.8 Table 1: Spontaneous behaviour test results (locomotion), period 1 (0-20 min). M= male mice, F= female mice, time= mice age when test, * significant different with its respective control (p 0.01). 1. Data taken from the spontaneous behaviour test result of article Eriksson et al Data taken from the spontaneous behaviour test result of article Ankarberg et al Data taken from the spontaneous behaviour test result of Paper Ⅲ from Emma Ankarberg s thesis Data taken from the spontaneous behaviour test result of Master thesis from Adam Söderberg Data taken from the spontaneous behaviour test result of Master thesis from Feiya Luo (the first section of this result part) -31-

36 Period month,M 2. 5-month,M 2. 7-month,M 3. 4-month,M 4. 2-month,F 5. 2-month,F 5. 2-month+1-week,F 5. 4-month,F Saline-Saline 170.5± ± ± ± ± ± ± ±67.1 Saline-OP1 (0.17mg/kg b.w.) 318.3± ±90.9 Saline-OP2 (0.3 mg/kg b.w.) 265.4± ± ± ± ±93.5 Saline-Nicotine 40μg 120.4± ± ±75.1 Saline-Nicotine 80μg 148.4± ± ±75.1 Nicotine 33μg-Saline 107.6± ± ± ± ± ± ± ±43.4 Nicotine 33μg-OP ± ±62.1 Nicotine 33μg-OP ± ± ± ± ±65.0 Nicotine 33μg-Nicotine 40μg 127.5± ± ±111.5 Nicotine 33μg-Nicotine 80μg 155.1± ± ±111.5 Nicotine 66μg-Saline 141.0± ± ± ± ± ±76.0 Nicotine 66μg-OP ± ± ±90.0 Nicotine 66μg-Nicotine 40μg 126.8± ± ±74.7 Nicotine 66μg-Nicotine 80μg 109.1± ± ±74.7 Table 2: Spontaneous behaviour test results (locomotion), period 2 (20-40 min). M= male mice, F= female mice, time= mice age when test. 1. Data taken from the spontaneous behaviour test result of article Eriksson et al Data taken from the spontaneous behaviour test result of article Ankarberg et al Data taken from the spontaneous behaviour test result of Paper Ⅲ from Emma Ankarberg s thesis Data taken from the spontaneous behaviour test result of Master thesis from Adam Söderberg Data taken from the spontaneous behaviour test result of Master thesis from Feiya Luo (the first section of this result part) -32-

37 Period month,M 2. 5-month,M 2. 7-month,M 3. 4-month,M 4. 2-month,F 5. 2-month,F 5. 2-month+1-week,F 5. 4-month,F Saline-Saline 50.4± ± ± ± ± ± ± ±7.3 Saline-OP1 (0.17mg/kg b.w.) 11.0± ±17.7 Saline-OP2 (0.3 mg/kg b.w.) 6.0± ± ± ± ±16.0 Saline-Nicotine 40μg 57.0± ± ±4.0 Saline-Nicotine 80μg 44.1± ± ±4.0 Nicotine 33μg-Saline 45.3± ± ± ± ± ± ± ±9.1 Nicotine 33μg-OP1 10.2± ±67.1 * Nicotine 33μg-OP2 10.0± ±85.0 * 8.8± ± ±8.1 Nicotine 33μg-Nicotine 40μg 52.5± ± ±4.8 Nicotine 33μg-Nicotine 80μg 41.0± ± ±4.8 Nicotine 66μg-Saline 33.1± ± ± ± ± ±12.9 Nicotine 66μg-OP2 7.8± ± ±44.9 * Nicotine 66μg-Nicotine 40μg 33.1± ± ±4.2 Nicotine 66μg-Nicotine 80μg 39.5± ± ±4.2 Table 3: Spontaneous behaviour test results (locomotion), period 3 (40-60 min). M= male mice, F= female mice, time= mice age when test, * significant different with its respective control (p 0.01). 1. Data taken from the spontaneous behaviour test result of article Eriksson et al Data taken from the spontaneous behaviour test result of article Ankarberg et al Data taken from the spontaneous behaviour test result of Paper Ⅲ from Emma Ankarberg s thesis Data taken from the spontaneous behaviour test result of Master thesis from Adam Söderberg Data taken from the spontaneous behaviour test result of Master thesis from Feiya Luo (the first section of this result part) -33-

