Radioprotective Effect of Ocimum sanctumand Amifostine on the Salivary Gland of Rats

After Therapeutic Radioiodine Exposure

Lebana J. Joseph,1 Uma S. Bhartiya,1 Yogita S. Raut,1 Rohini W. Hawaldar,2 Yogendra Nayak,3

Yogita P. Pawar,1 Nirmala A. Jambhekar,2 and M.G.R. Rajan1

Abstract

The current study investigated the radioprotective effect of Ocimum sanctum on the salivary gland of ratsadministered radioiodine (131I) and compared its efficacy with a known radioprotectant, amifostine. The ex-perimental rats were divided in four groups and sacrificed in three different batches at 1, 3, and 6 months of timeinterval after 18.5 MBq/100g (i.p.) 131I exposure. Six months duration batch received 131I exposure twice with thegap of 3 months. Two groups of experimental rats were presupplemented with O. sanctum (40 mg/kg for 5 days,orally) and amifostine (200 mg/kg, s.c) before 131I exposure separately. Increased Technetium-99m-pertechnetate(99mTcO4

- ) uptake at 30 minutes post injection in salivary glands of only 131I exposed rats may imply delay inclearance at 6 months of exposure in comparison to their counterparts sacrificed at 1 month. Parotid glandhistology showed atrophy with lipomatosis in only 131I exposed rats at 3 and 6 months of duration. O. sanctumand amifostine presupplemented and subsequently exposed to 131I rats at 3 and 6 months duration exhibitedcomparable histopathology with controls. Our study indicates possible radioprotective effect of O. sanctum andamifostine against high-dose 131I exposure.

Key words: amifostine, Ocimum sanctum, radioiodine, rats, salivary gland

Introduction

High dose radioiodine (131I) therapy is a standard proce-dure in the treatment of differentiated thyroid cancer to

ablate remnant thyroid tissue post thyroidectomy or for me-tastasis. Due to the presence of sodium iodide symporter,iodide gets actively transported into the thyroid gland and anumber of nonthyroidal tissues such as salivary gland,stomach, lacrimal gland, and lactating mammary gland.1,2

Among these, salivary glands are known to exhibit irrevers-ible radiation damage such as sialadenitis, xerostomia, andneoplasia in patients with differentiated thyroid cancer afterhigh-dose 131I therapy.3,4 Therefore, high-dose 131I therapy isgenerally recommended under salivary gland stimulation inorder to minimize the salivary gland damage, however, with

limited success.5,6 Ionizing radiation damages DNA eitherdirectly or indirectly via the production of free radicals.Radioprotection can be achieved by use of free radical scav-engers during 131I therapy. So far, amifostine is the onlychemo and radioprotective drug available to reduce the sideeffects of radiation exposure in patients with cancer.7,8 Ami-fostine is preferentially accumulated in certain tissues in-cluding the salivary gland, thus making these tissues lesssensitive to radiation damage without protecting tumorcells.8–10 However, a major drawback of the use of amifostineis its severe adverse effects that sometimes result in discon-tinuation of the use of amifostine in some patients.11 How-ever, the use of amifostine as a radioprotectant is mainlyreported in patients with head and neck cancer receiving ex-ternal radiation exposure.8,11 Limited studies have reported

1Bio-Medical Group, Radiation Medicine Centre, Bhabha Atomic Research Centre, C/O Tata Memorial Hospital Annexe, Mumbai, India.2Tata Memorial Centre, Parel, Mumbai, India.3Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, India.

Address correspondence to: Uma S. Bhartiya; Bio-Medical Group, Radiation Medicine Centre, Bhabha Atomic Research Centre, C/O TataMemorial Hospital Annexe; Jerbai Wadia Road, Parel, Mumbai 400 012, IndiaE-mail: umapat@yahoo.com

CANCER BIOTHERAPY AND RADIOPHARMACEUTICALSVolume 26, Number 6, 2011ª Mary Ann Liebert, Inc.DOI: 10.1089/cbr.2011.1014

737

the usage of amifostine against internal radioiodine exposurein differentiated thyroid carcinoma and with controversialresults.7,12,13

At present, many traditional ayurvedic herbal formula-tions, plant extracts alone or in combination, are being ex-plored for their efficacy against radiation-induced celldamage. Among them, Ocimum sanctum, a medicinal plant inIndia, is known to have various beneficial effects includingradioprotection against 60Co gamma radiation.14–16 Ourearlier work has shown better radioprotection by oral sup-plementation of O. sanctum as compared with other naturalantioxidants in salivary glands of mice exposed to an internaltherapeutic dose of 131I at 24 hours.17–19 Since irreversiblesalivary gland damage is known to occur at later stages, wethought of exploring the possible beneficial effect ofO. sanctum on the salivary glands of rats after a long durationand the repeated therapeutic dose of 131I exposure andcompared its efficacy with a known radioprotectant, ami-fostine.

