Effects of Podophyllum hexandrum on radiation induced delay of postnatal appearance of reflexes and physiological markers in rats irradiated in utero.

Summary

Effect of 2.0 Gy gamma-dose delivered to rats in utero on 17th day of gestation was studied to monitor the radiation induced retardation of neurophysiological development in postnatal young ones. Rhizome of Podophyllum hexandrum which has been well documented for mitigating radiation injuries in adult mice was attempted for modifying radiation damage. Rats were observed from postnatal day 1 to 25 for the age of the appearance of physiological markers (pinna detachment, inscisor's eruption, eye opening) and acquisition of reflexes (surface righting, visual placing, reflex suspension, negative geotaxis). In irradiated groups there was a significant weight reduction in mother rats and offsprings throughout the experimental period. There was radiation-induced delay in the appearance of pinna detachment but not in eye opening and inscisor's eruption. Appearance of the reflexes were also delayed due to irradiation. Preirradiation administration of the extract of Podophyllum hexandrum (i.p., 200 mg/kg/b.w.) mitigate d radiation induced postnatal physiological alterations. These studies have implications in protection against damage (in utero) due to planned radiation exposure.

Key words: Podophyllum hexandrum, Radioprotection, Reflex suspension visual placing

* Introduction

It has long been recognized that developing neurons within the central nervous system are more sensitive to insult by ionizing radiation than mature neurons, and that sensitivity changes with developmental age (UNSCEAR, 1993). Different neuronal populations within the brain are produced at various times during and shortly after gestation (Shimada et al., 1993). It is therefore possible to use radiation to interfere with normal developmental processes of specific regions of brain. Numerous investigators have attempted to identify alteration of brain structures and underlying behavioral modifications in rats and mice.

Animals irradiated during the formation of the hippocampus show behavioral changes associated with hippocampectomy in adults, such as spontaneous perseverative turning (Mickely et al., 1989), increased locomotor activity, deficit in learning spatial tasks (Di Cicco et al., 1991) and delay in exploratory behavior (Bhaskar and Uma Devi, 1996). These data reveal that exposure to ionizing radiation at different periods during gestation can disrupt both the morphology of specific areas of brain such as hippocampus and the functions associated with those areas. Neonatal irradiation of brain can also reduce the number of granule cells of dentate gyrus and prevent the formation of mossy fibers without an effect on other cells in hippocampus area (Gaiarsa et al., 1994). During last several decades a large number of chemicals have been investigated to overcome radiation damage. Natural molecules like cystine, cysteamine, Vitamins like A, C, E, glutathione etc, and synthetic molecules like AET (Aminoethylisothiouronium bromide hydrobromide), WR2721 etc. (Prasad, 1995) are some of the worth mentioning agents. However, severe side effects such as nausea, vomiting, hypertension and neurotoxicity (Landauer et al., 1987) associated with most of the radioprotective agents tried at therapeutic levels have restrained their use. In fact no ideal radioprotective agent is available today. Therefore, search for newer more effective agents is inevitably continuing. Recently a number of plant products have been evaluated for protection against lethal dose of radiation (Goel and Kumar, 2000; Goel and Shobi, 2001) P. hexandrum has also been investigated quite extensively in this context (Goel et al., 1998). The ligans and other chemical moieties present in it have demonstrated antitumour activity while podophyllotoxin and several other related compounds have manifested antimitotic activity and capability of inhibiting DNA, RNA and protein synthesis (Singh and Shah, 1994).

The main aim of this study was to find out whether a radioprotector found effective against haemopoietic and gastrointestinal syndrome, can render radioprotection to the developing nervous system. The radioprotective action of such an agent against planned exposure should not entail any side effects to the developing nervous tissue. Cells of the embryonic nervous system are radiosensitive and are actively differentiating and maturing during the foetal and early postnatal periods (Reyners et al., 1992). The 17th day of gestation is one of the midgestational stages when the central nervous system can be affected by irradiation and is also a period when gross malformations in most organ systems would not be expected, hence it was used for this study (Jenish and Brent, 1988). The parameters selected were the onset (age) of acquisition of selected reflexes and appearance of selected physiological markers in terms of age.

