- Email: [email protected]
Protection of mouse jejunum against lethal irradiation by Podophyllum hexandrum
Phytomedicine, Vol. 8(6), pp. 413–422 © Urban & Fischer Verlag 2001 http://www.urbanfischer.de/journals/phytomed
Phytomedicine
Protection of mouse jejunum against lethal irradiation by Podophyllum hexandrum C.A. Salin, N. Samanta and H.C. Goel Department of Radiation Biology, Institute of Nuclear Medicine and Allied Science, Delhi, India
Summary Radiation induced gastrointestinal damage occurs due to the destruction of the clonogenic crypt cells and eventual depopulation and denudation of the villi. P. hexandrum, a plant, known for its antitumour activity, has been shown to protect the mice against whole body lethal (10 Gy) irradiation. Present study was undertaken to investigate the radioprotective effect of P. hexandrum on jejunal villi cells, crypt cells, their proliferative capacity and mitigation of apoptosis. In an in vivo micro colony survival assay, pre-irradiation administration of P. hexandrum (–2 h) increased the number of surviving crypts in the jejunum by a factor of 3.0 (P < 0.05) and villi cellularity by 2.7 (P < 0.05) fold in comparison to irradiated control. Pre-irradiation administration of P. hexandrum reduced the incidence of apoptotic bodies in the crypts (P < 0.05) in a time dependent manner and depicted a mitotic arrest till the 24 h. However, after 84 h the percentage of mitosis was observed to be nearly similar to that of unirradiated control. This study suggests that arrest of cell division may help in protecting the clonogenic cells against radiation. It would be interesting to investigate further the role of P. hexandrum in influencing various cell cycle regulators like bcl-2, TGF-β, Cyclin-E etc. Key words: Radioprotection, Podophyllum hexandrum, Microcolony assay, Crypts, Apoptosis, Mitosis
j Introduction Gastrointestinal epithelium is an important tissue responsible for digestion and acting as a barrier to luminal bacteria and toxins (Tien et al., 1997). Therefore, every three to six day’s intestinal epithelium renews itself with the help of crypts of lieberkuhn located at the base of the villi (Potten and Morris 1988). Crypts contains few stem cells which keep on proliferating in a regulated way and the transit time of cells from proliferative compartment in the crypts to the extrusion zone at the tip of the villus is between three to five days in the mouse. This rapid turnover makes crypts as one of the most radiosensitive tissues of the body. Stem cells of the crypts get sterilised by lethal irradiation, subsequently the crypts shrink and disappear within 2–3 days; transit cells however continue to divide for a few
divisions and migrate to the villus, which itself is lost within three to five days leading to manifestation of gastrointestinal syndrome (Potten 1990). Plants are known to produce plethora of secondary metabolites with wide array of pharmacological activities and therefore have been used both in the traditional as well as modern system of medicine. The isolated compounds or the active principle and the molecular drugs affect certain biochemical pathways and also interfere with other cellular mechanisms leading to the manifestation of toxic effects (Weiss et al., 1990). The natural combination of compounds present in the whole plant extract often contains several molecules, which take care of the side effects produced by the active principles. 0944-7113/01/08/06-413 $ 15.00/0
414
C. A. Salin et al.
Podophyllum hexandrum Royle known as Indian Podophyllum belongs to the family Podophyllaceae. The lignans and the other chemical moieties present in it display antitumour activity while podophyllotoxin and several other related compounds have antimitotic activity and inhibit DNA, RNA and protein synthesis (Singh and Shah 1994). A dose of 10 Gy causes total mortality by severely jeopardising the bone marrow function. Concurrently it also causes damage to gastrointestinal system and especially to the stem cells in the crypts. Pre-administration of aqueous extract of P. hexandrum has been shown to render significant radioprotection against whole body lethal irradiation (10 Gy) in mice (Goel et al., 1998). The effect of the radioprotective agent, P. hexandrum extract was therefore investigated for its capacity to protect crypt stem cells. This information could be of significant importance for even other gastrointestinal disorders.
j Materials and methods Experimental procedures were adopted as approved by the animal experimentation ethics committee and complied with the Standard Laboratory Guidelines of INMAS. Experimental Animals
12–13 weeks old inbred Swiss albino strain ’A’ male mice weighing 25 ± 3g, were maintained under controlled environment (25 ± 2 °C, photoperiod 12 h/day) and provided standard animal food pellet (Amrut laboratory animal feed, India) and water ad libitum. Not more than three animals were housed in a polyvinyl cage and each group had three animals. Herbal extraction
Aqueous extract of whole rhizome of P. hexandrum (Goel et al., 1998), was filtered and lyophilised to obtain a brownish black resin and stored at 4 °C. The aqueous extract of P. hexandrum was chromatographed according to Harborne (1973) using Silica gel G plates and Methanol-conc. NH4OH (200:3) as solvent and Podophyllotoxin 95% (ACROS , New Jersey, USA) as standard marker. The plates were detected with a) Dragendorff-, b) Iodoplatinate- and c) Marquis-reagents. For experiments, the resin was resuspended in triple distilled water and sterilised by passing through 0.2 µm filter (Minisart® NML). Dose of 200mg/kg b.w of herbal extract was administered to each mouse by i.p route; sham treated mice received distilled water.
Fig. 1a. Crypt cellularity, Mitotic bodies(MI), crypt (C) Apoptotic bodies(AB) in the jejunal crypts 84 h post irradiation (1000× magnification).
The experiments regarding whole body survival and crypt survival after radiation exposure conducted with lyophilised extract of P. hexandrum were repeated further with extracts from 2–3 samples of rhizomes collected within a period of 3 months. The data was pooled and averaged for atleast three different batches of the extract.
Jejunal radioprotection by P. hexandrum
415
Mice were individually placed in a plastic container and a whole body dose of 10 Gy was administered by using 60Co gamma cell (model 220, Atomic Energy Commission, Canada). The dose rate during the experimental period was 1.06 Gy/minute during the course of this study.
lying close together and appearing crowded. Nonviable crypts contained no cells and were sparsely populated by enlarged cells with prominent eosinophilic cytoplasm (Withers and Elkind 1970, Khan et al., 1997). The number of microcolonies was determined in ten cross sections per animal at 450× magnification.
