Protection against radiation-induced conditioned taste aversion by Centella asiatica

Physiology & Behavior 73 (2001) 19 ± 23

Protection against radiation-induced conditioned taste aversion by Centella asiatica V. Shobi1, H.C. Goel* Radiation Biology Division, Institute of Nuclear Medicine and Allied Sciences, DRDO, Lucknow Marg, New Delhi 110 054, India Received 7 May 2000; received in revised form 15 December 2000; accepted 2 January 2001

Abstract Radiations are known to cause behavioural perturbations like conditioned taste aversion (CTA), performance decrement, learning, etc., even at very low doses. The manifestation of radiation-induced behavioural degradation has not been understood well and requires further studies. Therefore, the effects of low-dose whole-body 60Co g-irradiation in male rats were studied in terms of body weight and CTA learning. For CTA, the consumption of saccharin solution was considered as a parameter. To protect against the adverse effects of radiation, Centella asiatica (aqueous extract) was tested and compared with ondansetron, a standard antiemetic drug. A dose of 2 Gy incurred significant body weight loss [t(9) = 9.00, P < .05] and induced CTA in rats [t(26) = 9.344, P < .01]. Administration of C. asiatica (100 mg/kg bw ip, 2 Gy, 1 h) rendered significant radioprotection against radiation-induced body weight loss and CTA that became evident on the second postirradiation day [t(7) = 0.917, P >> .05; t(7) = 4.016, P > .05]. Ondansetron (1 mg/kg bw) elicited higher degree of protection against CTA [t(7) = 3.641, P >.05] than C. asiatica [t(7) = 7.196, P >.05] on the first postirradiation day, but on the second postirradiation day, both were equally effective [t(7) = 3.38, P >.05; t(7) = 4.01, P >.05]. In case of C. asiatica-treated animals, however, there was a consistently declining CTA from the second to the fifth postirradiation day whereas in ondansetron-treated animals it was inconsistent. Present investigation suggests that C. asiatica could be useful in preventing radiation-induced behavioural changes during clinical radiotherapy. D 2001 Elsevier Science Inc. All rights reserved. Keywords: g-Radiation; CTA learning; Body weight; GABA; C. asiatica; Ondansetron

1. Introduction Mature neurons generally do not undergo cellular turnover, and brain has therefore been considered to be radioresistant [1]. For the same reason, radiotherapy has been considered to be relatively safe for treating brain tumours. However, later studies have clearly demonstrated that even whole-body exposure to very low dose (0.1 cGy) of electron beam [2] induces retrograde amnesia (RA). The 10 6-s pulse of low-intensity photoflash in place of electron beam exposure has also resulted in RA. Higher doses of radiation (10 ± 100 Gy) induce emesis, nausea, taste aversion, diarrhoea besides behavioural degradation in terms of coordination, performance, learning, memory, etc., at least in experimental animals [3 ±5]. * Corresponding author. Tel.: +91-11-3970081; fax: +91-11-3919509. E-mail address: [email protected] (H.C. Goel). 1 Present address: Institut fuer Zoologie, Lehrstuhl fuer Entwicklungsbiologie, Universitaet Regensburg, 93040 Regensburg, Germany.

In rodents, there is no emesis and taste aversion is an equivalent manifestation. There are many similarities between emesis and taste aversion [6]. Hence, conditioned taste aversion (CTA) in rats has been recognised as a highly reliable paradigm for evaluating behavioural alterations, induced either by radiation or other environmental agents/ toxins [7]. Though, the intricate mechanisms underlying manifestation of CTA are yet to be elucidated the role of various neurotransmitters in induction of emesis and CTA has already been reported. Exposing an organism to ionising radiation can affect biogenic amines in the brain [8]. Acetylcholine receptor antagonist atropine (15 mg/kg sc or 100 mg/kg ip) has offered protection against radiation or LiCl-induced CTA [9,10]. Selective antagonists of serotonin receptor 3 (5HT3, e.g., ondansetron) have also been reported to prevent emesis induced by chemotherapeutic agents like cisplatin in laboratory animals [11,12], as well as in clinics [13]. However, several side effects like drowsiness, sluggishness, headache, stomach cramp, disturbed vision, etc., have also been documented [14]. A radio-

