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Regulatory Toxicology and Pharmacology 54 (2009) 282–286
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Acute toxicity assessment of choline by inhalation, intraperitoneal and oral routes in Balb/c mice Amit Kumar Mehta a,b, Naveen Arora a, Shailendra Nath Gaur c, Bhanu Pratap Singh a,* a
Allergy and Immunology Section, Institute of Genomics and Integrative Biology, Delhi 110007, India Department of Biotechnology, University of Pune, Pune 411007, India c Department of Pulmonary Medicine, V. P. Chest Institute, University of Delhi, Delhi 110007, India b
a r t i c l e
i n f o
Article history: Received 6 February 2009 Available online 19 May 2009 Keywords: Animal model Asthma Choline Inhalation Oral gavage Toxicity
a b s t r a c t Studies suggest that choline has potential to be used as a dietary supplement and a drug for immune inflammatory diseases like asthma and rhinitis. But there are apprehensions regarding adverse effects of choline when given orally in high doses. To address this knowledge gap, toxicity assessment of choline chloride was carried out by intranasal (i.n.), oral and intraperitoneal (i.p.) routes in Balb/c mice for 28 days. Body weight, food and water consumption of mice were recorded daily. Hematology and clinical chemistry were assessed to check hepatocellular functions and morphological alterations of the cells. Splenocyte counts were analysed for evaluating cellular immunity. Liver function test was performed by assaying different enzyme systems in serum such as, urea, blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Body weight, food and water consumption did not differ between mice treated with choline and the saline control group. Hematologic and biochemical variables were not affected with any increase in serum toxicity marker enzymes indicating normal liver functioning. Choline administration did not affect total cholesterol and high density lipoprotein levels as compared to their respective controls. Urea and blood urea nitrogen levels in choline treated mice were not different than controls. Creatinine level was, however, higher than control in i.p. treatment group, but other parameters were normal. In conclusion, the repeated consumption of choline chloride via i.n. and oral or i.p. routes did not cause toxicity in mice in the toxicological endpoints examined. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Choline has had a wide use as a food additive since the early 1930s. It is a lipotropic factor, and plays a crucial role in mobilization of fats in liver, essential for acetylcholine (ACh) formation and used for phosphatidyl choline (PC) synthesis by de novo pathway (Pelech and Vance, 1984; Blusztajn, 1998). It has shown antiinflammatory activity in arthritis and also in an allergic asthma animal model (Ganley et al., 1958; Mehta et al., 2007). Choline has been shown to produce antinociceptive effects against inflammatory pain, which strongly supports the involvement of alpha-7 nAChRs in the antinociception of choline (Wang et al., 2005). Choline magnesium trisalicylate improved symptoms in patients with aspirin induced asthma (Szczeklik et al., 1990). Studies show that tricholine citrate reduces symptoms and drug requirement in asthma patients (Gupta and Gaur, 1997; Gaur et al., 1997).
* Corresponding author. Address: Allergy and Immunology Section, Institute of Genomics and Integrative Biology, Delhi University Campus, Room No. 509, Mall Road, Delhi 110007, India. Fax: +91 11 27667471. E-mail address: [email protected] (B.P. Singh). 0273-2300/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2009.05.009
Choline is a dietary component, occurs both as free choline and as phosphatidylcholine (PC), such as lecithin, in egg yolk, vegetables and animal fat (Zeisel, 2000). It is important for the structural integrity of cell membranes, methyl-group metabolism, cholinergic neurotransmission, transmembrane signalling, lipid and cholesterol transport and general metabolism. Human cells grown in culture deprived of choline died due to apoptosis (Yen et al., 1999). Choline deficiency also results in production of reactive oxygen species (ROS) and increase in oxidative stress in liver and plasma as one of the pathogenic mechanism of mitochondrial and oxidative damage (Yoshida et al., 2006; Ossani et al., 2007). There is an endogenous pathway for the de novo biosynthesis of the choline moiety via sequential methylation of phosphatidyl-ethanolamine using S-adenosyl-methionine as the methyl donor (Bremer and Greenberg, 1961). Thus, the demand for dietary choline is modified by metabolic methyl-exchange relationships between choline and three nutrients namely methionine, folate, and vitamin B12 (Zeisel and Blusztajn, 1994). There is debate, whether choline is an essential component of the diet, due to the de novo synthesis in the body (Zeisel, 2000). The evidence suggests that such synthesis is not always sufficient
A.K. Mehta et al. / Regulatory Toxicology and Pharmacology 54 (2009) 282–286
to meet human requirement. The U.S. Institute of Medicine (IOM) established an adequate intake (AI) for adults i.e. 550 mg/day of choline for men and 425 mg/day for women and tolerable upper intake level (UL) for adults is 3.5 g/day (IOM, DRI, 1998). The primary criterion used to estimate the AI for choline is the prevention of liver damage as assessed by measuring serum alanine aminotransferase levels in normal healthy subjects. However, the symptoms of hypotension and trimethylaminuria (fishy body odour) were selected as the critical effect in deriving the UL for choline in patients treated with oral administration of choline chloride (Boyd et al., 1977; Growdon et al., 1977; Lawrence et al., 1980). Despite beneficial effects, choline intake as a dietary supplement requires safety assessment. High doses of choline have been associated with trimethylaminuria, vomiting, salivation, sweating and diarrhoea (Busby et al., 2004). The animal data provide supportive evidence for a low degree of toxicity of choline. Further, animal studies have indicated growth suppression at high intake of choline (LSRO/FASEB, 1975). But due to large doses and i.v./i.p. routes of administration in these reports, they were not considered relevant to human intakes from food and supplements. The present study was aimed to assess the toxicity of choline through intranasal (i.n.) route and compared with oral and intraperitoneal (i.p.) route in Balb/c mice.
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study provides information on the possible health hazards likely to arise from repeated exposure over a relative period of time (OECD, 1981, 1995). The present study was undertaken following good laboratory practices under OECD guidelines. 2.2. Hematology and clinical chemistry At the end of the study, mice were deprived of food overnight and sacrificed on day 29 by overdose of sodium pentobarbital (100 mg/kg; i.p.) and the blood was collected. Blood was centrifuged at 1500g for 15 min, sera separated and stored at 20 °C for immunoassays. Total and differential cell counts were made using Leishman stain. Urea, blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were determined for liver and kidney functioning using commercial kits (Span Diagnostics Pvt. Ltd., India). Total cholesterol (TC) and high density lipoprotein (HDL) levels were measured for normal cardiac functioning (Monozyme Pvt. Ltd., India). 2.3. Cell counts Spleen was removed aseptically from mice and single cell suspension was made in RPMI-1640 medium. Viable splenic cells in each case were counted using hemocytometer by trypan blue exclusion test.