* Materials and Methods

Animals

200-250 days old inbred Sprague-Dawley rats and their off spring kept under standard laboratory conditions (photoperiod of 12-hr/day; 25 [+ or -] 2 [degrees]C were fed standard animal food pellets (Amrut Laboratory Animal Feed, India) ad libitum. Rats weighed 260 [+ or -] 10 g (females), 300 [+ or -] 14 g (males). One male and one female rat were housed together in each cage and kept overnight, and checked next day at 9:00 A.M for sperms in the vaginal smear to confirm pregnancy. The day was counted as 0 day of pregnancy or the first day of gestation. Therefore, embryos that were 18 days old were considered to be in their 17th day of prenatal life (Jenish and Brent, 1988). 24 female rats were recruited for this study. The mother rats were allowed to deliver and raise their young ones, litters of less than four offspring was not considered for the study. All procedures on experimental animals were carried out in strict compliance with the Animal Ethics Committee rules and regulations adopted at this institute.

Herbal extract

Aqueous whole extract of rhizome of P hexandrum was lyophilized and stored at 4 [degrees] C as described earlier (Goel et al., 1996). For experiments the extract was resuspended in triple distilled water and sterilized by passing through 0.2-[micro]m filter (Minisart [R] NML). Dose of 200 rug in 0.8 ml/kg b.w. of the extract was administered i.p. to each pregnant rat 2 h prior to irradiation; sham treated rats received equal volume of physiological saline.

Irradiation

On 17th day of gestation each pregnant rat was placed in a wire gauze container and put in [Co.sup.60] Gamma cell (Model 220, Atomic Energy of Canada Limited; dose rate of about 0.977 cGy/sec. during the course of investigation) to deliver 2 Gy whole body dose.

Treatment groups

The mother rats divided into four groups of 6 each were given various treatments on 17th day of pregnancy and the delivered pups were studied group-wise up to 25th postnatal day; Animals were afterwards used for other experimental studies.

* Control (Sham treated): 6 + 8 + 9 + 8 + 7 + 7 = 45 pups (25 [male], 20 [female])

* Podophyllum treated: 7 + 8 + 8 + 9 + 5 + 6 = 43 pups (23 [male], 20 [female])

* Irradiated: 7 + 6 + 5 + 8 + 7 + 5 = 38 pups (22 [male], 16 [female]; 8 [female] & 4 [male] died within Ist week.

* Podophyllum + Irradiation: (7 + 6 + 7 + 8 + 8 + 5 = 41 pups; 27 [male], 14 [female]: (2 [female] & 1 [male] died on Ist postnatal day)

Body weight of mother rats

The body weight of all the treated mother rats was observed every day from day one before irradiation (16th day of gestation) until the day of parturition (usually 22nd day of gestation).

Parameters for observation:

* Postnatal survival rate: first week of postnatal life.

* Postnatal body weight: every 5th day from postnatal day 1 to 25.

* Pinna detachment: Observations were recorded in days when the pinna of both ears unfolded to a fully erect position and continued until the entire litter exhibited it.

* Incisor's eruption: Pups were examined on 8, 9 & 10th postnatal days.

* Eye opening: Any visible streak appearing in the membrane covering the eye from 12th postnatal day was examined until both eyes of each animal in each litter were open.

* Surface righting: Each rat from 5th to 7th postnatal day was held momentarily on its back and released. The capability of attaining the normal dorso-ventral posture within 2 s (3 successive trials) was considered positive for integration of the motor and vestibular system.

* Reflex suspension: The muscular and motor coordination was tested on 7th postnatal day by placing them on a 3 mm diameter bar hanging 45 cm above the base; the time, a pup could hang on that bar, was noted.

* Negative geotaxis: The gravitational (stationary) positioning ability of a pup was tested from 6th postnatal day onwards by placing it with its head directed downward on a platform inclined at 30[degrees] and considered positive if the animal was able to turn and climb up the inclined surface against gravity within 15 s. Entire litter was checked until each neonate achieved 3 out of 3 positive responses successively during daily session.

* Visual placing ability: young ones on 17th postnatal day and onwards were held by the tail with head down over a horizontal rope placed 18 cm above a padded surface. Vibrissae were not allowed to touch the rope. Extension of its head and forelimbs toward the rope in two successive trials was considered positive.

Analysis of data

CHI square test and ANOVA were applied for different physiological markers and reflexes and p < 0.05 was considered significant.