Materials and Methods

Animals

Female adult Wistar rats weighing 250–300 g were ob-tained from Animal House Facility, BARC, Trombay,Mumbai. They were maintained in an air-conditioned roomwith free access to water and food. Animal studies wereperformed in compliance with BARC Institutional AnimalEthics Committee’s guidelines.

Experimental design

Totally, 72 rats used for the experiment were divided intothe following four groups with six animals each. Group I:Control. Group II: 131I exposed (18.5 MBq/100 g 131I, i.p). 131Ias Na131I was obtained from the Board of Radiation andIsotope Technology. Group III: oral O. sanctum (40 mg/kg)for 5 consecutive days before 18.5 MBq/100g 131I exposure.O. sanctum used in the study was a generous gift fromDr. Yogendra Nayak, Department of Pharmacology, ManipalUniversity, Manipal, India. Group IV: administration ofamifostine (200 mg/kg, s.c.) 15 minutes before 18.5 MBq/100g 131I exposure. Amifostine was obtained from NATCOPharma Limited. Totally, three such batches of 1, 3, and 6months of duration receiving 131I exposure were studied.

The animals were weighed and sacrificed after 1, 3, and 6months of duration of 131I exposure. Six-month experimentalanimals were exposed twice with 18.5 MBq/100g 131I with 3months gap between the two exposures.

The experimental rats were supplemented with 1.5 lg/100 g of thyroxine (T4) and 1% calcium lactate in drinkingwater to maintain the euthyroid state.

Parameters

Biodistribution studies using 99mTc-pertechnetate. The99mTc-pertechnetate (99mTcO4

- ) uptake was studied by i.p.administration of 1.85 MBq of 99mTcO4

- to all the animalsfrom the 1 and 6 months duration group, 30 minutes beforesacrifice. 99mTcO4

- was prepared in-house from a 99Mo-lybdenum - 99mTc generator.

The rats were sacrificed by cardiac puncture under etheranesthesia. Tissues excised for counting radioactivity in-cluded liver, stomach, kidneys, bladder, and salivary glands(parotid and submandibular from both the sides).The wetweights of the parotid and submandibular glands were re-corded before measuring their 99mTcO4

- activity. In addi-tion, blood was also collected and counted for its 99mTcO4

-

activity. The percent uptake in blood was calculated bycorrecting it for the volume. The carcass was separatelycounted for its radioactive content.

The 99mTcO4- activity was counted by using a broad,

well-type scintillation counter designed for animal tissues,and the percent 99mTcO4

- uptake was calculated by con-sidering injected activity as 100%. In case of salivary glands,the % uptake was calculated as percent injected dose/g tis-sue weight/kg body weight.

Histopathology. The parotid glands of 3 rats from eachgroup at 1, 3, and 6 months of duration were fixed in 10%neutral buffered formalin. Sections at 3–5 lm were preparedand stained with hematoxylin/eosin for histopathology in aconventional manner.

Statistics

Statistical analysis was performed using ANOVA withBonferroni correction. Within-group comparison for99mTcO4

- uptake was done by using a paired t-test with asignificance level < 0.05, whereas between-group compari-son was done by using Student’s t- test with a statisticalsignificance level < 0.01(with continuity correction).

Results

Total body weight as well as parotid and submandibularweights in all the experimental rats did not show any sig-nificant change in comparison to control animals at 1, 3, and6 months of duration (Table 1). After administration of

Table 1. Body Weight, Parotid, and Submandibular Gland Weight of the Experimental Rats

at 1, 3, and 6 Months After131I Exposure

Body weight (g) Parotid (mg) Submandibular (mg)

Time (month) 1 3 6 1 3 6 1 3 6

Gr. I 332 – 32 325 – 30 348 – 31 466 – 62 454 – 103 509 – 63 580 – 39 633 – 141 758 – 90Gr. II 326 – 18 295 – 23 307 – 21 432 – 52 364 – 80 520 – 104 537 – 86 480 – 75 556 – 63Gr. III 326 – 13 310 – 22 323 – 22 506 – 114 419 – 92 544 – 54 563 – 38 505 – 42 640 – 59Gr. IV 316 – 14 310 – 35 317 – 21 427 – 69 434 – 46 576 – 88 562 – 55 523 – 40 610 – 84

Total number of animals = 72 (mean – SD).Gr. I, control; Gr. II, 131I; Gr. III, O. sanctum + 131I; Gr. IV, amifostine + 131I; SD, standard deviation.