Tissue preparation and light microscopy
Apoptosis in the crypts
Animals were sacrificed by cervical dislocation and small intestine was taken out while flushing the peritoneal cavity with normal saline. 3 to 4 cm of jejunum was isolated and its lumen was flushed with normal saline to remove particulate debris. The jejunum was divided into three segments each of 1 cm size and fixed in 10% buffered formaldehyde and processed for routine histopathological studies using H&E stain.
Apoptosis in the jejunal crypt cells was monitored and scored on the basis of pycnotic nuclei, marginal condensation of the chromatin, fragmentation of the nuclear material and fragments extruding into the crypt lumen (Potten et al., 1994, Meritt et al., 1995). Total number of apoptotic cells were scored in each crypt and expressed as the mean number of apoptotic bodies per crypt. Four transverse sections were scored for each mouse (Pritchard et al., 1998).
Radiation treatment
Morphometry
Non serial transverse sections of jejunum were studied for the number of villi, cells/villous, mitotic figures, apoptotic bodies and microcolonies. Each section was separated from the previous one by a minimum of 50 µm of tissue; a second observer regularly crosschecked counts. Crypt microcolony survival assay
Jejunum was taken out of the animals sacrificed 84 h after irradiation and number of microcolonies (regenerating clones or surviving crypts) per section were studied (Withers and Elkind 1970). Each viable colony was considered to have 10 or more clustered cells with prominent nucleus and little cytoplasm
Fig. 1b. Microcolonies counts in T.S. jejunum 84 h after various treatments. * P < 0.05 P. hexandrum + irradiated mice were compared with irradiated mice; each point represented the mean ± S.E of data from three mice.
Number of cells per crypt and per villus
Each transverse section of jejunum displayed crypts and villi in longitudinal section and their cells were counted at 450× magnification. Only complete longitudinal sections including the opening of the crypt and the full length of the villi from base to the tip were counted. Data were pooled from three to four separate transverse sections of jejunum from each mouse (Ruifrok et al., 1997). Mitotic Index /crypt
Cryptal mitotic figures appearing darkly stained and out of alignment with the nuclei of other cells (Ettarh et al. 2000), were counted from eight different crypts
416
C. A. Salin et al.
Fig. 1c. Crypt microcolonies (C), muscular layer (Mu), Villi (V) and Crypt Ghost (CG) in the jejunal mucosae 84 h post irradiation (100× magnification).
along with crypt cell number selected from three separate transverse sections of jejunum of each mouse and the result is expressed as percentage mitosis per crypt. From each sample, counts were done on atleast three non-serial, coded sections sufficient to minimise the subjectivity of the observer. Analysis of data
Data with respect to different parameters obtained from mice belonging to various treatment groups were sub-
jected to student-t test and significance was assessed at 95% confidence intervals.
j Results In the aqueous extract of P. hexandrum by TLC no Podophyllotoxin could be detected. The cross sections of jejunum of the control mice displayed normal histological features. Mice treated with
Jejunal radioprotection by P. hexandrum Podophyllum only were similar to the sham treated group. Irradiated mice (10 Gy) at 84 h stage manifested denuded and eroded villi, shrunken villus stromal cores, elongated and dilated crypts, depopulated crypts, decrease in crypt cell number, decrease in mitotic index and apoptotic bodies in the crypts (Fig. 1a). Pre-irradiation administration of Podophyllum substantially reduced the damage in comparison to the irradiated control.
417
Crypt microcolony survival
The average viable crypts per T.S. of jejunum in the untreated control were about 138 ± 2 (Fig. 1b and 1c). Podophyllum alone rendered significant decrease in the number of crypts (120 ± 2.9) than the control (P > 0.05). Irradiation reduced the number of viable crypts to 34 ± 2 (80% reduction) as seen at 84 h post-irradia-
Fig. 2a. Effect of pretreatment of Podophyllum on radiation induced apoptosis in crypt cells observed in T.S. jejunum at different post-irradiation intervals. * P < 0.05 when compared with 10 Gy irradiated mice at all time intervals; each point represented the mean ± S.E of data from three mice.
Fig. 2b. Apoptotic bodies (AB) in the crypts (C) of jejunum 6 h post irradiation, Muscular layer (mu) (450× magnification).
418
C. A. Salin et al.
Table 1. Total number of cell population per crypt sections observed in T.S. of jejunum of strain ‘A’ mice. 6h Control Podophyllum Irradiation (10 Gy) Podophyllum +10 Gy irradiation
12 h
35 ± 3.0 36 ± 1.6 36 ± 5.1 39 ± 3.4 30 ± 3.2 20 ± 2.9
24 h
84 h
35 ± 2.0 33 ± 2.4 34 ± 3.4 34 ± 3.1 18 ± 2.5 17 ± 1.9
36 ± 1.9* 31 ± 2.0* 31 ± 2.4* 35 ± 2.7*
Each value is the mean ± S.E of results from three mice. * P < 0.05, (Drug treated irradiated mice were compared with radiation treated mice).
tion period. Pre-irradation administration of Podophyllum showed significantly higher number of viable crypts (101 ± 1.3) as compared to irradiated group (P < 0.05). Pharmacodynamics of Apoptosis in the crypts
The apoptotic frequency in the unirradiated untreated control was about 0.06 ± 0.02 (Fig. 2a and 2b). Podophyllum treatment rendered a significant increase (P < 0.05) in the frequency of apoptotic bodies at 6 and 12 h after the treatment; however at 24 and 84 h it remained insignificant (Fig. 2a). Irradiation enhanced apoptotic yield which was at its maximum (4.8 ± 0.4) 6 h after irradiation. However, after 12 h the number of apoptotic cells remained decreased significantly (2.1 ±
0.3). Podophyllum treatment before irradiation rendered significant reduction (P < 0.05) in the apoptotic bodies in comparison to irradiated control. This trend though was distinctly evident at all time intervals studied here, yet was very prominent at 84 h-stage (0.3 ± 0.06). Mitotic index
Significant decrease in the number of mitotic bodies per crypt was observed in Podophyllum treated, radiation treated and Podophyllum treated irradiated groups (Fig. 3a and 3b) in comparison to untreated control. In Podophyllum treated group the mitotic activity decreased sharply within 6 h, after which it increased steeply from 24 h onwards and became almost normal at 84 h. In irradiated group the mitotic index remained at its lowest ebb upto 12 h; with passage of time, the mitotic activity continued to increase yet the cellularity in the crypts remained distinctly low even at 84 h stage. Podophyllum treatment given before 10 Gy irradiation manifested low mitotic activity till 24 h but later it increased attaining nearly the untreated control value by 84 h. Crypt cell number
Podophyllum treatment did not affect the crypt cellularity, which was similar to that of untreated control (Table 1). 6 h after irradiation apoptosis and loss of stem cells were observed to reduce the crypt cellularity, which continued to deplete further significantly, and the P value remained less than 0.05 at 12, 24 and
Fig. 3a. Radioprotective effect of Podophyllum on mitotic activity in crypt cells observed in T.S. jejunum at different time intervals. * P < 0.05 compared with 10 Gy irradiated mice; each point represented the mean ± S.E of data from three mice.