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protector without these side effects is presently lacking. Therefore, for planned as well as unplanned radiation exposures there is a need to develop certain agents to mitigate radiation-induced behavioural degradation. Several natural compounds from plants exhibit radioprotection mainly due to antioxidant properties [15], so we have focused on Centella asiatica (Linn.) because of its neuromodulating properties as cited in Susruta Samhita and the wide medicinal uses mentioned in Ayurveda since prehistoric times [16]. C. asiatica is a herbaceous plant coming under Umbelliferae found throughout India, at an altitude up to 610 m. The plant has been trivially known as Brahmi in Indian villages and is traditionally used for improving the mental ability [17], wound healing, and skin lesions. In pharmacological studies, the plant extract has shown to have central nervous system depressant activity [18] and it also has reported to improve maze learning capability in rat [19]. There are many reports about the modulating effect of C. asiatica on central nervous system but protective effects against radiation-induced behavioural changes and performance deficits have not been available in the literature. Therefore, we designed this study to investigate the effect of C. asiatica on radiation-induced CTA. The efficiency of C. asiatica was compared with a standard antiemetic drug, ondansetron. 2. Materials and methods 2.1. Animals Inbred Sprague ±Dawley male rats (8 months old) were used for the experiments. Animals were kept in standard laboratory conditions (photoperiod of 12 h/day and air temperature 25°C ‹ 2°C). Rats were housed in individual polyvinyl cages and fed standard animal food pellets (Amrut Laboratory Animal Feed, India) and regular tap water ad libitum. Animals were weighing 323 ‹ 25 g. All procedures involving animals were carried out in strict compliance with the Animal Ethics Committee rules and regulations followed in this institute. 2.2. Irradiation Each rat was placed in a wire gauze container and put in the 60Co Gamma Cell (Model 220, Atomic Energy, Canada), having a dose rate of about 9.96 ±9.90 cGy/min during the course of the investigation, to deliver the desired wholebody dose. Dosimetry was carried out with Baldwin Farmer secondary dosimeter and Fricke dosimeter. 2.3. Plant extract C. asiatica plants (leaf and stem) collected from local source and identified with the help of an ethnobotanist, were extracted in water, filtered, and lyophilised and stored at

4°C. It was resuspended in triple distilled water and filtered through a 0.2-mm filter (Minisart NML) for sterilisation. Maximum tolerance dose was investigated on the basis of acute toxicity and even 500 mg/kg bw did not manifest any toxic symptoms. Hence, 100 mg/kg bw of the extract was administered intraperitoneally for experimental purposes. 2.4. Reference agent Emeset (Cipla, India), a commercial preparation, containing ondansetron hydrochloride dihydrate equivalent to ondansetron as its active ingredient, dissolved in sterilised triple-distilled water was administered 1 mg/kg bw ip as a reference compound for comparison with C. asiatica extract. 2.5. Body weight Body weight of the animals was recorded daily before beginning the experiments. Body weight was expressed as percentage change from the `conditioning day'. 2.6. Conditioned taste aversion and experimental protocol A two-bottle test to study taste aversion learning was employed [20]. Eighty-six rats were trained for 23.5-h water deprivation, scheduled for 10 days, and each rat during this period received tap water only for 30 min (10:00± 10:30 a.m.). On the tenth day of training, all the rats participating in the experiment were `conditioned' by offering them a choice between 0.1% saccharin solution (`conditioned stimulus') and tap water for 30 min, and the intake was recorded. Twenty rats showing no preference to `conditioned stimulus' were excluded from the experiment. Immediately following the conditioning session, the rats were divided into six groups: Group 1 Ð vehicle ( 1 h)/ sham irradiation (n = 8), Group 2 Ð vehicle ( 1 h)/2 Gy (n = 27), Group 3 Ð C. asiatica ( 1 h)/sham (n = 7), Group 4 Ð ondansetron ( 1 h)/sham (n = 8), Group 5 Ð C. asiatica ( 1 h)/2 Gy (n = 8), Group 6 Ð ondansetron ( 1 h)/2 Gy (n = 8). Twenty-four hours after the treatment, all rats were again offered the choice of 0.1% saccharin solution and tap water, and intake of each fluid was measured. The taste aversion was expressed as a percent of consumption of saccharin solution relative to total fluid intake on the conditioning day. Measurements were recorded up to five postirradiation days. 2.7. Statistical analysis 2.7.1. Body weight and conditioned taste aversion Animals consuming saccharin solution to the extent of 50% or more of the total fluid intake on `conditioning day', were only included in this study. Change in body weight and intake of saccharin solution was analysed separately. Bartlett test [21] was applied to test for significance of variation in variances. Normality was assumed since minor