738 JOSEPH ET AL.

18.5 MBq/100g 131I, the rats from all the three groups ex-hibited an ablation of the thyroid gland as early as 1 month.This was evident by the absence of thyroid tissue at the timeof sacrifice as well as the absence of 99mTcO4

- uptake in thethyroid gland of these animals. Adequacy of the T4 supple-mentation was observed by normalized levels of T4 in bloodsamples of the experimental rats. At 1 month duration, the T4

levels were 4.5 – 2.5, 3.6 – 1.7, 5.2 – 3.1, and 3.4 – 1.1 lg/dL inGr. I, Gr. II, Gr. III, and Gr. IV, respectively. Similarly, at 6months, the levels were 4.6 – 0.57, 5.0 – 1.5, 4.3 – 0.35, and3.7 – 1.0 lg/dL in Gr. I, Gr. II, Gr. III, and Gr. IV, respec-tively. No toxic effects were observed in the group of ratsreceiving O. sanctum presupplementation, whereas 17%mortality was seen in the group of rats receiving a single s.c.injection of amifostine. However, we were not able to as-certain the exact cause of the death in amifostine-treated rats.

Biodistribution studies were performed using 99mTcO4- at

1 and 6 months of 131I exposure in two separate batches ofexperimental animals. The main intention of 99mTcO4

- studywas to assess the parenchymal function of the salivaryglands. Figure 1 shows the percent uptake of the radioiso-tope in various organs after 30 minutes of 99mTcO4

- admin-

istration in the experimental animal batch sacrificed at 1 and6 months of duration. No significant change was observed inpercent uptake in the liver, stomach, kidneys, bladder, car-cass, and blood.

Table 2 shows percent 99mTcO4- uptake expressed as in-

jected 99mTcO4- dose/g tissue weight/kg body weight at 30

minutes in parotid and submandibular glands from both thesides in two batches of rats sacrificed at 1 and 6 months ofduration of 131I exposure. At 1 month, the percent 99mTcO4

-

uptake in all the four groups did not demonstrate any sig-nificant alteration in parotid as well as submandibularglands. At 6 months of duration, Gr. II animals exposed to131I exhibited an increase in 99mTcO4

- uptake in the parotidgland when compared with Gr. I animals at 6 months;however, the increase was not statistically significant. On theother hand, a significant increase in 99mTcO4

- uptake wasobserved in Gr. II animals at a 6 months time interval whencompared with Gr. II at a 1 month interval ( p = 0.010). Asignificant elevation in 99mTcO4

- uptake was also observedin O. sanctum pretreated group (Gr. III) at 6 months incomparison to its 1 month counter part ( p = 0.039). Similarly,99mTcO4

- uptake in the submandibular gland at 6 months in

FIG. 1. Biodistributionstudies at 30 minutes using99mTcO4

- in experimental rats(mean – standard deviation,n = 6).

Table 2. Percentage99m

Tc Pertechnetate Uptake in Parotid and Submandibular Glands

at 30 Minutes in Experimental Rats

Parotid (%ID/g/kg body weight) Submandibular (%ID/g/kg body weight)

Time (month) 1 6 1 6

Gr. I 0.645 – 0.14 0.657 – 0.48 0.539 – 0.07 0.579 – 0.30Gr. II 0.635 – 0.17 1.389 – 0.42a 0.633 – 0.27 1.219 – 0.30c,d

Gr. III 0.698 – 0.28 1.110 – 0.27b 0.618 – 0.24 0.834 – 0.30Gr. IV 0.641 – 0.47 0.646 – 0.49 0.578 – 0.28 0.788 – 0.29

Total number of animals = 72 (mean – SD).ap = 0.010 vs. Gr. II (1 month).bp = 0.039 vs. Gr. III (1 month).cp = 0.015 vs. Gr. III (1 month).dp = 0.004 vs. Gr. I (6 months).