38 Period month,M 2. 5-month,M 2. 7-month,M 3. 4-month,M 4. 2-month,F 5. 2-month,F 5. 2-month+1-week,F 5. 4-month,F Saline-Saline 131.8± ± ± ± ±45.3 Saline-OP1 (0.17mg/kg b.w.) 312.5±55.6 Saline-OP2 (0.3 mg/kg b.w.) 265.0± ±57.6 Saline-Nicotine 40μg 208.6± ± ±62.1 Saline-Nicotine 80μg 366.0± ±136.8 * 350.5±134.2 * Nicotine 33μg-Saline 109.5± ± ± ± ±62.5 Nicotine 33μg-OP ±48.9 * Nicotine 33μg-OP ±72.9 * 217.1±43.7 Nicotine 33μg-Nicotine 40μg 140.4± ± ±66.2 Nicotine 33μg-Nicotine 80μg 61.1± ±147.1 * 410.1±154.2 * Nicotine 66μg-Saline 150.1± ± ± ±73.7 Nicotine 66μg-OP ±27.6 Nicotine 66μg-Nicotine 40μg 31.0± ± ±21.7 Nicotine 66μg-Nicotine 80μg 25.4± ± ±20.7 Table 4: Spontaneous behaviour test results (locomotion), period 4 (60-80 min). M= male mice, F= female mice, time= mice age when test, * significant different with its respective control (p 0.01). 1. Data taken from the spontaneous behaviour test result of article Eriksson et al Data taken from the spontaneous behaviour test result of article Ankarberg et al Data taken from the spontaneous behaviour test result of Paper Ⅲ from Emma Ankarberg s thesis Data taken from the spontaneous behaviour test result of Master thesis from Adam Söderberg Data taken from the spontaneous behaviour test result of Master thesis from Feiya Luo (the first section of this result part) -34-

39 Discussion The results from the present study shows that neonatal exposure to a substance known to affect the cholinergic system, such as nicotine, at doses that apparently do not cause direct effects on spontaneous behaviour, can make the animals more susceptible to another cholinergic agent, such as paraoxon, at adult age. Furthermore, the adults were exposed to doses that when given to untreated animals have no significant effects. In the spontaneous behaviour in 4-month-old animals after adult exposure to paraoxon at age of 2 months, the effects on spontaneous behaviour, was manifested as a lack of habituation to a novel home environment. This development of alternations in spontaneous behaviour indicates a delayed effect. In comparing females and males, there were some differences observed, female mice appeared to be less sensitive in this alternation than male mice. This sex-related difference was seen by comparing male mice neonatally exposed to nicotine and their response to paraoxon at 5-month-old while female mice neonatally exposed to nicotine their response to paraoxon at 2 month of age. Despite this difference, two months after paraoxon exposure, the change in spontaneous behaviour is the same for male mice and female mice. The effect on the spontaneous behaviour in the adult female mice neonatally exposed to nicotine indicated two different responses to paraoxon, one acute response and also a delayed effect. The first observed in the nicotine-paraoxon groups at 2 months of age, and the second at 4 months of age. At 2 months of age there was a decrease in activity in the spontaneous behaviour in the control animals and the mice neonatally receiving nicotine 33 μg or nicotine 66 μg, and no significant difference between control animals and the neonatally nicotine -35-