2. Materials and methods 2.4. Pathology 2.1. Animals Balb/c mice of both sexes (6–8 weeks), weighing 18–20 g, obtained from National Institute of Virology, Pune, India were quarantined for 10 days to get acclimatized in experimental conditions. They were placed in cages at 22–25 °C, with 40–70% relative humidity and controlled 12-h light:dark cycle. Water and standard chow diet were given ad libitum. The study protocol was approved by the animal ethics committee of Institute of Genomics and Integrative Biology (IGIB), Delhi. All animal experiments were carried out in the morning to minimize the effects of circadian rhythm. Mice were randomly divided into six groups of 10 animals each of both sexes. In three groups (Groups 1, 3 and 5), choline was administered through oral, i.p. and i.n. routes, separately. As control(s), saline (0.9% sodium chloride) was given in other three groups (Groups 2, 4 and 6) by the same routes as above. For oral route, choline chloride (200 mg/kg) was administered once daily till 28 day by oral gavage (Group 1). Mice received choline chloride (200 mg/kg) in 100 ll saline by i.p. at every alternate day for 28 days (Group 3). However, for i.n. dose (200 mg/kg), choline chloride was given in 50 ll vehicle for every alternate day for 28 days (Group 5). Control mice were given 100 ll normal saline by oral gavage and or i.p. or 50 ll saline through i.n. route for 28 days by the same protocol. Mice receiving i.n. dose were lightly anesthetized (3% isoflurane) before administering each dose. The maximum level of daily tolerable upper intake level (UL) that is likely to pose no risk of adverse effects is 3.5 g/day for 19 years and older subjects (IOM, DRI, 1998), which is equivalent to approx. 60 mg/kg for a normal adult human of approx. 60 kg. In the present study, dose of 200 mg/kg was chosen, which is approx. 3.5 times higher as compared to tolerable upper intake level (UL) for adults to assess the toxicity by inhalation (i.n.), oral and intraperitoneal (i.p.) routes. Body weight, food and water consumption of mice were recorded daily along with signs of distress, if any. Skin, eyes, mucous membrane and behaviour of mice were examined daily. Growth rate was calculated as the difference between the final weight and the initial weight divided by 28 days. The 28-day repeat dose
Detailed gross necropsy, including careful examination of the body’s external surfaces, orifices and cranial, thoracic and abdominal cavities and their content was performed in all the groups. The lungs, heart, liver, spleen, and kidney were excised, trimmed of any adherent tissue and their wet weights were recorded. 2.5. Histopathology The excised organs were fixed in 10% neutral buffered formalin (pH 7.4) and coded samples were given to a pathologist for the study. After fixation, lung sections of 4 lm were cut and stained with hematoxylin and eosin (H&E). Histological evaluation of vital organs like lung, heart, liver, spleen and kidney sections was done using light microscope. 2.6. Statistics Results were expressed as means ± SD. Statistical differences between means were compared by Student’s t-test. Multigroup comparisons of the means were carried out by ANOVA. The P < 0.05 was considered as significant.
3. Results 3.1. Body weight, food and water consumption Daily administration of choline chloride via three different routes at 200 mg/kg caused no adverse effects or mortality during the experimental period of 28 days. There were no statistically significant differences between choline treated groups and their respective controls in the body weight (Table 1; P > 0.05). Growth rates of choline treated groups were not significantly different with their respective controls (Table 1; P > 0.05). The mice did not differ in food or water consumption in choline or saline (control) groups. The mean body weight increase, and mean food consumption of the mice given choline via different routes did not differ significantly from their respective controls (P > 0.05).
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Table 1 Effect of choline chloride on body weight of Balb/c mice. Days
Oral
i.p.
i.n.
Choline
Control
Choline
Control
Choline
Control
0 7 14 21 28
21.0 ± 0.70 22.70 ± 0.52 23.52 ± 0.29 24.64 ± 0.22 25.38 ± 1.70
20.82 ± 0.87 22.68 ± 0.61 23.57 ± 0.24 24.24 ± 0.16 24.98 ± 2.15
21.18 ± 1.02 22.82 ± 0.57 22.96 ± 0.05 24.96 ± 0.21 25.14 ± 0.98
21.05 ± 0.38 22.14 ± 0.81 22.36 ± 0.15 25.12 ± 0.11 25.05 ± 1.28
20.86 ± 0.63 22.2 ± 0.63 22.66 ± 0.37 24.95 ± 0.19 25.24 ± 2.22
20.97 ± 0.42 21.89 ± 0.41 22.49 ± 0.25 24.88 ± 0.29 25.19 ± 1.33
Growth rate
0.156 ± 0.09
0.148 ± 0.07
0.141 ± 0.10
0.143 ± 0.11
0.156 ± 0.07
0.151 ± 0.12
Control, saline.
3.2. Hematology and clinical bio-chemistry
3.3. Splenic cell counts
Red blood cell counts of choline treated groups were not significantly different to their respective controls (P > 0.05). Haemoglobin levels were also not different in controls and choline treated mice by all the three routes (P > 0.05). White blood cell counts (WBC) and neutrophils, lymphocytes, eosinophils and monocytes were not altered after the choline treatment and were not statistically different to their respective control groups (P > 0.05). Thus, the hematologic parameters did not indicate any adverse changes due to choline administration (Table 2). After 28 days of choline treatment, there were no significant differences (P > 0.05) in ALT levels of choline treated groups as compared to their respective saline control groups (Table 3). Choline administration did not result in increased activity of serum toxicity marker enzymes (ALT, AST), indicating normal liver functioning. However, 28-day treatment with choline through i.p. route led to a increase in creatinine level (0.35 ± 0.07 mg/dl) compared to control group (0.24 ± 0.06 mg/dl; P < 0.05). The creatinine levels in mice treated with choline by oral (0.23 ± 0.09 mg/dl) or i.n. (0.25 ± 0.12 mg/dl) were not statistically different to their respective controls (P > 0.05). Urea and blood urea nitrogen levels in choline treated mice were not statistically different than saline treated mice through all the three routes. Choline administration did not affect total cholesterol and HDL levels as compared to their respective controls (P > 0.05).
The effect of choline on growth inhibition of mononuclear cells was examined to evaluate the choline induced immuno-suppression (Fig. 1). In the i.n. choline treated mice, the number of mononuclear cells was 170.2 106 cells/ll ± 2.16 which was not significantly different to its control as 169.4 106 cells/ll ± 2.49 (P > 0.05). Choline administration did not affect mononuclear cells in oral and i.p. groups as compared to their respective controls (P > 0.05). 3.4. Pathology On day 29, mice were subjected to a detailed post-mortem examination of the internal organs. There were no macroscopic differences in size, colour or texture of the organs. The repeated choline administration did not affect the weight of lungs, heart, liver, spleen or kidney (Table 4). The weight of other organs was comparable to their respective controls. 3.5. Histopathology No pathological signs were observed in the vital organs examined for the study (data not shown). Choline administration did not affect liver tissue of mice with no signs of morphological variations in its lining with respect to their controls. Kidney sections of mice treated with choline were indistinguishable from kidney of
Table 2 Hematological parameters in mice treated with choline or saline control. Parameters
Hb (g/dl) RBC (106/mm3) WBC (106/mm3) Monocytes Lymphocytes Neutrophils Eosinophils
Oral
i.p.
i.n.