* Results

Body weight of mother rats

The control group manifested a steady increase in the mean body weight from the 16th to 22nd pregnancy day (Table. 1). P. hexandrum treatment had no adverse effect on the mean body weight gain as compared to the control group. On 17th day of pregnancy when irradiation (2Gy) was done, the increase in the mean body weight was adversely affected (p <0.05). The difference in the mean body weight of the irradiated and control group, however, did not widen further although the irradiated group continued to have decreased body weight than controls up to 22nd pregnancy day. Pre-irradiation treatment with P. hexandrum rendered the mean body weight of mother rats comparable to the control group on all days (16-22).

Postnatal body weight of offspring treated in utero

The offsprings of the control group manifested a steady increase in mean body weight from postnatal day 1 to 25 (Fig. 1). P. hexandrum treatment (200 mg/kg b.w.) had no adverse effect on the m.b.w gain as compared to the control group. The mean body weight of the irradiated offsprings (in utero) was significantly less than that of the control groups (p < 0.05). Preirradiation treatment with P. hexandrum rendered the m.b.w of offspring comparable to the control group on all days (1-25). No effect of sex on m.b.w loss was observed in any group.

Still birth & Postnatal survival of offspring treated in utero

P. hexandrum administration on 17th day of pregnancy did not affect the postnatal survival of the offsprings. Irradiation of offsprings in utero led to a steady decrease in survival (67%) up to 4th postnatal day after which a plateau was observed. Mother rats of control and P. hexandrum treated groups did not deliver any stillbirth and the postnatal survival remained 100%. Irradiated mothers delivered 8 still births and 30 live births, while preirradiation administration of P. hexandrum to mother rats rendered 3 still births and 38 live births. Postnatal survival among the live births was expressed as percent survival.

Pinna detachment, incisor's eruption & eye opening among offspring treated in utero

Pinna detachment in control was completed during 3 & 4th post-natal days and P. hexandrum treated in utero did not manifest any effect as compared to control. Irradiation delayed pinna detachment significantly on 3rd (p < 0.01) and 4th day (p < 0.05) as compared to control. P. hexandrum treatment before irradiation reduced significantly (p < 0.05) the radiation induced delay of pinna detachment which was attained in all groups by 5th postnatal day (Table 2).

The P. hexandrum treatment had no effect on incisor eruption and eye opening as compared to control. On 8th postnatal day there was a significant increase in incisor's eruption (p < 0.01) in the neonates irradiated in utero in comparison to control group (0% incidence). On the 9th postnatal day all irradiated offsprings had their incisors erupted (Table 2). The preirradiation administration of P. hexandrum significantly delayed radiation induced incisor's eruption on 8th day yet was significantly higher than control (p < 0.01) having 0% incidence. On 9th day onwards the incidence of incisor eruption was comparable to control and on 10th postnatal day incisor's eruption was attained in all groups.

In the neonates, which received radiation exposure in utero in comparison to control (0% incidence on 12th day) there was a significant acceleration in eye opening (p < 0.05) on 12th and 13th postnatal days (Table 2). The preirradiation administration of P. hexandrum significantly reduced the radiation induced incidence of eye opening on 12th day (p < 0.05) and it remained comparable to the controls on 13th postnatal day. On 14th day all groups attained full eye opening.

Surface righting, reflex suspension, negative geotaxis and visual placing ability

Offsprings of P. hexandrum treated mother rats were slightly less efficient in surface righting on 5th and 6th postnatal day but were comparable to controls on 7th postnatal day (Table 3). In comparison to the control, irradiation reduced surface righting efficiency significantly (p < 0.01) on all postnatal days (5-7). P. hexandrum treatment before irradiation enhanced the surface righting efficiency significantly (p < 0.05) as compared to the irradiated group on all postnatal days and remained significantly (p < 0.05) lower than the control on 5th & 6th postnatal day.

The offsprings of P. hexandrum treated mother rats were more efficient in negative geotaxis than control on 6th day, but on later days the efficiency was comparable to control (Table 3). Irradiation reduced negative geotaxis significantly in comparison to control on 6th postnatal day (p < 0.01) and onwards (p < 0.05 on 7-9 days) all postnatal days (6-9). Preirradiation treatment with P. hexandrum significantly protected (p < 0.05) against radiation-induced decrease in negative geotaxis but remained quite comparable to the control on all postnatal days (Table 3).