RADIOPROTECTION BY OCIMUM SANCTUM IN RAT SALIVARY GLANDS 739

Gr. II animals demonstrated significant elevation in com-parison to their counterpart at 1 month ( p = 0.015) as well asGr. I animals at 6 months ( p = 0.004). In contrast, rats pre-treated with O. sanctum and amifostine exhibited markedreduction in 99mTcO4

- uptake in the submandibular glandsin comparison to Gr. II rats. The 99mTcO4

- uptake in Gr. IIIand Gr. IV rats was almost comparable to controls (Table 2).A histopathological change in the parotid gland was studiedin 3 rats from each group at 1, 3, and 6 months of duration.Controls exhibited a well-defined lobular structure withdensely packed acini and a well-developed duct. Parotidgland samples were purely serous. One month after 131I ex-posure, the parotid gland did not exhibit any significanthistopathological changes in any of the experimental groupsin comparison to control animals. At 3 months of duration,Gr. II rats exposed to only 131I showed progressive lipoma-tosis with atrophic acinar cells, whereas no significant al-terations except slight vacuolization were observed in theparotid histopathology of the rats presupplemented withO. sanctum and amifostine followed by 131I exposure (Fig. 2).

At 6 months with two 131I exposures, multifocal areas oflipomatosis and atrophy were observed in Gr. II animals,whereas the rats presupplemented with O. sanctum andamifostine remained comparable to controls (Fig. 3).

Discussion

Salivary gland dysfunction is a known complication of 131Itherapy that can have a serious impact on the quality of lifeof a patient with thyroid cancer.2,20 Due to the damagingeffect of 131I on the salivary gland, high-dose radioiodinetherapy is generally performed under salivary gland stimu-lation. Sialogogues are thought to have an important func-tion in preventing the side effects of 131I therapy.20 However,this approach has its limitations, as it requires continuousadministration of sialogogues.21 Lemon products are knownto decrease the transit time of 131I through the salivary glandso as to reduce the amount of radiation exposure to the tis-sues, however, without much beneficial effect in the patientpopulation.5,6 In view of good prognosis of differentiated

FIG. 2. (A–D) Rat parotid gland histology at 3 months after 131I exposure. (A) Control parotid gland showing normallobular structure with intact cell membrane and intact duct (original magnification 400 · ). (B) Parotid gland of the ratexposed to 131I exhibiting complete destruction of lobular structure and loss of parenchymal cells with progressive lipo-matosis (original magnification 100 · ). (C) Parotid gland of the rat presupplemented with Ocimum sanctum followed by 131Iexposure exhibiting well-maintained lobular structure with intact duct and presence of slight acinar vacuolization (originalmagnification 400 · ). (D) Parotid gland of the rat presupplemented with amifostine followed by 131I exposure showingnormal acini with intact duct along with focal vacuolization (original magnification 400 · ).

740 JOSEPH ET AL.

thyroid carcinoma after 131I therapy, it is important to avoidthe salivary gland damage, ultimately improving the qualityof life of these patients. Currently, amifostine is the only drugapproved by the Food and Drug Administration for thepurpose of radioprotection in patients with cancer. However,it is known to exhibit adverse effects that may lead to thediscontinuation of the same in some patients.11,22 In addition,its beneficial effects in patients with thyroid cancer under-going 131I therapy are yet to be established.12,13 Hence, themajor focus of our experiment was to develop a betterradioprotectant for the salivary gland against therapeutic 131Iexposure that would be nontoxic and highly effective. Ourearlier work in mice had given us encouraging preliminaryresults for salivary gland radioprotection against 3.7 MBq of131I exposure for 24 hours by using O. sanctum pre-supplementation.17,19

Our current experiment compares the effect of O. sanctumand amifostine presupplementation on the body weight andsalivary gland weight of the rats. Biodistribution of99mTcO4

- after 30 minutes of administration was comparedin various organs including salivary glands. No significantchange in the body weight and salivary gland weight wasobserved at 1, 3, and 6 months after radioiodine exposure.Nagler et al. have observed significant reduction in bodyweights as well as salivary gland weight after 60Co externalradiation exposure of 10 and 15 Gy at 3, 6, 9, and 12 monthstime interval.23,24 However, our experimental design wasdifferent, where the animals were exposed to a single inter-nal therapeutic dose of 131I of 18.5 MBq.

The batch of experimental animals sacrificed at 6 monthstime interval receiving two 131I exposures showed 2.19 and1.59 times increase in 99mTcO4

- uptake within groups in theparotid gland of Gr. I, Gr. III animals. However, in the sub-mandibular gland, the increase was 1.92, 1.35 and 1.36 timesin Gr. II, Gr. III, and Gr. IV, respectively, when compared with1 month exposure. The observed increase was statisticallysignificant in 131I exposed group in parotid as well as sub-mandibular glands. O. sanctum pretreated and 131I exposedgroup also demonstrated a significant increase in 99mTcO4

-

uptake in parotid glands. Trapping of 99mTcO4- by the sali-

vary gland generally begins at 1 minute and reaches a peak inabout 5–10 minutes. At a later stage, it also provides theinformation regarding the excretion pattern. The observedincrease in the uptake could be the indication of slow excre-tion due to the parenchymal damage, thus resulting indelayed 99mTcO4