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deviations from normality do not greatly affect the results. Welch statistic (WL) was applied to test for equality of means in spite of heterogeneous variances amongst the treatment groups [22] using ni/si2 as weights, where ni and si2 are, respectively, the sample size and the variance in i-th treatment group. This test was preferred since in most of the treatment groups sample size was less than 10. Paired t test was applied to test the significance of rise/fall in saccharin intake on any subsequent day after initiation of experiment. Welch's test was repeated to test for significance of rise/fall in saccharin intake amongst the treatment groups. The probability level of 5% was considered as significant. Random allocation according to some statistical technique was assumed. Heterogeneity in variances imposed restriction to apply ANOVA. 3. Results 3.1. Body weight The body weight of the rats was observed to be 323 ‹ 25 g ranging from 304 to 342 g. There was no evidence of significant variations in the body weight (WL = 2.11; 5.15, 8, P >> .05) on the initial day, indicating that the treatment groups were homogeneous with respect to body weight. Body weight increased significantly on the first follow-up day in all the groups (except Groups 1 and 4), Group 2 [t(9) = 14.29, P < .01], Group 3 [t(6) = 2.75, P < .05], Group 5 [t(7) = 6.80, P < .01], and Group 6 [t(7) = 13.09, P < .01]. After 24 h, the body weight reverted to `initial day' weight in all the groups without any significant difference ( P >> .05). However, in the irradiated group, the decline beginning on the second postirradiation day continued till the fifth postirradiation day [t(9) = 9.00, P < .01], which was the last day of observation (Fig. 1).

Fig. 2. Radioactive effect of C. asiatica (100 mg/kg bw) and ondansetron (1 mg/kg bw) against 2-Gy-induced CTA in rats. The drugs were administered 1 h before irradiation and consumption of 0.1% saccharin solution was measured in milliliters and expressed as percentage of control value on `conditioning day'.

CTA in rats [t(26) = 9.34, P < .01]; saccharin intake was zero in 60% of the rats in this group [vehicle ( 1 h)/sham: t(7) = 0.005, P >.01]. Ondansetron or C. asiatica administered alone did not produce any CTA [t(7) = 1.17, P >.05; t(6) = 0.31, P >.05]. Aqueous extract of C. asiatica (100 mg/ kg bw ip) 1 h before irradiation rendered significant protection against CTA [t(7) = 4.02, P >.05] on the second postirradiation day (Fig. 2). There was a steady recovery in the group C. asiatica ( 1 h)/2 Gy. Ondansetron (1 mg/kg bw ip) showed higher degree of protection [t(7) = 3.64, P >.05] than C. asiatica on the first postirradiation day. However, on the second postirradiation day C. asiatica was as effective as ondansetron [t(7) = 4.02; t(7) = 3.381, P >.05] and the recovery was approaching near to control value (Group 1) on the fifth postirradiation day [t(7) = 1.587, P >.05].

3.2. Conditioned taste aversion The variances of the treatment groups differed significantly [c2(5) = 27.319; P < .01] in respect of saccharin intake. A dose of 2 Gy (60Co g-rays) inflicted significant

Fig. 1. Effect of C. asiatica (100 mg/kg bw) and ondansetron (1 mg/kg bw) administered 1 h before 2-Gy whole-body irradiation in rats.

4. Discussion There was significant increase in body weight up to 24 h after irradiation in all groups except in Groups 1 and 4. Thereafter, the body weight reverted to normal in all groups. However, in the group given radiation dose only the decline in body weight continued up to the fifth postirradiation day, which was the last day of observation (Fig. 1). As per experimental plan, body weight was recorded in `preconditioned' situation. On the `conditioning day' after C. asiatica and ondansetron treatment and irradiation, the experimental animals were provided 0.1% saccharin solution/water. Next, weight was recorded 24 h later. On `conditioning day', novel saccharin solution consumption hiked total fluid intake by rats. This could possibly be responsible for increased body weight in all the groups recorded 24 h after `conditioning'.