Jejunal radioprotection by P. hexandrum
419
Fig. 3b. Mitotic bodies (MI) in the crypts of jejunum 84h post irradiation (450× magnification). Muscular layer (Mu), Crypt ghost (CG).
84 h stage when compared with corresponding unirradiated control values. Pre-irradiation administration of Podophyllum did not show decrease in the crypt cellularity at 6 h stage. However at 12 and 24 h post-irradiation period it remained lower than the unirradiated control but was about 40–50% higher than the irradiated control; at 84 h stage the crypt cell number recovered almost to the unirradiated control value.
Villus Cellularity
The numbers of cells along the length of the villi section after 84 h of various treatments have been shown in Fig. 4. Cellularity in the villi reduced to 29% and 79% in the radiation treated and in the Podophyllum treated irradiated groups respectively as compared to the unirradiated control group considered as 100%. Podophyllum treatment alone did not adversely affect villus cellularity as seen at 84 h post-treatment period.
420
C. A. Salin et al.
j Discussion The major component of radiation injury is mediated by free radical generation which interact with various bio-macromolecules like DNA, phospholipids and other cell organelle. Radioprotective agents minimise this damage by reducing generation of free radicals. It is well known that proliferating cells are more susceptible to radiation damage (Jonathan et al., 1999). Crypts of Lieberkuhn continuously generate cells in a highly regulated manner to provide appropriate villi architecture and are therefore highly sensitive to radiation injury. Pre-irradiation administration of Podophyllum ameliorated the acute radiation damage in the mouse jejunum as seen through the significantly higher number of crypt microcolonies (Fig. 1b). In comparison to untreated irradiated control, Podophyllum treated irradiated mice rendered higher number of microcolonies which may be attributed possibly to two fold reasons: (a). the protection of crypt cells against radiation induced apoptosis (Fig. 2a) and (b). accelerated recruitment of cells by enhanced rate of proliferation as seen through increased mitotic index of crypt cellularity at 84 h stage (Fig. 3a and Table 1). The increased microcolonies in the Podophyllum treated irradiated mice was helpful in replenishment of the damaged and apoptotic cells at a faster rate resulting in the protection and maintenance of villi (Fig. 4). At molecular level the stem cells of the crypt were protected against radiation damage due to free radical scavenging (Premkumar and Goel 2000) and possibly by enhancing repair of target molecules. Iron is consid-
ered an important contributor in the generation of free radicals (Stevens et al., 2000) and agents which can chelate iron can reduce free radical generated damage (Morel et al., 1993). Flavonoids present in various medicinal plants have been well documented for iron chelation properties (Bars et al., 1994, Sgaragli et al., 1993). Podophylloquercetin present in Podophyllum extract is a flavanoid (Singh and Shah 1994) and may play a pivotal role in iron chelation and free radical scavenging (Premkumar and Goel 2000). The causation of damage during irradiation could also be reduced if DNA synthesis was inhibited and cells were arrested during reproductive activity (Booth et al., 2000). Substances, which can chelate iron such as mimosine, are known to reversibly block cell cycle progression in mammalian cells (Kulp and Vulliet 1996) through inhibition of cyclin dependent kinase activity (Kulp et al., 1996). Since Podophyllum contains compounds, which can chelate iron (Premkumar and Goel 2000), it can possibly contribute in arresting the cell cycle at G1 stage, and therefore administration of Podophyllum extract before irradiation could help in reducing the damage to the stem cells. Podophyllum extract also possesses compounds that inhibit topoisomerase-II activity (Cragg and Suffness 1988) and this leads to the arrest of cells in G2 phase. However, the cell cycle arrest was reversible in nature, since the mitotic index was at its nadir by 24 h in the Podophyllum treated and in Podophyllum+10 Gy treated mice. However the mitotic index in these two groups increased thereafter indicating the commencement of cell cycle. This allowed the mitotic index to reach the control value at 84 h stage in both the groups
Fig. 4. Effect of pretreatment of Podophyllum on the maintenance of cell number along the villi observed 84 h after irradiation observed in T.S. jejunum. * P < 0.05 compared with 10 Gy irradiated mice; each point represented the mean ± S.E of data from three mice.
Jejunal radioprotection by P. hexandrum (Fig. 3a). Thus, pre-irradiation administration of Podophyllum arrested the cell cycle temporarily and its free radical scavenging action protected the clonogenic cells in the crypts against radiation damage. This could have significantly protected the crypt cellularity (Table 1) thereby contributing to an increase in microcolonies (Fig. 1b). Further insight into the role of P. hexandrum on various gene expressions such as bcl-2, implicated in suppression of apoptosis, (Reed 1997, Kroemer and Reed 2000) and TGF-β responsible for arresting cells at G1 phase and rendering radioprotection in epithelial cells, (Booth et al., 2000, Blobe et al., 2000, Potten et al., 1997) could be useful in understanding explicitly the mechanism of radioprotection of stem cells by P. hexandrum.