- clearance ultimately increasing 99mTcO4-

uptake. In contrast, Bohuslavizki et al.25 have observed pro-gressive decline in 99mTcO4

- uptake from 4 to 24 weeks after2 GBq of 131I exposure by acquiring sequential images of eachup to 25 minutes in the same batch of animals. However, wehave studied the percent uptake of 99mTcO4

- at 30 minutes offixed time interval in parotid and submandibular glands ofthe experimental rats. We could not perform the sequentialimaging in the same batch of animals, as they were sacrificedat fixed time intervals. Detailed sequential imaging studies inthese groups particularly in O. sanctum pretreated (Gr. III) andamifostine (Gr. IV) pretreated groups are required to addressthe reason for the difference in clearance of the 99mTcO4

- inthe salivary glands in these two groups.

The histopathological studies have shown no significantchanges in parotid gland architecture at 1 month. The lack ofsignificant morphological alteration at 1 month is in

FIG. 3. (A–C) Rat parotid gland histology of the experi-mental animal after two 131I exposures with 3 months of gapbetween the two exposures. (A) Parotid gland of the rat aftertwo 131I exposures exhibiting destroyed lobular structurewith multifocal areas of lipomatosis with proliferation of fi-brous connective tissue resulting in constriction of the duct(original magnification 100 · ). (B) Parotid gland of the ratpresupplemented with O. sanctum followed by two 131I ex-posures exhibiting normal acini with duct (original magni-fication 400 · ). (C) Parotid gland of rat presupplementedwith amifostine followed by two exposures of 131I showingnormal acini with duct (original magnification 400 · ).

RADIOPROTECTION BY OCIMUM SANCTUM IN RAT SALIVARY GLANDS 741

agreement with many earlier reports.26 - 29 Our study differsfrom those with regard to the type of radiation used. Theyhave externally irradiated the head and neck region of theexperimental animals with 60Co, whereas in the present ex-periment, the rats were exposed to 131I in vivo. At later stagessuch as 3 and 6 months of duration, 131I exposed rats showcellular atrophy in the parotid gland accompanied by lipo-matosis. On the other hand, rats pretreated with O. sanctumand amifostine showed comparable cellular histology tocontrol animals. This is in agreement with the observationreported by Bohuslavizki et al.,25 wherein rabbits were ex-posed to a high dose of 131I in vivo to study the radiopro-tective effect of amifostine. Kutta et al.30 studied theradioprotective effect of amifostine on the histology as wellas electron microscopy of the salivary gland of rabbits afterexposure to 131I. They have observed marked lipomatosisand apoptosis in the salivary gland of the rabbits exposed to1 GBq radioiodine exposure at 3 and 6 months of duration.Similarly, Yan et al. have also observed severe salivary glanddamage after external duel field irradiation at 16 weeks oftime interval in the pig parotid gland in comparison to asingle-field irradiation of the same dose.31

The radioprotective effect of amifostine is attributed to itspreferential localization in the salivary glands and ability toscavenge free radicals.11 In the current study, the beneficialeffect of amifostine was observed on the glandular histologyof rats receiving 131I exposure as well as uptake studies doneat 30 minutes after administration of 99mTcO4

- . This indi-cates the radioprotective ability of the amifostine againstinternal 131I exposure in salivary glands. Nonetheless, theadverse side effects, limited route of administration, narrowtime window, cost, and need of medical supervision haslimited its use in patient population.8,22 In this context, wehave observed 17% mortality in amifostine-treated group,whereas no such adverse effect was found in the grouppresupplemented with O. sanctum, which is easily availableand a cost-effective natural antioxidant. In addition,O. sanctum presupplementation also exhibits beneficial ef-fects on the histopathology of the salivary gland, thus indi-cating radioprotective ability against 131I exposure.

In case of differentiated thyroid carcinoma, the usage ofamifostine as a radioprotectant has not been unequivocallyaccepted. Controversial reports regarding its efficacy un-derline the need of exploring a better radioprotectant for thesalivary gland protection after high-dose 131I therapy. In thelight of this, our study reports the radioprotective ability ofO. sanctum on the salivary gland in rats exposed to 131I. Theuse of O. sanctum should be explored in detail for its effect onthe functional ability of the salivary gland as a safe radio-protectant that can be effectively used in patients with dif-ferentiated thyroid carcinoma receiving 131I therapy.

Disclosure Statement

The authors declare that there are no financial or otherconflicting interests in the present work.

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