Choline
Control
Choline
Control
Choline
Control
16.28 ± 0.70 7.08 ± 0.52 4.92 ± 0.29 2.44 ± 0.22 72.67 ± 1.70 23.45 ± 1.64 0.97 ± 0.17
16.58 ± 0.87 6.98 ± 0.61 4.59 ± 0.24 2.92 ± 0.16 73.53 ± 2.15 22.97 ± 1.63 0.94 ± 0.19
16.26 ± 1.02 7.05 ± 0.57 4.64 ± 0.05 2.94 ± 0.21 72.54 ± 0.98 23.87 ± 1.41 0.89 ± 0.09
16.23 ± 0.38 6.94 ± 0.81 4.78 ± 0.15 3.51 ± 0.11 72.76 ± 1.28 23.41 ± 1.31 0.95 ± 0.14
16.67 ± 0.63 6.67 ± 0.63 4.71 ± 0.37 3.20 ± 0.19 72.85 ± 2.22 23.65 ± 1.33 0.94 ± 0.18
16.84 ± 0.42 6.98 ± 0.41 4.48 ± 0.25 3.61 ± 0.29 73.21 ± 1.33 22.93 ± 0.98 0.97 ± 0.19
Table 3 Clinical bio-chemistry parameters in mice treated with choline or saline control. Parameters
Urea (mg/dl) BUN (mg/dl) HDL (mg/dl) TC (mg/dl) ALT (IU/L) AST (IU/L) Creatinine (mg/dl) *
Oral
i.p.
i.n.
Choline
Control
Choline
Control
Choline
Control
59.35 ± 1.59 26.98 ± 1.48 266 ± 0.29 20.44 ± 0.22 47.2 ± 4.22 88.42 ± 4.29 0.23 ± 0.09
58.59 ± 1.03 27.00 ± 0.71 262.9 ± 0.24 21.92 ± 0.16 46.6 ± 5.68 88.26 ± 4.97 0.21 ± 0.10
53.46 ± 1.54 25.26 ± 1.64 264.6 ± 0.05 20.94 ± 0.21 43.54 ± 1.89 91.51 ± 3.04 0.35 ± 0.07*
55.20 ± 2.57 27.17 ± 2.01 263.9 ± 0.15 22.51 ± 0.11 46.04 ± 3.64 90.49 ± 4.28 0.24 ± 0.06
57.11 ± 3.00 27.88 ± 1.79 264.1 ± 0.37 21.20 ± 0.19 47.51 ± 3.36 88.90 ± 6.21 0.25 ± 0.12
57.22 ± 1.55 28.46 ± 1.24 265.7 ± 0.25 22.61 ± 0.29 48.72 ± 2.95 90.33 ± 3.8 0.22 ± 0.08
Represent significance difference from the control group.
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6
splenic cells (×10 cells/ul)
174
171
168
165 oral choline oral control i.p. choline i.p. control i.n. choline i.n. control Fig. 1. Spleen cell counts in mice treated with choline or saline control.
control groups. All the other vital organs (lung, heart, spleen) appeared normal and did not show any alterations in structure due to choline treatment by all the three routes. 4. Discussion Asthma and allergies afflict a sizeable population worldwide (Umetsu et al., 2002). Despite the progress made in pharmacotherapy, the incidence of asthma has increased during the last three decades. Pharmacotherapy has adverse effects on long term use and the symptoms reoccur on drug withdrawal. Therefore, new drugs with minimum side effects are required for the prophylactic or symptomatic treatment. Asthma has been hypothesized as being a metabolic disorder since the patient with asthma has altered fat metabolism (Agarwal et al., 1986; Agarwal, 1987; Johnson et al., 2007; Shore, 2008). Choline administration in asthmatics has shown lysophosphatidyl choline lowering effect (Agarwal, 1987). It was also effective in inhibiting airway inflammation in the mouse model of airway hyperresponsiveness (Mehta et al., 2007). There are apprehensions regarding adverse effects of choline since some of the patients showed trimethylaminuria, nausea and diarrhoea during the treatment (Boyd et al., 1977; LSRO/FASEB, 1981; Gaur et al., 1997; Busby et al., 2004). In the present study, choline chloride has been evaluated for acute toxicity in Balb/c mice via oral, i.p. and i.n. routes for 28 days, according to OECD guidelines (OECD, 1995). Toxicity resulting from exposure could occur at the various portal of entry, such as the lungs and skin, or at other distant sites. Inhalation is an important route of exposure in clinical practice for asthma, rhinitis, etc., since the drugs can be delivered directly into the target organ. Pulmonary inflammation is a common response to various particles and associated with other pathological outcomes after exposure to these particles (Bajpai et al., 1992). In the present study, intranasal administration of choline in mice did not cause pulmonary inflammation as determined by blood and spleen cells counts and lung histopathology. The examination of vital organs carried out during the autopsy did not reveal any alterations in i.n. choline group and were comparable with their respective control as well as with other choline treatment groups
(oral or i.p.). In fact, many drugs administered intranasally are often absorbed faster and more efficiently than those from oral administration translating into a quick uptake of drug into the bloodstream and often resulting in a faster onset of action (O’Hagan and Illum, 1990). In the present study, oral, i.p. or i.n. choline treatment for 28 days did not affect the body weight or the mean growth rate of mice compared to their respective controls. These results indicate that in terms of growth, choline is well tolerated by animals at the doses and routes of administration tested. The UL of choline in adults is 3.5 g/day. However, the present study was performed using a dose of choline (200 mg/kg) in excess of the dose as used in previous study (Mehta et al., 2007). High doses of oral choline (more than 10 g/day and or 150–300 mg/kg/day) administered in certain diseases have been associated with adverse effects like trimethylaminuria, vomiting, salivation, sweating and diarrhoea as observed in previous human studies (Boyd et al., 1977; Growdon et al., 1977; Gelenberg et al., 1979; Lawrence et al., 1980; Busby et al., 2004). A study by Sahu et al., reported adverse effects on cellularity of spleen, thymus, peripheral lymph node and pathological lesions in lungs and lymph node, when choline chloride was given via i.p. route for 8 months (Sahu et al., 1986). However, in the present study no adverse effects were observed in disease free mice administered with high doses of choline for 28 days. Furthermore, the present experiments were carried out in rodent species and the response may not be always comparable with humans. The peripheral cell counting is used to indicate a change in quality and quantity of precursor cells, and to determine the toxicity based on hematological parameters (Schofeld, 1986). The destruction of RBCs indicates the abnormality in hepatocellular functions and morphological alterations of the cells indicate the changes in phospholipid ratio and contents (Sherlock and Dooley, 1993). The increase in neutrophils and eosinophils leads to the production of reactive oxygen species during inflammatory reactions and damage the surrounding tissues. In the present study, hematological parameters (WBCs, Hb, lymphocytes, monocytes, etc.) were not affected with choline treatment by any of the three routes tested. Liver plays an essential role in the synthesis of plasma proteins and different globulins. The elevated levels of ALT and AST are the conventional indicator of hepatic damage (Fu et al., 2009). ALT is the most reliable variable for the evaluation of hepatic toxicity whereas AST is not as useful because of its wide distribution (Ringler and Dabich, 1979). In the present study, the high density lipoprotein profile was not affected by choline administration. ALT levels indicate hepatic integrity that did not differ between control and choline treated mice. AST too did not differ between choline treated mice compared to control groups. The enzyme levels suggest no alteration of hepatic function in choline treated mice than controls. Creatinine levels were, however, higher in i.p. choline treated animals as compared to control. This indicates that the drug had an effect on the metabolism and functioning of the kidney might be due to the route (i.p.) tested as the same was not observed in i.n. or oral routes. However, other parameters were normal in i.p. treated group as compared to control. In conclusion, the choline administration by i.n., oral or i.p. route did not cause harmful effects in terms of hematology, clinical bio-
Table 4 Organ weight of mice administrated with choline and or saline control for 28 days. Organ weights
Lung Heart Liver Spleen Kidney
Oral
i.p.