Acknowledgments
The authors are thankful to Director INMAS for his support during this study and to Singh, S., Prasad, J., Kumar, P., Shobi, V., Prakash,H., Premkumar, I. and Arora, R., for their help during the experiments and Prof. C. K. Gupta, Consultant, INMAS, for statistical analysis of the data.
j References Bars, W., Michael, C., Saran, M.: Flavonoids anti oxidants: Rate constants for reactions with oxygen radicals. Methods Enzymol. 234: 426–429, 1994. Blobe, G.C., Schiemann, W.P., Lodish, H.F.: Role of transforming growth factor-β in human disease. New Eng. J. Med. 342 (18): 1350–1358, 2000. Booth, D., Haley, J.D., Bruskin, A.M., Potten, C.S.: Transforming growth factor-B3 protects murine small intestinal crypt stem cells and animal survival after irradiation possibly by reducing stem-cell cycling. Int. J. Cancer. 86 (1): 53–59, 2000. Cragg, G., Suffness, M.: Metabolism of plant–derived anticancer agents. Pharmacol. Ther. 37: 425–461, 1988. Ettarh, R.R., Hodgel, G.M., Carr, K.E.: Radiation effects in the small bowel of the diabetic mouse. Int. J. Radiat. Biol. 76: 241–248, 2000. Goel, H.C., Prasad,J., Ashok Sharma., Brahma Singh: Antitumour and radioprotective action of Podophyllum hexandrum. Ind. J. Exp. Biol. 36: 583–587, 1998. Harborne J.B.: Alkaloids, Phytochemical Methods, Chapman and Hall, London, 1973, p. 177–182. Jonathan, E.C., Bernhard, E.J., McKenna, W.G.: How does radiation kill cells? Curr. Opin. Chem. Biol. 3: 77–83, 1999. Khan, W.B., Chaoxiang Shui, Shoucheng Nung, Susan, J. Knox: Enhancement of murine intestinal stem cell survival after irradiation by keratinocyte growth factor. Radiat. Res. 148: 248–253, 1997. Kroemer,G., Reed, J.C.: Mitochondrial control of cell death. Nature Med. 6 (5): 513–519, 2000.
421
Kulp, K.S., Green, S.L., Vulliet, P.R.: Iron deprivation inhibits cyclin-dependent kinase activity and decreases cyclin D/CDK4 protein levels in asynchronous MDA-MB543 human breast cancer cells. Exp. Cell Res. 229 (1): 60–68, 1996. Kulp, K.S., Vulliet, P.R.: Mimosine blocks cell cycle progression by chelating iron in asynchronous human breast cancer cells. Toxicol. Appl. Pharmacol. 139 (2): 356–364, 1996. Meritt, A.J., Potten, C.S., Watson, A.J.M., Loh, D.Y., Kei-ichi Nakayama, Keiko Nakayama, Hickman, J.A.: Differential expression of bcl-2 in intestinal epithelia – correlation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia. J. Cell. Sci. 108: 2261–2271, 1995. Morel, I., Lescoat, G., Gogrel, P., Sergent, O., Pasdeloup, N., Brissot, P., Cillard, P., Cillard, J.: Antioxidant and ironchelating activities of the flavonoids catechin, quercetin and diosmetin on iron loaded rat hepatocyte cultures. Biochem. Pharmacol. 7: 13–19, 1993. Potten, C.S, Morris, R.J.: Epithelial stem cells in vivo. J. Cell. Sci. Suppl. 10: 45–62, 1988. Potten, C.S.: A Comprehensive study of the radiobiological response of the murine (BDF1) small intestine. Int. J. Radiat. Biol. 58 (6): 925–973, 1990. Potten, C.S., Meritt, A.J., Hickman, J.A., Hall, P.A. Faranda, A.: Characterisation of radiation induced apoptosis in the small intestine and its biological implications. Int. J. Radiat. Biol. 65: 71–78, 1994. Potten, C.S., Booth, D., Haley, J.D.: Pretreatment with transforming growth factor beta-3 protects small intestinal stem cells against radiation damage in vivo. Br. J. Cancer. 75 (10): 1454–1459, 1997. Premkumar, I., Goel, H.C.: Metal chelation and related properties of P. hexandrum, a possible role in radioprotection. Ind. J. Exp. Biol. 38 (10): 1003–1006, 2000. Pritchard, M.D., Potten, C.S., Hickman, J.H.: The relationships between P53 dependent apoptosis, Inhibition of proliferation and 5-fluorouracil induced histopathology in murine intestinal epithelia. Cancer Res. 58 (23): 5453–5465, 1998. Reed, J.C.: Bcl-2 family proteins in cancer; regulators of cell death involved in resistance to therapy. In: Robert, R., Ruffolo Jr., George Poste, Brian, W. Metcalf (eds.): Cell Cycle Regulation. Harwood Academic Publishers, Netherlands, pp. 151–167, 1997. Ruifrok, A.C.C., Mason, K.A., Lozano, G., Thames, H.D.: Spatial and temporal patterns of expression of epidermal growth factor, transforming growth factor alpha and transforming growth factor beta 1–2 and their receptors in mouse jejunum after radiation treatment. Radiat. Res. 147 (1): 1–12, 1997. Sgaragli, G.P., Valoti, M., Gorelli, B., Fusi, F., Palmi, M., Mantovani, P.: Calcium antagonist and antioxidant properties of some hindered phenols. Br. J. Pharmacol. 119: 369–377, 1993. Singh, J., Shah, N.C.: Podophyllum: A Review. Curr. Res. Med. Aromat. Plants. 16 (2): 53–83, 1994. Stevens, G.R., Morris, J.E., Anderson, E.: Hemochromatosis heterozygotes may constitute a radiation-sensitive sub population. Radiat. Res. 153: 844–847, 2000. Tien, C.Ko, Wade, A. Bresnahan, E., Thompson, A.: Intestinal cell cycle regulation. In: Meijer, L, Guidet, S.,
422
C. A. Salin et al.
Philippe,M. (eds.): Progress in cell cycle research. Plenum Press, New York, pp. 43–52, 1997. Weiss, J.F., Kumar, K.S., Walden, T.L., Neta, R., Landauer, M.R, Clark, E.P.: Advances in radioprotection through the use of combined agent regimens. Int.J.Radiat.Biol. 57(4): 709–722, 1990. Withers, H.R., Elkind, M.M.: Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation. Int. J. Radiat. Biol. 17: 261–267, 1970.