40 treated animals were seen. When the animals were given the first paraoxon injection, the neonatally nicotine 66μg-treated animals responded with hypoactive behaviour. These results are similar to those results shown in earlier studies where male mice were exposed to nicotine neonatally (33 or 66 μg/kg b.w.) and response to nicotine or OP at adult age (Ankarberg et al 2001; Eriksson et al. 2000; Ankarberg et al. 2004). This type of behaviour may be the result of an increased concentration of ACh, as nicotine stimulates an increased release of transmitters including ACh (Eriksson 2000) and also by direct activity on nicotinic receptors. OPs will increase the amount of ACh by inhibiting AChE and thereby reduce the degradation of ACh. The acute reaction to paraoxon and change in spontaneous behaviour was also seen after exposure to paraoxon for one week. These animals showed the same differences in spontaneous behaviour during the first period, 24 hours after the last administration of paraoxon. The second and delayed effect was observed when the animals were 4 months old. The neonatally nicotine 66μg- and adult paraoxon-treated animals were hypoactive at the beginning of the test period and hyperactive at the end of the 60 minutes test period, when compared to the control animals. This indicates that with increasing age the animals had developed a persistent difficulty to habituate to a novel home environment. It has earlier been seen that neonatal male mice exposed to a low dose of DDT, a substance that increases neuronal activity, caused behavioural changes in adult animals (Johansson et al. 1996). These animals developed additional behavioural disturbances when they were exposed to paraoxon at adult age, and the disturbances worsened with age, as the spontaneous behaviour was additional changed, especially the rearing variable, together with significantly reduced performance in swim maze -36-

41 test of Morris water maze type. In the present study, the additional behaviour defects were not seen directly after receiving all the paraoxon injections, but appeared 2 months later. This indicates that different environmental agency can alter the adult susceptibility to OPs, and obviously via changes of the function of the cholinergic system. Behaviour is a major function whereby animals adapt to changes in the environment and changes in behaviour may reveal effects on the nervous system caused by the influence of a toxicant. Spontaneous behaviour is especially meaningful as it reflects a function dependent on the integration of a sensory input into a motor output, thus revealing the ability of animals to habituate to a novel home environment and integrate new information with previously attained. It can thereby also be used in order to measure cognitive functions. Cholinergic dysfunctions have been presented to cause impairments in learning and memory and cognitive functions (Bartus et al. 1982). Ageing is also known to cause impairments in learning and memory functions (Gage et al. 1984; Gallagher et al. 1993; Lamberty and Gower 1990). In Lamberty and Gower study, normal NMRI mice exhibited changes in spontaneous behaviour and learning from the age of 9 months. In the present study, the behavioural changes appear earlier in life and worsen over time, it seems therefore possible that neonatal nicotine exposure and adult paraoxon treatment may have brought this ageing process forward. The comparison between male and female mice in reaction to nicotine and paraoxon indicate that females might be less susceptible. Female mice neonatally exposed to nicotine (66 μg/ kg b.w.) and receiving paraoxon exposure at 2 month of age showed a hypoactive condition in the spontaneous behaviour -37-

42 during the fourth test period (60-80 min), while male mice showed this change already at nicotine 33 μg. In 2 monthold mice, no significant difference was seen in female mice receiving 33 μg nicotine and adult exposure to paraoxon. This difference between males and females might be sex-related difference and indicate that female mice neonatally exposed to nicotine might be less susceptible to paraoxon at adult age. However, the observed sex difference might also be due to that the studies in male mice were conducted in 2 month older animals, compared to the studies in female mice. Smolen et al. (1987) have suggested an increased susceptibility in adult males to neurotoxicity and longterm behavioural changes following OP poisoning. In the present comparison study, male mice show an increased susceptibility in response to paraoxon when neonatally exposed to nicotine. Whether the observed changes may be due to sex-related pharmacokinetic differences of OPs or endocrine differences are not known and remain further studies. -38-

43 Concluding Remarks In conclusion, this study indicates that low dose exposure to paraoxon which causing low AChE inhibition can induce permanent effects on behaviour in adult animals neonatally exposed to low doses of nicotine. No effects on spontaneous behaviour were seen in adult mice neonatally exposed to saline vehicle and response to the same paraoxon doses at adult age. This indicates that differences in adult susceptibility to environmental pollutants are not necessarily inherited. It might just as well be acquired by low dose exposure to different toxic agents during a critical period of brain development in early life. This study also indicates that female mice neonatally exposed to nicotine appeared to be less sensitive than male mice in alternation of susceptibility to adult paraoxon exposure or nicotine exposure. Since the observed sex-related difference might be due to that the data taken from the studies in male mice were from 2 months older animals compared to the studies in female mice, further comparisons need to be done. -39-