i.n.
Choline
Control
Choline
Control
Choline
Control
0.209 ± 0.03 0.150 ± 0.02 1.134 ± 0.09 0.228 ± 0.02 0.192 ± 0.01
0.206 ± 0.02 0.151 ± 0.01 1.118 ± 0.08 0.230 ± 0.01 0.194 ± 0.02
0.217 ± 0.04 0.156 ± 0.03 1.151 ± 0.08 0.220 ± 0.02 0.202 ± 0.01
0.211 ± 0.02 0.152 ± 0.02 1.129 ± 0.09 0.231 ± 0.01 0.198 ± 0.01
0.207 ± 0.03 0.152 ± 0.04 1.122 ± 0.07 0.231 ± 0.03 0.193 ± 0.01
0.210 ± 0.02 0.149 ± 0.02 1.113 ± 0.08 0.225 ± 0.04 0.196 ± 0.02
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chemistry and histopathology of various organs in mice. Inhalation is the preferred route for drug delivery in asthma. The knowledge generated here on acute toxicity assessment of choline chloride will support for clinical trial with choline (inhalation and oral) in asthma patients. However, additional toxicological assessments involving longer inhalation exposures will be helpful for better understanding of the potential toxicity of inhaled choline chloride. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments A.K.M. is thankful to Indian Council of Medical Research, New Delhi for senior research fellowship. Authors thank CSIR task force for providing funds to this study. References Agarwal, K.P., Mehta, D., Gupta, S., Chhabra, S.K., 1986. Metabolic basis for asthma and rhinitis: an integrated approach. Lung 164, 279–291. Agarwal, K.P., 1987. Metabolic basis of asthma: a united hypothesis. Chest 91, 148s– 151s. Bajpai, R., Waseem, M., Gupta, G.S., Kaw, J.L., 1992. Ranking toxicity of industrial dusts by bronchoalveolar lavage fluid analysis. Toxicology 73, 161–167. Blusztajn, J.K., 1998. Choline, a vital amine. Science 281, 794–795. Boyd, W.D., Graham, W.J., Blackwood, G., Glen, I., McQueen, J., 1977. Clinical effects of choline in Alzheimer senile dementia. Lancet 2, 7–11. Bremer, J., Greenberg, D., 1961. Methyl transferring enzyme system of microsomes in the biosynthesis of lecithin (phosphatidylcholine). Biochem. Biophys. Acta 46, 205–216. Busby, M.G., Fischer, L., da Costa, K.A., Thompson, D., Mar, M.H., Zeisel, S.H., 2004. Choline and betaine-defined diets for use in clinical research and for the management of trimethylaminuria. J. Am. Diet. Assoc. 104, 1836–1845. Fu, X., Ji, R., Dam, J., 2009. Acute, subacute toxicity and genotoxic effect of BioQuinone Q10 in mice and rats. Regul. Toxicol. Pharmacol. 53, 1–5. Ganley, O.H., Graessle, O.E., Robinson, H.J., 1958. Anti-inflammatory activity of components obtained from egg-yolk, peanut oil and soya-bean lecithin. J. Lab. Clin. Med. 51, 709–714. Gaur, S.N., Agarwal, G., Gupta, S.K., 1997. Use of LPC antagonist, choline, in the management of bronchial asthma. Ind. J. Chest Dis. Allied Sci. 39, 107–113. Gelenberg, A.J., Doller-Wojcik, J.C., Growdon, J.H., 1979. Choline and lecithin in the treatment of tardive dyskinesia: preliminary results from a pilot study. Am. J. Psychiatry 136, 772–776. Growdon, J.H., Hirsch, M.J., Wurtman, R.J., Wiener, W., 1977. Oral choline administration to patients with tardive dyskinesia. N. Engl. J. Med. 297, 524–527. Gupta, S.K., Gaur, S.N., 1997. A placebo controlled trial of two dosage of LPC antagonist-choline in the management of bronchial asthma. Ind. J. Chest Dis. Allied Sci. 39, 149–156. Institute of Medicine, National Academy of Sciences USA, 1998. Dietary reference intakes for folate, thiamin, riboflavin, niacin, vitamin B12, panthothenic acid, biotin, and choline. National Academy Press, Washington, DC.