j Address H.C. Goel, Head, Radiation Biology Division, Institute of Nuclear Medicine and Allied sciences, Lucknow Marg, Delhi-110 054, India Tel: ++91-011- 3970081; Fax: ++91-011-3919509; e- mail: [email protected]
Phytomedicine
Protection of mouse jejunum against lethal irradiation by Podophyllum hexandrum C.A. Salin, N. Samanta and H.C. Goel Department of Radiation Biology, Institute of Nuclear Medicine and Allied Science, Delhi, India
Summary Radiation induced gastrointestinal damage occurs due to the destruction of the clonogenic crypt cells and eventual depopulation and denudation of the villi. P. hexandrum, a plant, known for its antitumour activity, has been shown to protect the mice against whole body lethal (10 Gy) irradiation. Present study was undertaken to investigate the radioprotective effect of P. hexandrum on jejunal villi cells, crypt cells, their proliferative capacity and mitigation of apoptosis. In an in vivo micro colony survival assay, pre-irradiation administration of P. hexandrum (–2 h) increased the number of surviving crypts in the jejunum by a factor of 3.0 (P < 0.05) and villi cellularity by 2.7 (P < 0.05) fold in comparison to irradiated control. Pre-irradiation administration of P. hexandrum reduced the incidence of apoptotic bodies in the crypts (P < 0.05) in a time dependent manner and depicted a mitotic arrest till the 24 h. However, after 84 h the percentage of mitosis was observed to be nearly similar to that of unirradiated control. This study suggests that arrest of cell division may help in protecting the clonogenic cells against radiation. It would be interesting to investigate further the role of P. hexandrum in influencing various cell cycle regulators like bcl-2, TGF-β, Cyclin-E etc. Key words: Radioprotection, Podophyllum hexandrum, Microcolony assay, Crypts, Apoptosis, Mitosis
j Introduction Gastrointestinal epithelium is an important tissue responsible for digestion and acting as a barrier to luminal bacteria and toxins (Tien et al., 1997). Therefore, every three to six day’s intestinal epithelium renews itself with the help of crypts of lieberkuhn located at the base of the villi (Potten and Morris 1988). Crypts contains few stem cells which keep on proliferating in a regulated way and the transit time of cells from proliferative compartment in the crypts to the extrusion zone at the tip of the villus is between three to five days in the mouse. This rapid turnover makes crypts as one of the most radiosensitive tissues of the body. Stem cells of the crypts get sterilised by lethal irradiation, subsequently the crypts shrink and disappear within 2–3 days; transit cells however continue to divide for a few
divisions and migrate to the villus, which itself is lost within three to five days leading to manifestation of gastrointestinal syndrome (Potten 1990). Plants are known to produce plethora of secondary metabolites with wide array of pharmacological activities and therefore have been used both in the traditional as well as modern system of medicine. The isolated compounds or the active principle and the molecular drugs affect certain biochemical pathways and also interfere with other cellular mechanisms leading to the manifestation of toxic effects (Weiss et al., 1990). The natural combination of compounds present in the whole plant extract often contains several molecules, which take care of the side effects produced by the active principles. 0944-7113/01/08/06-413 $ 15.00/0
414
C. A. Salin et al.
Podophyllum hexandrum Royle known as Indian Podophyllum belongs to the family Podophyllaceae. The lignans and the other chemical moieties present in it display antitumour activity while podophyllotoxin and several other related compounds have antimitotic activity and inhibit DNA, RNA and protein synthesis (Singh and Shah 1994). A dose of 10 Gy causes total mortality by severely jeopardising the bone marrow function. Concurrently it also causes damage to gastrointestinal system and especially to the stem cells in the crypts. Pre-administration of aqueous extract of P. hexandrum has been shown to render significant radioprotection against whole body lethal irradiation (10 Gy) in mice (Goel et al., 1998). The effect of the radioprotective agent, P. hexandrum extract was therefore investigated for its capacity to protect crypt stem cells. This information could be of significant importance for even other gastrointestinal disorders.
j Materials and methods Experimental procedures were adopted as approved by the animal experimentation ethics committee and complied with the Standard Laboratory Guidelines of INMAS. Experimental Animals
12–13 weeks old inbred Swiss albino strain ’A’ male mice weighing 25 ± 3g, were maintained under controlled environment (25 ± 2 °C, photoperiod 12 h/day) and provided standard animal food pellet (Amrut laboratory animal feed, India) and water ad libitum. Not more than three animals were housed in a polyvinyl cage and each group had three animals. Herbal extraction
Aqueous extract of whole rhizome of P. hexandrum (Goel et al., 1998), was filtered and lyophilised to obtain a brownish black resin and stored at 4 °C. The aqueous extract of P. hexandrum was chromatographed according to Harborne (1973) using Silica gel G plates and Methanol-conc. NH4OH (200:3) as solvent and Podophyllotoxin 95% (ACROS , New Jersey, USA) as standard marker. The plates were detected with a) Dragendorff-, b) Iodoplatinate- and c) Marquis-reagents. For experiments, the resin was resuspended in triple distilled water and sterilised by passing through 0.2 µm filter (Minisart® NML). Dose of 200mg/kg b.w of herbal extract was administered to each mouse by i.p route; sham treated mice received distilled water.
Fig. 1a. Crypt cellularity, Mitotic bodies(MI), crypt (C) Apoptotic bodies(AB) in the jejunal crypts 84 h post irradiation (1000× magnification).
The experiments regarding whole body survival and crypt survival after radiation exposure conducted with lyophilised extract of P. hexandrum were repeated further with extracts from 2–3 samples of rhizomes collected within a period of 3 months. The data was pooled and averaged for atleast three different batches of the extract.
Jejunal radioprotection by P. hexandrum
415
Mice were individually placed in a plastic container and a whole body dose of 10 Gy was administered by using 60Co gamma cell (model 220, Atomic Energy Commission, Canada). The dose rate during the experimental period was 1.06 Gy/minute during the course of this study.
lying close together and appearing crowded. Nonviable crypts contained no cells and were sparsely populated by enlarged cells with prominent eosinophilic cytoplasm (Withers and Elkind 1970, Khan et al., 1997). The number of microcolonies was determined in ten cross sections per animal at 450× magnification.