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Irradiation is known to cause body weight loss as a consequence of cytotoxicity [23]. We also observed reduction in body weight up to the fifth postirradiation day, the last day of observation. Postirradiation release of serotonin (5HT) from the gastric enterochromaffin cells could manifest nausea in man and taste aversion in rats, thus affecting the body weight adversely [20]. Moreover, 5HT also affects the food intake both in man and animals [24]. Ondansetron is a 5HT3 receptor antagonist and may be counteracting the 5HT-mediated suppression of food intake in postirradiation situation and thus helps in maintaining the body weight. Contrary to this, C. asiatica, which increases GABA levels in rat brain [18], plays a role in regulating feeding and running behaviour in rats thereby influencing body weight [25]. Thus, C. asiatica administered 1 h before irradiation may be protecting against body weight loss in comparison to the group exposed to radiation only. The role of 5HT and GABA in connection with the food intake and body weight gain has been subjected to much investigation. The mechanisms underlying food intake and 5HT release in lateral hypothalamus is more complex [26] and needs to be investigated further. The mechanisms involving CTA learning are possibly mediated through several control mechanisms. Many potent inhibitors of emesis are not able to attenuate emesis induced by wide variety of emetogens. Cisplatin-induced emesis could be attenuated by dexamethasone but not by zacopride or GR38032F [27]. Prochlorperazine, trimethobenzamide, or cyclizine were not effective against radiation- or lithium chloride-induced CTA [28]. The 5HT3 antagonist, ondansetron, has been effective against CTA induced by radiation, morphine, etc., but not against nicotine-induced one [29,30]. On the contrary, it has been reported that 5HT3 receptors are not involved in drug-induced taste aversion in rats [31] though well established in man [32]. In rats, the CTA possibly involves brainstem areas that are known for emesis in other species [33]. It is probable that ondansetron acts by blocking 5HT3 receptors, while C. asiatica extract may be acting through multiple mechanisms. The present study indicates that C. asiatica was able to provide significant protection against 2-Gy-induced CTA in rats. It is well-documented [34] that the central mechanism is responsible for CTA learning, which mediates through cholinergic pathway. Acetylcholine receptor antagonist atropine (15 mg/kg sc or 100 mg/kg ip) has been shown to protect against radiation- or LiCl-induced CTA [10]. Since GABA and its agonists inhibit the central cholinergic mechanism by affecting the turnover rate of acetylcholine in rat brain [35], it was expected that increase in GABA levels in rat brain will offer protection against radiationinduced CTA. This information may be usefully exploited for benefit of patients undergoing radiotherapy. It is presumed that C. asiatica may be taking more time to act at the area postrema in brainstem, the centre involved in taste aversion response, since it may have to cross the blood ± brain barrier. This may explain the maximal protec-

tion achieved by C. asiatica about 48 h after irradiation (Fig. 2). Ondansetron, on the other hand, acted faster by blocking 5HT3 receptor in gastric mucosa thereby stopping the visceral pathway involved in CTA [11,12]. This explains that, initially, C. asiatica rendered slower pace of recovery in comparison to ondansetron, but later, it became more effective than ondansetron. In neurofunctional hierarchy, visceral pathway precedes central neural pathway [35]. It is known that free radicals also lead to taste aversion in rats [36]. It has also been reported that C. asiatica contains many antioxidant molecules like carotenoids, ascorbic acid, and terpenoids [37]. These antioxidants may scavenge the free radicals produced by irradiation, thus protecting the organism against the free radical-induced cytotoxic and genotoxic effects, especially in radiosensitive gastrointestinal system. The present study demonstrates that C. asiatica offers good behavioural radioprotection against CTA in rats by its multitargeted action that need to be unravelled by further studies. Acknowledgments The authors are thankful to Dr. C.K. Gupta, former professor in the Department of Biostatistics, V.P. Chest Institute, Delhi University, Delhi, for the help and guidance in the statistical analysis. We are also grateful to Surendar Singh, Jadish Prasad, P.K. Agrawala, I. Prem Kumar, Damodar Gupta, and Namita Samanta for extending timely help during experiments and to Rajesh Arora for providing the plant extract. References [1] Kimeldrof DJ, Garcia J, Rubadeau DO. Radiation-induced conditioned avoidance behaviour in rats, mice and cats. Radiat Res 1966;12:710 ± 8. [2] Wheeler TG, Hardy KA. Retrograde amnesia produced by electron beam exposure: causal parameters and duration of memory loss. Radiat Res 1985;101:74 ± 80. [3] Bogo V. Effects of bremsstrahlung and electron radiation on rat motor performance. Radiat Res 1984;100:313 ± 20. [4] Burghardt WB, Hunt WA. Characterization of radiation-induced performance decrement using a two-lever shock-avoidance task. Radiat Res 1985;103:149 ± 57. [5] Franz CG. Effects of mixed neutron-gamma total-body irradiation on physical activity performance of rhesus monkeys. Behav Neural Biol 1985;40:114 ± 8. [6] Rabin BM, Hunt WA, Lee J. Effects of dose and of partial body ionizing radiation on taste aversion learning in rats with lesions of the area postrema. Physiol Behav 1984;32:119 ± 22. [7] Rabin BM, Hunt WA. Mechanisms of radiation-induced conditioned taste aversion learning. Neurosci Biobehav Rev 1986;10(1):55 ± 65. [8] Hunt WA, Dalton TK, Darden JH. Transient alterations in neurotransmitter activity in the caudate nucleus of rat brain after a high dose of ionizing radiation. Radiat Res 1979;80:556 ± 62. [9] Deutsch R. Effects of atropine on conditioned taste aversion. Pharmacol, Biochem Behav 1978;8:685 ± 94. [10] Gould MN, Yatvin NB. Atropine-caused central nervous system inter-

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