Johnson, J.B., Summer, W., Cutler, R.G., Martin, B., Hyun, D.H., Dixit, V.D., Pearson, M., Nassar, M., Telljohann, R., Maudsley, S., Carlson, O., John, S., Laub, D.R., Mattson, M.P., 2007. Alternate day calorie restriction improves clinical findings and reduces markers of oxidative stress and inflammation in overweight adults with moderate asthma. Free Radic. Biol. Med. 42, 665–674. Lawrence, C.M., Millac, P., Stout, G.S., Ward, J.W., 1980. The use of choline chloride in ataxic disorders. J. Neurol. Neurosurg. Psychiatry 43, 452–454. LSRO/FASEB (Life Sciences Research Office/Federation of American Societies for Experimental Biology), 1975. Evaluation of the Health Aspects of Choline Chloride and Choline Bitartarte as Food Ingredients. Report #PB-223845/9. Department of Health, Education and Welfare Washington, DC. LSRO/FASEB (Life Sciences Research office/Federation of American Societies for Experimental Biology), 1981. Effects of Consumption of Choline and Lecithin on Neurological and Cardiovascular Systems. Report #PB-82-133257. Bethesda, MD. Mehta, A.K., Gaur, S.N., Arora, N., Singh, B.P., 2007. Effect of choline chloride in allergen induced mice model of airway inflammation. Eur. Respir. J. 30, 662– 671. O’Hagan, D.T., Illum, L., 1990. Absorption of peptides and proteins from the respiratory tract and the potential for development of locally administered vaccine. Crit. Rev. Ther. Drug Carrier Syst. 7, 35–97. Organization for Economic Cooperation and Development, OECD, 1981. Guideline for Testing of Chemicals. Repeated Dose Inhalation Toxicity: 28-day or 14-day Study Test. Guideline 412. Organization for Economic Cooperation and Development, OECD, 1995. Guidelines for Testing Chemicals. Repeated Dose 28-d Oral Toxicity Study in Rodents, No. 407. Paris, France. Ossani, G., Dalghi, M., Repetto, M., 2007. Oxidative damage lipid peroxidation in the kidney of choline-deficient rats. Front. Biosci. 12, 1174–1183. Pelech, S.L., Vance, D.E., 1984. Regulation of phosphatidyl choline synthesis. Biochem. Biophys. Acta 779, 217–251. Ringler, D.H., Dabich, L., 1979. Haematology and clinical biochemistry. In: Baker, H.J., Lindsey, J.R., Weisbroth, S.H. (Eds.), The Laboratory Rat, vol. I. Biology and Diseases, Academic Press Inc., San Diego, pp. 105–121. Sahu, A.P., Saxena, A.K., Singh, K.P., Shanker, R., 1986. Effect of chronic choline administration in rats. Ind. J. Exp. Biol. 24, 91–96. Schofeld, R., 1986. Assessment of cytotoxicity injury to bone marrow. Br. J. Cancer 53, 115–125. Sherlock, S., Dooley, J., 1993. Disease of the Liver and Biliary System. ninth ed. Blackwell Scientific Publication, Oxford. Shore, S.A., 2008. Obesity and asthma: possible mechanisms. J. Allergy Clin. Immunol. 121, 1087–1093. Szczeklik, A., Nizankowska, E., Dworski, R., 1990. Choline magnesium trisalicylate in patients with aspirin-induced asthma. Eur. Respir. J. 3, 535–539. Umetsu, D.T., McIntire, J.J., Akbari, O., Macaubas, C., DeKruyff, R.H., 2002. Asthma: an epidemic of dysregulated immunity. Nat. Immunol. 3, 715–720. Wang, Y., Su, D.M., Wang, R.H., Liu, Y., Wang, H., 2005. Antinociceptive effects of choline against acute and inflammatory pain. Neuroscience 132, 49–56. Yen, C.L., Mar, M.H., Zeisel, S.H., 1999. Choline deficiency-induced apoptosis in PC12 cells is associated with diminished membrane phosphatidylcholine and sphingomyelin, accumulation of ceramide and diacylglycerol, and activation of a caspase. FASEB J. 13, 135–142. Yoshida, Y., Itoh, N., Hayakawa, M., Habuchi, Y., Inoue, R., Chen, ZH., Cao, J., Cynshi, O., Niki, E., 2006. Lipid peroxidation in mice fed a choline-deficient diet as evaluated by total hydroxyoctadecadienoic acid. Nutrition 22, 303–311. Zeisel, S.H., Blusztajn, J.K., 1994. Choline and human nutrition. Annu. Rev. Nutr. 14, 269–296. Zeisel, S.H., 2000. Choline, needed for normal development of memory. J. Am. Coll. Nutr. 19, 528S–531S.
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Acute toxicity assessment of choline by inhalation, intraperitoneal and oral routes in Balb/c mice Amit Kumar Mehta a,b, Naveen Arora a, Shailendra Nath Gaur c, Bhanu Pratap Singh a,* a
Allergy and Immunology Section, Institute of Genomics and Integrative Biology, Delhi 110007, India Department of Biotechnology, University of Pune, Pune 411007, India c Department of Pulmonary Medicine, V. P. Chest Institute, University of Delhi, Delhi 110007, India b
a r t i c l e
i n f o
Article history: Received 6 February 2009 Available online 19 May 2009 Keywords: Animal model Asthma Choline Inhalation Oral gavage Toxicity
a b s t r a c t Studies suggest that choline has potential to be used as a dietary supplement and a drug for immune inflammatory diseases like asthma and rhinitis. But there are apprehensions regarding adverse effects of choline when given orally in high doses. To address this knowledge gap, toxicity assessment of choline chloride was carried out by intranasal (i.n.), oral and intraperitoneal (i.p.) routes in Balb/c mice for 28 days. Body weight, food and water consumption of mice were recorded daily. Hematology and clinical chemistry were assessed to check hepatocellular functions and morphological alterations of the cells. Splenocyte counts were analysed for evaluating cellular immunity. Liver function test was performed by assaying different enzyme systems in serum such as, urea, blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Body weight, food and water consumption did not differ between mice treated with choline and the saline control group. Hematologic and biochemical variables were not affected with any increase in serum toxicity marker enzymes indicating normal liver functioning. Choline administration did not affect total cholesterol and high density lipoprotein levels as compared to their respective controls. Urea and blood urea nitrogen levels in choline treated mice were not different than controls. Creatinine level was, however, higher than control in i.p. treatment group, but other parameters were normal. In conclusion, the repeated consumption of choline chloride via i.n. and oral or i.p. routes did not cause toxicity in mice in the toxicological endpoints examined. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Choline has had a wide use as a food additive since the early 1930s. It is a lipotropic factor, and plays a crucial role in mobilization of fats in liver, essential for acetylcholine (ACh) formation and used for phosphatidyl choline (PC) synthesis by de novo pathway (Pelech and Vance, 1984; Blusztajn, 1998). It has shown antiinflammatory activity in arthritis and also in an allergic asthma animal model (Ganley et al., 1958; Mehta et al., 2007). Choline has been shown to produce antinociceptive effects against inflammatory pain, which strongly supports the involvement of alpha-7 nAChRs in the antinociception of choline (Wang et al., 2005). Choline magnesium trisalicylate improved symptoms in patients with aspirin induced asthma (Szczeklik et al., 1990). Studies show that tricholine citrate reduces symptoms and drug requirement in asthma patients (Gupta and Gaur, 1997; Gaur et al., 1997).