Tissue preparation and light microscopy
Apoptosis in the crypts
Animals were sacrificed by cervical dislocation and small intestine was taken out while flushing the peritoneal cavity with normal saline. 3 to 4 cm of jejunum was isolated and its lumen was flushed with normal saline to remove particulate debris. The jejunum was divided into three segments each of 1 cm size and fixed in 10% buffered formaldehyde and processed for routine histopathological studies using H&E stain.
Apoptosis in the jejunal crypt cells was monitored and scored on the basis of pycnotic nuclei, marginal condensation of the chromatin, fragmentation of the nuclear material and fragments extruding into the crypt lumen (Potten et al., 1994, Meritt et al., 1995). Total number of apoptotic cells were scored in each crypt and expressed as the mean number of apoptotic bodies per crypt. Four transverse sections were scored for each mouse (Pritchard et al., 1998).
Radiation treatment
Morphometry
Non serial transverse sections of jejunum were studied for the number of villi, cells/villous, mitotic figures, apoptotic bodies and microcolonies. Each section was separated from the previous one by a minimum of 50 µm of tissue; a second observer regularly crosschecked counts. Crypt microcolony survival assay
Jejunum was taken out of the animals sacrificed 84 h after irradiation and number of microcolonies (regenerating clones or surviving crypts) per section were studied (Withers and Elkind 1970). Each viable colony was considered to have 10 or more clustered cells with prominent nucleus and little cytoplasm
Fig. 1b. Microcolonies counts in T.S. jejunum 84 h after various treatments. * P < 0.05 P. hexandrum + irradiated mice were compared with irradiated mice; each point represented the mean ± S.E of data from three mice.
Number of cells per crypt and per villus
Each transverse section of jejunum displayed crypts and villi in longitudinal section and their cells were counted at 450× magnification. Only complete longitudinal sections including the opening of the crypt and the full length of the villi from base to the tip were counted. Data were pooled from three to four separate transverse sections of jejunum from each mouse (Ruifrok et al., 1997). Mitotic Index /crypt
Cryptal mitotic figures appearing darkly stained and out of alignment with the nuclei of other cells (Ettarh et al. 2000), were counted from eight different crypts
416
C. A. Salin et al.
Fig. 1c. Crypt microcolonies (C), muscular layer (Mu), Villi (V) and Crypt Ghost (CG) in the jejunal mucosae 84 h post irradiation (100× magnification).
along with crypt cell number selected from three separate transverse sections of jejunum of each mouse and the result is expressed as percentage mitosis per crypt. From each sample, counts were done on atleast three non-serial, coded sections sufficient to minimise the subjectivity of the observer. Analysis of data
Data with respect to different parameters obtained from mice belonging to various treatment groups were sub-
jected to student-t test and significance was assessed at 95% confidence intervals.
j Results In the aqueous extract of P. hexandrum by TLC no Podophyllotoxin could be detected. The cross sections of jejunum of the control mice displayed normal histological features. Mice treated with
Jejunal radioprotection by P. hexandrum Podophyllum only were similar to the sham treated group. Irradiated mice (10 Gy) at 84 h stage manifested denuded and eroded villi, shrunken villus stromal cores, elongated and dilated crypts, depopulated crypts, decrease in crypt cell number, decrease in mitotic index and apoptotic bodies in the crypts (Fig. 1a). Pre-irradiation administration of Podophyllum substantially reduced the damage in comparison to the irradiated control.
417
Crypt microcolony survival
The average viable crypts per T.S. of jejunum in the untreated control were about 138 ± 2 (Fig. 1b and 1c). Podophyllum alone rendered significant decrease in the number of crypts (120 ± 2.9) than the control (P > 0.05). Irradiation reduced the number of viable crypts to 34 ± 2 (80% reduction) as seen at 84 h post-irradia-
Fig. 2a. Effect of pretreatment of Podophyllum on radiation induced apoptosis in crypt cells observed in T.S. jejunum at different post-irradiation intervals. * P < 0.05 when compared with 10 Gy irradiated mice at all time intervals; each point represented the mean ± S.E of data from three mice.
Fig. 2b. Apoptotic bodies (AB) in the crypts (C) of jejunum 6 h post irradiation, Muscular layer (mu) (450× magnification).
418
C. A. Salin et al.
Table 1. Total number of cell population per crypt sections observed in T.S. of jejunum of strain ‘A’ mice. 6h Control Podophyllum Irradiation (10 Gy) Podophyllum +10 Gy irradiation
12 h
35 ± 3.0 36 ± 1.6 36 ± 5.1 39 ± 3.4 30 ± 3.2 20 ± 2.9
24 h
84 h
35 ± 2.0 33 ± 2.4 34 ± 3.4 34 ± 3.1 18 ± 2.5 17 ± 1.9
36 ± 1.9* 31 ± 2.0* 31 ± 2.4* 35 ± 2.7*
Each value is the mean ± S.E of results from three mice. * P < 0.05, (Drug treated irradiated mice were compared with radiation treated mice).
tion period. Pre-irradation administration of Podophyllum showed significantly higher number of viable crypts (101 ± 1.3) as compared to irradiated group (P < 0.05). Pharmacodynamics of Apoptosis in the crypts
The apoptotic frequency in the unirradiated untreated control was about 0.06 ± 0.02 (Fig. 2a and 2b). Podophyllum treatment rendered a significant increase (P < 0.05) in the frequency of apoptotic bodies at 6 and 12 h after the treatment; however at 24 and 84 h it remained insignificant (Fig. 2a). Irradiation enhanced apoptotic yield which was at its maximum (4.8 ± 0.4) 6 h after irradiation. However, after 12 h the number of apoptotic cells remained decreased significantly (2.1 ±
0.3). Podophyllum treatment before irradiation rendered significant reduction (P < 0.05) in the apoptotic bodies in comparison to irradiated control. This trend though was distinctly evident at all time intervals studied here, yet was very prominent at 84 h-stage (0.3 ± 0.06). Mitotic index
Significant decrease in the number of mitotic bodies per crypt was observed in Podophyllum treated, radiation treated and Podophyllum treated irradiated groups (Fig. 3a and 3b) in comparison to untreated control. In Podophyllum treated group the mitotic activity decreased sharply within 6 h, after which it increased steeply from 24 h onwards and became almost normal at 84 h. In irradiated group the mitotic index remained at its lowest ebb upto 12 h; with passage of time, the mitotic activity continued to increase yet the cellularity in the crypts remained distinctly low even at 84 h stage. Podophyllum treatment given before 10 Gy irradiation manifested low mitotic activity till 24 h but later it increased attaining nearly the untreated control value by 84 h. Crypt cell number
Podophyllum treatment did not affect the crypt cellularity, which was similar to that of untreated control (Table 1). 6 h after irradiation apoptosis and loss of stem cells were observed to reduce the crypt cellularity, which continued to deplete further significantly, and the P value remained less than 0.05 at 12, 24 and
Fig. 3a. Radioprotective effect of Podophyllum on mitotic activity in crypt cells observed in T.S. jejunum at different time intervals. * P < 0.05 compared with 10 Gy irradiated mice; each point represented the mean ± S.E of data from three mice.