P. hexandrum administration did not influence the visual placing efficiency as compared to the controls on 17th to 19th postnatal day (Table 3). Irradiation significantly (p < 0.01) retarded the visual placing ability as compared to the control on 17th & 18th day but was less significant (p < 0.05) on 19th day. P. hexandrum treatment before irradiation significantly improved the visual placing ability in comparison to the irradiated group; (p < 0.01 on 17th & 18th day while p < 0.05 on 19th postnatal day). However, it remained comparable to control from 17th to 19th to postnatal day.

The reflex suspension time of P. hexandrum treated group was comparable to the control during 7th-10th postnatal days. The offsprings, which were irradiated in utero, exhibited significantly reduced time of reflex suspension as compared to control on 7th day (p < 0.05) and on 8-10 postnatal days (p < 0.01). Preirradiation treatment of P. hexandrum significantly protected (p < 0.05) against radiation-induced decrease in reflex suspension on all postnatal days (Table 4); however it remained comparable to control on 7th & 8th day but significantly different on 9th and 10th postnatal days.

* Discussion

Teratogenic effects of ionizing (gamma or X) radiations have been widely reported in mammalian embryo and fetus. The damaging effect of irradiation on developing brain cells during prenatal (Altman, 1971) and postnatal conditions (Norton and Kilmer, 1987) has also been amply demonstrated. Damage to neuronal cells during development leads to structural and functional deficiencies (Hayashi et al., 1978). For the postnatal study various parameters were selected to evaluate the functional attributes of the developing brain adversely affected by irradiation during prenatal stage. The relative extent of radiation-induced damage in postnatal young ones may also unravel the differential radiosensitivity of the developing neuronal cells irradiated during prenatal life. P hexandrum that has already been found to protect against radiation-induced mortality was studied with respect to the radioprotection of the developing brain.

The radiation-induced loss of mean body weight (m.b.w) of mother rats (Table. 1) has already been reported (Goel et al., 1996) and could be attributed to radiation induced necrosis of crypt cells (Jonathan et al., 1999) leading to denudation of villi (Salin et al., 2001) affecting the absorption of food adversely and loss of m.b.w. subsequently. Radiation induces taste aversion, which leads to reduced food intake and subsequently reduced body weight. Preirradiation administration of C. asiatica reduced radiation induced taste aversion and loss of m.b.w. (Goel et al., 2001). Whether preirradiation administration of herbal extract of P. hexandrum could also protect against radiation induced loss of m.b.w by reducing taste aversion needs to be confirmed. The depressed haemopoetic system may also indirectly reduce the m.b.w of mother and fetus (Mole, 1993). The mother rats administered P. hexandrum alone did not manifest any body weight loss indicating that the P. hexandrum treatment did not affect the normal cel ls and tissues. Administration of P. hexandrum prior to irradiation prevented radiation induced m.b.w loss due to one or several mechanisms mentioned above which need to be investigated furthur.

Radiation is known to damage the fetal erythropoietic system (Mole, 1993) leading to depleted oxygen transport to various functional regions including the brain which may lead to hypoactivity in the postnatal life. Administration of P. hexandrum before irradiation rendered the m.b.w of offsprings comparable to the control (Fig. 1). It implies that P. hexandrum interfered with the mechanism of damage induction by irradiation.

Radiation induced deviations in behavior and delays in the attainment of some physiological markers and reflexes in early life may be sensitive indicators of toxicity and are associated with normal cellular differentiation, growth, and migration (Coyle et al., 1976). Alterations in physiological markers and reflexes, as shown in the present study, indicate that cellular development and differentiation have been disturbed in irradiated groups, but was mitigated by P. hexandrum if administered before irradiation.

The delay in the pinna detachment (Table 2) of prenatally irradiated offsprings is due to the reduced cell proliferation rate. The radiation is known to adversely affect the proliferation rate of dividing cells including dermal and epidermal cells (Cassarett, 1980). The exposure of 2 Gy was sublethal and could have rendered inhibition of cell proliferation temporarily for one to two cell cycles. Later the proliferation might have become comparable to control. Pinna detachment was therefore delayed by a day or two. The improvement in pinna detachment observed in the offspring which were administrated P. hexandrum prior to irradiation may be due to its effects in minimizing radiation induced temporary suspension of dermal or epidermal cell proliferation. The eye opening and incisor's eruption are associated with apoptotic process. Since irradiation accelerates apoptosis (Jonathan et al., 1999) and reduces cell proliferation, the gums and eye lids were thinner leading to early eruption of incisor's and opening o f eye lid. Since P. hexandrum treatment protected against radiation induced apoptosis and cell proliferation, the eye opening and incisor's eruption followed normal pattern (Table 2).