* Corresponding author. Address: Allergy and Immunology Section, Institute of Genomics and Integrative Biology, Delhi University Campus, Room No. 509, Mall Road, Delhi 110007, India. Fax: +91 11 27667471. E-mail address: [email protected] (B.P. Singh). 0273-2300/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2009.05.009
Choline is a dietary component, occurs both as free choline and as phosphatidylcholine (PC), such as lecithin, in egg yolk, vegetables and animal fat (Zeisel, 2000). It is important for the structural integrity of cell membranes, methyl-group metabolism, cholinergic neurotransmission, transmembrane signalling, lipid and cholesterol transport and general metabolism. Human cells grown in culture deprived of choline died due to apoptosis (Yen et al., 1999). Choline deficiency also results in production of reactive oxygen species (ROS) and increase in oxidative stress in liver and plasma as one of the pathogenic mechanism of mitochondrial and oxidative damage (Yoshida et al., 2006; Ossani et al., 2007). There is an endogenous pathway for the de novo biosynthesis of the choline moiety via sequential methylation of phosphatidyl-ethanolamine using S-adenosyl-methionine as the methyl donor (Bremer and Greenberg, 1961). Thus, the demand for dietary choline is modified by metabolic methyl-exchange relationships between choline and three nutrients namely methionine, folate, and vitamin B12 (Zeisel and Blusztajn, 1994). There is debate, whether choline is an essential component of the diet, due to the de novo synthesis in the body (Zeisel, 2000). The evidence suggests that such synthesis is not always sufficient
A.K. Mehta et al. / Regulatory Toxicology and Pharmacology 54 (2009) 282–286
to meet human requirement. The U.S. Institute of Medicine (IOM) established an adequate intake (AI) for adults i.e. 550 mg/day of choline for men and 425 mg/day for women and tolerable upper intake level (UL) for adults is 3.5 g/day (IOM, DRI, 1998). The primary criterion used to estimate the AI for choline is the prevention of liver damage as assessed by measuring serum alanine aminotransferase levels in normal healthy subjects. However, the symptoms of hypotension and trimethylaminuria (fishy body odour) were selected as the critical effect in deriving the UL for choline in patients treated with oral administration of choline chloride (Boyd et al., 1977; Growdon et al., 1977; Lawrence et al., 1980). Despite beneficial effects, choline intake as a dietary supplement requires safety assessment. High doses of choline have been associated with trimethylaminuria, vomiting, salivation, sweating and diarrhoea (Busby et al., 2004). The animal data provide supportive evidence for a low degree of toxicity of choline. Further, animal studies have indicated growth suppression at high intake of choline (LSRO/FASEB, 1975). But due to large doses and i.v./i.p. routes of administration in these reports, they were not considered relevant to human intakes from food and supplements. The present study was aimed to assess the toxicity of choline through intranasal (i.n.) route and compared with oral and intraperitoneal (i.p.) route in Balb/c mice.
283
study provides information on the possible health hazards likely to arise from repeated exposure over a relative period of time (OECD, 1981, 1995). The present study was undertaken following good laboratory practices under OECD guidelines. 2.2. Hematology and clinical chemistry At the end of the study, mice were deprived of food overnight and sacrificed on day 29 by overdose of sodium pentobarbital (100 mg/kg; i.p.) and the blood was collected. Blood was centrifuged at 1500g for 15 min, sera separated and stored at 20 °C for immunoassays. Total and differential cell counts were made using Leishman stain. Urea, blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were determined for liver and kidney functioning using commercial kits (Span Diagnostics Pvt. Ltd., India). Total cholesterol (TC) and high density lipoprotein (HDL) levels were measured for normal cardiac functioning (Monozyme Pvt. Ltd., India). 2.3. Cell counts Spleen was removed aseptically from mice and single cell suspension was made in RPMI-1640 medium. Viable splenic cells in each case were counted using hemocytometer by trypan blue exclusion test.
2. Materials and methods 2.4. Pathology 2.1. Animals Balb/c mice of both sexes (6–8 weeks), weighing 18–20 g, obtained from National Institute of Virology, Pune, India were quarantined for 10 days to get acclimatized in experimental conditions. They were placed in cages at 22–25 °C, with 40–70% relative humidity and controlled 12-h light:dark cycle. Water and standard chow diet were given ad libitum. The study protocol was approved by the animal ethics committee of Institute of Genomics and Integrative Biology (IGIB), Delhi. All animal experiments were carried out in the morning to minimize the effects of circadian rhythm. Mice were randomly divided into six groups of 10 animals each of both sexes. In three groups (Groups 1, 3 and 5), choline was administered through oral, i.p. and i.n. routes, separately. As control(s), saline (0.9% sodium chloride) was given in other three groups (Groups 2, 4 and 6) by the same routes as above. For oral route, choline chloride (200 mg/kg) was administered once daily till 28 day by oral gavage (Group 1). Mice received choline chloride (200 mg/kg) in 100 ll saline by i.p. at every alternate day for 28 days (Group 3). However, for i.n. dose (200 mg/kg), choline chloride was given in 50 ll vehicle for every alternate day for 28 days (Group 5). Control mice were given 100 ll normal saline by oral gavage and or i.p. or 50 ll saline through i.n. route for 28 days by the same protocol. Mice receiving i.n. dose were lightly anesthetized (3% isoflurane) before administering each dose. The maximum level of daily tolerable upper intake level (UL) that is likely to pose no risk of adverse effects is 3.5 g/day for 19 years and older subjects (IOM, DRI, 1998), which is equivalent to approx. 60 mg/kg for a normal adult human of approx. 60 kg. In the present study, dose of 200 mg/kg was chosen, which is approx. 3.5 times higher as compared to tolerable upper intake level (UL) for adults to assess the toxicity by inhalation (i.n.), oral and intraperitoneal (i.p.) routes. Body weight, food and water consumption of mice were recorded daily along with signs of distress, if any. Skin, eyes, mucous membrane and behaviour of mice were examined daily. Growth rate was calculated as the difference between the final weight and the initial weight divided by 28 days. The 28-day repeat dose
Detailed gross necropsy, including careful examination of the body’s external surfaces, orifices and cranial, thoracic and abdominal cavities and their content was performed in all the groups. The lungs, heart, liver, spleen, and kidney were excised, trimmed of any adherent tissue and their wet weights were recorded. 2.5. Histopathology The excised organs were fixed in 10% neutral buffered formalin (pH 7.4) and coded samples were given to a pathologist for the study. After fixation, lung sections of 4 lm were cut and stained with hematoxylin and eosin (H&E). Histological evaluation of vital organs like lung, heart, liver, spleen and kidney sections was done using light microscope. 2.6. Statistics Results were expressed as means ± SD. Statistical differences between means were compared by Student’s t-test. Multigroup comparisons of the means were carried out by ANOVA. The P < 0.05 was considered as significant.