Jejunal radioprotection by P. hexandrum
419
Fig. 3b. Mitotic bodies (MI) in the crypts of jejunum 84h post irradiation (450× magnification). Muscular layer (Mu), Crypt ghost (CG).
84 h stage when compared with corresponding unirradiated control values. Pre-irradiation administration of Podophyllum did not show decrease in the crypt cellularity at 6 h stage. However at 12 and 24 h post-irradiation period it remained lower than the unirradiated control but was about 40–50% higher than the irradiated control; at 84 h stage the crypt cell number recovered almost to the unirradiated control value.
Villus Cellularity
The numbers of cells along the length of the villi section after 84 h of various treatments have been shown in Fig. 4. Cellularity in the villi reduced to 29% and 79% in the radiation treated and in the Podophyllum treated irradiated groups respectively as compared to the unirradiated control group considered as 100%. Podophyllum treatment alone did not adversely affect villus cellularity as seen at 84 h post-treatment period.
420
C. A. Salin et al.
j Discussion The major component of radiation injury is mediated by free radical generation which interact with various bio-macromolecules like DNA, phospholipids and other cell organelle. Radioprotective agents minimise this damage by reducing generation of free radicals. It is well known that proliferating cells are more susceptible to radiation damage (Jonathan et al., 1999). Crypts of Lieberkuhn continuously generate cells in a highly regulated manner to provide appropriate villi architecture and are therefore highly sensitive to radiation injury. Pre-irradiation administration of Podophyllum ameliorated the acute radiation damage in the mouse jejunum as seen through the significantly higher number of crypt microcolonies (Fig. 1b). In comparison to untreated irradiated control, Podophyllum treated irradiated mice rendered higher number of microcolonies which may be attributed possibly to two fold reasons: (a). the protection of crypt cells against radiation induced apoptosis (Fig. 2a) and (b). accelerated recruitment of cells by enhanced rate of proliferation as seen through increased mitotic index of crypt cellularity at 84 h stage (Fig. 3a and Table 1). The increased microcolonies in the Podophyllum treated irradiated mice was helpful in replenishment of the damaged and apoptotic cells at a faster rate resulting in the protection and maintenance of villi (Fig. 4). At molecular level the stem cells of the crypt were protected against radiation damage due to free radical scavenging (Premkumar and Goel 2000) and possibly by enhancing repair of target molecules. Iron is consid-
ered an important contributor in the generation of free radicals (Stevens et al., 2000) and agents which can chelate iron can reduce free radical generated damage (Morel et al., 1993). Flavonoids present in various medicinal plants have been well documented for iron chelation properties (Bars et al., 1994, Sgaragli et al., 1993). Podophylloquercetin present in Podophyllum extract is a flavanoid (Singh and Shah 1994) and may play a pivotal role in iron chelation and free radical scavenging (Premkumar and Goel 2000). The causation of damage during irradiation could also be reduced if DNA synthesis was inhibited and cells were arrested during reproductive activity (Booth et al., 2000). Substances, which can chelate iron such as mimosine, are known to reversibly block cell cycle progression in mammalian cells (Kulp and Vulliet 1996) through inhibition of cyclin dependent kinase activity (Kulp et al., 1996). Since Podophyllum contains compounds, which can chelate iron (Premkumar and Goel 2000), it can possibly contribute in arresting the cell cycle at G1 stage, and therefore administration of Podophyllum extract before irradiation could help in reducing the damage to the stem cells. Podophyllum extract also possesses compounds that inhibit topoisomerase-II activity (Cragg and Suffness 1988) and this leads to the arrest of cells in G2 phase. However, the cell cycle arrest was reversible in nature, since the mitotic index was at its nadir by 24 h in the Podophyllum treated and in Podophyllum+10 Gy treated mice. However the mitotic index in these two groups increased thereafter indicating the commencement of cell cycle. This allowed the mitotic index to reach the control value at 84 h stage in both the groups
Fig. 4. Effect of pretreatment of Podophyllum on the maintenance of cell number along the villi observed 84 h after irradiation observed in T.S. jejunum. * P < 0.05 compared with 10 Gy irradiated mice; each point represented the mean ± S.E of data from three mice.
Jejunal radioprotection by P. hexandrum (Fig. 3a). Thus, pre-irradiation administration of Podophyllum arrested the cell cycle temporarily and its free radical scavenging action protected the clonogenic cells in the crypts against radiation damage. This could have significantly protected the crypt cellularity (Table 1) thereby contributing to an increase in microcolonies (Fig. 1b). Further insight into the role of P. hexandrum on various gene expressions such as bcl-2, implicated in suppression of apoptosis, (Reed 1997, Kroemer and Reed 2000) and TGF-β responsible for arresting cells at G1 phase and rendering radioprotection in epithelial cells, (Booth et al., 2000, Blobe et al., 2000, Potten et al., 1997) could be useful in understanding explicitly the mechanism of radioprotection of stem cells by P. hexandrum.