3. Results 3.1. Body weight, food and water consumption Daily administration of choline chloride via three different routes at 200 mg/kg caused no adverse effects or mortality during the experimental period of 28 days. There were no statistically significant differences between choline treated groups and their respective controls in the body weight (Table 1; P > 0.05). Growth rates of choline treated groups were not significantly different with their respective controls (Table 1; P > 0.05). The mice did not differ in food or water consumption in choline or saline (control) groups. The mean body weight increase, and mean food consumption of the mice given choline via different routes did not differ significantly from their respective controls (P > 0.05).
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Table 1 Effect of choline chloride on body weight of Balb/c mice. Days
Oral
i.p.
i.n.
Choline
Control
Choline
Control
Choline
Control
0 7 14 21 28
21.0 ± 0.70 22.70 ± 0.52 23.52 ± 0.29 24.64 ± 0.22 25.38 ± 1.70
20.82 ± 0.87 22.68 ± 0.61 23.57 ± 0.24 24.24 ± 0.16 24.98 ± 2.15
21.18 ± 1.02 22.82 ± 0.57 22.96 ± 0.05 24.96 ± 0.21 25.14 ± 0.98
21.05 ± 0.38 22.14 ± 0.81 22.36 ± 0.15 25.12 ± 0.11 25.05 ± 1.28
20.86 ± 0.63 22.2 ± 0.63 22.66 ± 0.37 24.95 ± 0.19 25.24 ± 2.22
20.97 ± 0.42 21.89 ± 0.41 22.49 ± 0.25 24.88 ± 0.29 25.19 ± 1.33
Growth rate
0.156 ± 0.09
0.148 ± 0.07
0.141 ± 0.10
0.143 ± 0.11
0.156 ± 0.07
0.151 ± 0.12
Control, saline.
3.2. Hematology and clinical bio-chemistry
3.3. Splenic cell counts
Red blood cell counts of choline treated groups were not significantly different to their respective controls (P > 0.05). Haemoglobin levels were also not different in controls and choline treated mice by all the three routes (P > 0.05). White blood cell counts (WBC) and neutrophils, lymphocytes, eosinophils and monocytes were not altered after the choline treatment and were not statistically different to their respective control groups (P > 0.05). Thus, the hematologic parameters did not indicate any adverse changes due to choline administration (Table 2). After 28 days of choline treatment, there were no significant differences (P > 0.05) in ALT levels of choline treated groups as compared to their respective saline control groups (Table 3). Choline administration did not result in increased activity of serum toxicity marker enzymes (ALT, AST), indicating normal liver functioning. However, 28-day treatment with choline through i.p. route led to a increase in creatinine level (0.35 ± 0.07 mg/dl) compared to control group (0.24 ± 0.06 mg/dl; P < 0.05). The creatinine levels in mice treated with choline by oral (0.23 ± 0.09 mg/dl) or i.n. (0.25 ± 0.12 mg/dl) were not statistically different to their respective controls (P > 0.05). Urea and blood urea nitrogen levels in choline treated mice were not statistically different than saline treated mice through all the three routes. Choline administration did not affect total cholesterol and HDL levels as compared to their respective controls (P > 0.05).
The effect of choline on growth inhibition of mononuclear cells was examined to evaluate the choline induced immuno-suppression (Fig. 1). In the i.n. choline treated mice, the number of mononuclear cells was 170.2 106 cells/ll ± 2.16 which was not significantly different to its control as 169.4 106 cells/ll ± 2.49 (P > 0.05). Choline administration did not affect mononuclear cells in oral and i.p. groups as compared to their respective controls (P > 0.05). 3.4. Pathology On day 29, mice were subjected to a detailed post-mortem examination of the internal organs. There were no macroscopic differences in size, colour or texture of the organs. The repeated choline administration did not affect the weight of lungs, heart, liver, spleen or kidney (Table 4). The weight of other organs was comparable to their respective controls. 3.5. Histopathology No pathological signs were observed in the vital organs examined for the study (data not shown). Choline administration did not affect liver tissue of mice with no signs of morphological variations in its lining with respect to their controls. Kidney sections of mice treated with choline were indistinguishable from kidney of
Table 2 Hematological parameters in mice treated with choline or saline control. Parameters
Hb (g/dl) RBC (106/mm3) WBC (106/mm3) Monocytes Lymphocytes Neutrophils Eosinophils
Oral
i.p.
i.n.
Choline
Control
Choline
Control
Choline
Control
16.28 ± 0.70 7.08 ± 0.52 4.92 ± 0.29 2.44 ± 0.22 72.67 ± 1.70 23.45 ± 1.64 0.97 ± 0.17
16.58 ± 0.87 6.98 ± 0.61 4.59 ± 0.24 2.92 ± 0.16 73.53 ± 2.15 22.97 ± 1.63 0.94 ± 0.19
16.26 ± 1.02 7.05 ± 0.57 4.64 ± 0.05 2.94 ± 0.21 72.54 ± 0.98 23.87 ± 1.41 0.89 ± 0.09
16.23 ± 0.38 6.94 ± 0.81 4.78 ± 0.15 3.51 ± 0.11 72.76 ± 1.28 23.41 ± 1.31 0.95 ± 0.14
16.67 ± 0.63 6.67 ± 0.63 4.71 ± 0.37 3.20 ± 0.19 72.85 ± 2.22 23.65 ± 1.33 0.94 ± 0.18
16.84 ± 0.42 6.98 ± 0.41 4.48 ± 0.25 3.61 ± 0.29 73.21 ± 1.33 22.93 ± 0.98 0.97 ± 0.19
Table 3 Clinical bio-chemistry parameters in mice treated with choline or saline control. Parameters
Urea (mg/dl) BUN (mg/dl) HDL (mg/dl) TC (mg/dl) ALT (IU/L) AST (IU/L) Creatinine (mg/dl) *
Oral
i.p.
i.n.
Choline
Control
Choline
Control
Choline
Control
59.35 ± 1.59 26.98 ± 1.48 266 ± 0.29 20.44 ± 0.22 47.2 ± 4.22 88.42 ± 4.29 0.23 ± 0.09
58.59 ± 1.03 27.00 ± 0.71 262.9 ± 0.24 21.92 ± 0.16 46.6 ± 5.68 88.26 ± 4.97 0.21 ± 0.10
53.46 ± 1.54 25.26 ± 1.64 264.6 ± 0.05 20.94 ± 0.21 43.54 ± 1.89 91.51 ± 3.04 0.35 ± 0.07*
55.20 ± 2.57 27.17 ± 2.01 263.9 ± 0.15 22.51 ± 0.11 46.04 ± 3.64 90.49 ± 4.28 0.24 ± 0.06
57.11 ± 3.00 27.88 ± 1.79 264.1 ± 0.37 21.20 ± 0.19 47.51 ± 3.36 88.90 ± 6.21 0.25 ± 0.12
57.22 ± 1.55 28.46 ± 1.24 265.7 ± 0.25 22.61 ± 0.29 48.72 ± 2.95 90.33 ± 3.8 0.22 ± 0.08
Represent significance difference from the control group.
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6
splenic cells (×10 cells/ul)
174
171
168
165 oral choline oral control i.p. choline i.p. control i.n. choline i.n. control Fig. 1. Spleen cell counts in mice treated with choline or saline control.
control groups. All the other vital organs (lung, heart, spleen) appeared normal and did not show any alterations in structure due to choline treatment by all the three routes. 4. Discussion Asthma and allergies afflict a sizeable population worldwide (Umetsu et al., 2002). Despite the progress made in pharmacotherapy, the incidence of asthma has increased during the last three decades. Pharmacotherapy has adverse effects on long term use and the symptoms reoccur on drug withdrawal. Therefore, new drugs with minimum side effects are required for the prophylactic or symptomatic treatment. Asthma has been hypothesized as being a metabolic disorder since the patient with asthma has altered fat metabolism (Agarwal et al., 1986; Agarwal, 1987; Johnson et al., 2007; Shore, 2008). Choline administration in asthmatics has shown lysophosphatidyl choline lowering effect (Agarwal, 1987). It was also effective in inhibiting airway inflammation in the mouse model of airway hyperresponsiveness (Mehta et al., 2007). There are apprehensions regarding adverse effects of choline since some of the patients showed trimethylaminuria, nausea and diarrhoea during the treatment (Boyd et al., 1977; LSRO/FASEB, 1981; Gaur et al., 1997; Busby et al., 2004). In the present study, choline chloride has been evaluated for acute toxicity in Balb/c mice via oral, i.p. and i.n. routes for 28 days, according to OECD guidelines (OECD, 1995). Toxicity resulting from exposure could occur at the various portal of entry, such as the lungs and skin, or at other distant sites. Inhalation is an important route of exposure in clinical practice for asthma, rhinitis, etc., since the drugs can be delivered directly into the target organ. Pulmonary inflammation is a common response to various particles and associated with other pathological outcomes after exposure to these particles (Bajpai et al., 1992). In the present study, intranasal administration of choline in mice did not cause pulmonary inflammation as determined by blood and spleen cells counts and lung histopathology. The examination of vital organs carried out during the autopsy did not reveal any alterations in i.n. choline group and were comparable with their respective control as well as with other choline treatment groups
(oral or i.p.). In fact, many drugs administered intranasally are often absorbed faster and more efficiently than those from oral administration translating into a quick uptake of drug into the bloodstream and often resulting in a faster onset of action (O’Hagan and Illum, 1990). In the present study, oral, i.p. or i.n. choline treatment for 28 days did not affect the body weight or the mean growth rate of mice compared to their respective controls. These results indicate that in terms of growth, choline is well tolerated by animals at the doses and routes of administration tested. The UL of choline in adults is 3.5 g/day. However, the present study was performed using a dose of choline (200 mg/kg) in excess of the dose as used in previous study (Mehta et al., 2007). High doses of oral choline (more than 10 g/day and or 150–300 mg/kg/day) administered in certain diseases have been associated with adverse effects like trimethylaminuria, vomiting, salivation, sweating and diarrhoea as observed in previous human studies (Boyd et al., 1977; Growdon et al., 1977; Gelenberg et al., 1979; Lawrence et al., 1980; Busby et al., 2004). A study by Sahu et al., reported adverse effects on cellularity of spleen, thymus, peripheral lymph node and pathological lesions in lungs and lymph node, when choline chloride was given via i.p. route for 8 months (Sahu et al., 1986). However, in the present study no adverse effects were observed in disease free mice administered with high doses of choline for 28 days. Furthermore, the present experiments were carried out in rodent species and the response may not be always comparable with humans. The peripheral cell counting is used to indicate a change in quality and quantity of precursor cells, and to determine the toxicity based on hematological parameters (Schofeld, 1986). The destruction of RBCs indicates the abnormality in hepatocellular functions and morphological alterations of the cells indicate the changes in phospholipid ratio and contents (Sherlock and Dooley, 1993). The increase in neutrophils and eosinophils leads to the production of reactive oxygen species during inflammatory reactions and damage the surrounding tissues. In the present study, hematological parameters (WBCs, Hb, lymphocytes, monocytes, etc.) were not affected with choline treatment by any of the three routes tested. Liver plays an essential role in the synthesis of plasma proteins and different globulins. The elevated levels of ALT and AST are the conventional indicator of hepatic damage (Fu et al., 2009). ALT is the most reliable variable for the evaluation of hepatic toxicity whereas AST is not as useful because of its wide distribution (Ringler and Dabich, 1979). In the present study, the high density lipoprotein profile was not affected by choline administration. ALT levels indicate hepatic integrity that did not differ between control and choline treated mice. AST too did not differ between choline treated mice compared to control groups. The enzyme levels suggest no alteration of hepatic function in choline treated mice than controls. Creatinine levels were, however, higher in i.p. choline treated animals as compared to control. This indicates that the drug had an effect on the metabolism and functioning of the kidney might be due to the route (i.p.) tested as the same was not observed in i.n. or oral routes. However, other parameters were normal in i.p. treated group as compared to control. In conclusion, the choline administration by i.n., oral or i.p. route did not cause harmful effects in terms of hematology, clinical bio-
Table 4 Organ weight of mice administrated with choline and or saline control for 28 days. Organ weights
Lung Heart Liver Spleen Kidney
Oral
i.p.
i.n.
Choline
Control
Choline
Control
Choline
Control
0.209 ± 0.03 0.150 ± 0.02 1.134 ± 0.09 0.228 ± 0.02 0.192 ± 0.01
0.206 ± 0.02 0.151 ± 0.01 1.118 ± 0.08 0.230 ± 0.01 0.194 ± 0.02
0.217 ± 0.04 0.156 ± 0.03 1.151 ± 0.08 0.220 ± 0.02 0.202 ± 0.01
0.211 ± 0.02 0.152 ± 0.02 1.129 ± 0.09 0.231 ± 0.01 0.198 ± 0.01
0.207 ± 0.03 0.152 ± 0.04 1.122 ± 0.07 0.231 ± 0.03 0.193 ± 0.01
0.210 ± 0.02 0.149 ± 0.02 1.113 ± 0.08 0.225 ± 0.04 0.196 ± 0.02
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