Acknowledgments
The authors are thankful to Director INMAS for his support during this study and to Singh, S., Prasad, J., Kumar, P., Shobi, V., Prakash,H., Premkumar, I. and Arora, R., for their help during the experiments and Prof. C. K. Gupta, Consultant, INMAS, for statistical analysis of the data.
j References Bars, W., Michael, C., Saran, M.: Flavonoids anti oxidants: Rate constants for reactions with oxygen radicals. Methods Enzymol. 234: 426–429, 1994. Blobe, G.C., Schiemann, W.P., Lodish, H.F.: Role of transforming growth factor-β in human disease. New Eng. J. Med. 342 (18): 1350–1358, 2000. Booth, D., Haley, J.D., Bruskin, A.M., Potten, C.S.: Transforming growth factor-B3 protects murine small intestinal crypt stem cells and animal survival after irradiation possibly by reducing stem-cell cycling. Int. J. Cancer. 86 (1): 53–59, 2000. Cragg, G., Suffness, M.: Metabolism of plant–derived anticancer agents. Pharmacol. Ther. 37: 425–461, 1988. Ettarh, R.R., Hodgel, G.M., Carr, K.E.: Radiation effects in the small bowel of the diabetic mouse. Int. J. Radiat. Biol. 76: 241–248, 2000. Goel, H.C., Prasad,J., Ashok Sharma., Brahma Singh: Antitumour and radioprotective action of Podophyllum hexandrum. Ind. J. Exp. Biol. 36: 583–587, 1998. Harborne J.B.: Alkaloids, Phytochemical Methods, Chapman and Hall, London, 1973, p. 177–182. Jonathan, E.C., Bernhard, E.J., McKenna, W.G.: How does radiation kill cells? Curr. Opin. Chem. Biol. 3: 77–83, 1999. Khan, W.B., Chaoxiang Shui, Shoucheng Nung, Susan, J. Knox: Enhancement of murine intestinal stem cell survival after irradiation by keratinocyte growth factor. Radiat. Res. 148: 248–253, 1997. Kroemer,G., Reed, J.C.: Mitochondrial control of cell death. Nature Med. 6 (5): 513–519, 2000.
421
Kulp, K.S., Green, S.L., Vulliet, P.R.: Iron deprivation inhibits cyclin-dependent kinase activity and decreases cyclin D/CDK4 protein levels in asynchronous MDA-MB543 human breast cancer cells. Exp. Cell Res. 229 (1): 60–68, 1996. Kulp, K.S., Vulliet, P.R.: Mimosine blocks cell cycle progression by chelating iron in asynchronous human breast cancer cells. Toxicol. Appl. Pharmacol. 139 (2): 356–364, 1996. Meritt, A.J., Potten, C.S., Watson, A.J.M., Loh, D.Y., Kei-ichi Nakayama, Keiko Nakayama, Hickman, J.A.: Differential expression of bcl-2 in intestinal epithelia – correlation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia. J. Cell. Sci. 108: 2261–2271, 1995. Morel, I., Lescoat, G., Gogrel, P., Sergent, O., Pasdeloup, N., Brissot, P., Cillard, P., Cillard, J.: Antioxidant and ironchelating activities of the flavonoids catechin, quercetin and diosmetin on iron loaded rat hepatocyte cultures. Biochem. Pharmacol. 7: 13–19, 1993. Potten, C.S, Morris, R.J.: Epithelial stem cells in vivo. J. Cell. Sci. Suppl. 10: 45–62, 1988. Potten, C.S.: A Comprehensive study of the radiobiological response of the murine (BDF1) small intestine. Int. J. Radiat. Biol. 58 (6): 925–973, 1990. Potten, C.S., Meritt, A.J., Hickman, J.A., Hall, P.A. Faranda, A.: Characterisation of radiation induced apoptosis in the small intestine and its biological implications. Int. J. Radiat. Biol. 65: 71–78, 1994. Potten, C.S., Booth, D., Haley, J.D.: Pretreatment with transforming growth factor beta-3 protects small intestinal stem cells against radiation damage in vivo. Br. J. Cancer. 75 (10): 1454–1459, 1997. Premkumar, I., Goel, H.C.: Metal chelation and related properties of P. hexandrum, a possible role in radioprotection. Ind. J. Exp. Biol. 38 (10): 1003–1006, 2000. Pritchard, M.D., Potten, C.S., Hickman, J.H.: The relationships between P53 dependent apoptosis, Inhibition of proliferation and 5-fluorouracil induced histopathology in murine intestinal epithelia. Cancer Res. 58 (23): 5453–5465, 1998. Reed, J.C.: Bcl-2 family proteins in cancer; regulators of cell death involved in resistance to therapy. In: Robert, R., Ruffolo Jr., George Poste, Brian, W. Metcalf (eds.): Cell Cycle Regulation. Harwood Academic Publishers, Netherlands, pp. 151–167, 1997. Ruifrok, A.C.C., Mason, K.A., Lozano, G., Thames, H.D.: Spatial and temporal patterns of expression of epidermal growth factor, transforming growth factor alpha and transforming growth factor beta 1–2 and their receptors in mouse jejunum after radiation treatment. Radiat. Res. 147 (1): 1–12, 1997. Sgaragli, G.P., Valoti, M., Gorelli, B., Fusi, F., Palmi, M., Mantovani, P.: Calcium antagonist and antioxidant properties of some hindered phenols. Br. J. Pharmacol. 119: 369–377, 1993. Singh, J., Shah, N.C.: Podophyllum: A Review. Curr. Res. Med. Aromat. Plants. 16 (2): 53–83, 1994. Stevens, G.R., Morris, J.E., Anderson, E.: Hemochromatosis heterozygotes may constitute a radiation-sensitive sub population. Radiat. Res. 153: 844–847, 2000. Tien, C.Ko, Wade, A. Bresnahan, E., Thompson, A.: Intestinal cell cycle regulation. In: Meijer, L, Guidet, S.,
422
C. A. Salin et al.
Philippe,M. (eds.): Progress in cell cycle research. Plenum Press, New York, pp. 43–52, 1997. Weiss, J.F., Kumar, K.S., Walden, T.L., Neta, R., Landauer, M.R, Clark, E.P.: Advances in radioprotection through the use of combined agent regimens. Int.J.Radiat.Biol. 57(4): 709–722, 1990. Withers, H.R., Elkind, M.M.: Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation. Int. J. Radiat. Biol. 17: 261–267, 1970.
j Address H.C. Goel, Head, Radiation Biology Division, Institute of Nuclear Medicine and Allied sciences, Lucknow Marg, Delhi-110 054, India Tel: ++91-011- 3970081; Fax: ++91-011-3919509; e- mail: [email protected]