Negative geotaxis, surface righting and reflex suspension etc. are some of the responses reflecting functioning of the cerebellar and vestibular system. Prenatal irradiation is known to decrease the thickness of various layers in cerebellum (Mullinex et al.,1975). Irradiation on 17th prenatal day affects the intermingling of nuclear cells and transitory zones, formation of external germinal layer and its spreading on the surface of the cerebellum, the formation of future granular layer, the pale cells and golgi cells that develop from noncollapsing neuroepithelium of cerebellar ventricle (Altman, 1978). The delay in attaining negative geotaxis of prenatally irradiated offsprings is due to reduced population of neuronal cells in cerebellar and vestibular regions responsible for coordinating motor activity. Radiation induced delay in negative geotaxis is mitigated by preirradiation administration of P. hexandrum (Table 3) indicating its role in preventing radiation induced cerebellar and vestibular deficiencies .

The delay in attaining the surface righting capacity, reflex suspension and visual placing ability of prenatally irradiated rats is due to the damage or poor development of the motor, visual and vestibular system of brain. It has been reported (Kennedy, et al., 1973) that prenatal irradiation damages the development of telencephalic commissure that interferes with the function of control centers that organize specific motor acts. The surface righting (Table 3) and reflex suspension (Table 4) of offsprings that received P. hexandrum prior to irradiation was better than the irradiated group. Visual placing ability of offsprings that received P hexandrum prior to irradiation was also better (Table 3) implying that not only the cerebellum but also the visual areas of cerebrum were protected by P. hexandrum from radiation damage.

Our results reveal that R hexandrum treatment had no effects on parameters except surface righting ability and reflex suspension capability. The mechanism of radioprotection by P. hexandrum is multidirectional and further studies on various neurobehavioural, neurochemical, electrophysiological and histological parameters need further attention.

[FIGURE 1 OMITTED]

Table 1

Protective effect of P. hexandrum (200 mg/ kg b.wt.; -2 h) against
radiation (2 Gy; 17th day of pregnancy induced mean body weight loss in
pregnant rats.

Pregnancy day  Body weight(g) of mother rats

               Contorl (no treatment)  Podophyllum     2 Gy
               n = 6                   n = 6           n = 6

17             268 [+ or -] 9          267 [+ or -] 7  268 [+ or -] 7
18             278 [+ or -] 5          279 [+ or -] 6  271 [+ or -] 6
19             287 [+ or -] 6          288 [+ or -] 5  279 [+ or -] 9
20             296 [+ or -] 5          297 [+ or -] 7  285 [+ or -] 7
21             304 [+ or -] 7          305 [+ or -] 8  294 [+ or -] 8
22             309 [+ or -] 6          310 [+ or -] 5  299 [+ or -] 5

Pregnancy day  Body weight(g) of
               mother rats

               Podophyllum +2 Gy
               n = 6

17             270 [+ or -] 5
18             276 [+ or -] 7
19             285 [+ or -] 8
20             294 [+ or -] 5
21             302 [+ or -] 6
22             307 [+ or -] 5

All Values are Mean [+ or -] S. D
Table 2

Radioprotective effect of P. hexandrum on the appearance of various
physiological markers on specific postnatal days in offsprings which had
received various treatments in utero on 17th day of pregnancy.

Parameter   Postnatal  % offsprings in various treatment groups
observed    Day

                       Control         Irradiation  P. hexandru
                                                    + Irradiation

Pinna          3rd      78.57           30.76 **     66 *
detachment     4th     100              76.92 *      88 *
               5th     100             100          100
Incisor        8th       0              46.85 **     22.2 **
eruption       9th      42.85          100 **        44
              10th     100             100          100
Eye           12th       0              30.76 *      11.1 *
opening       13th      50              69.2 *       44
              14th     100             100          100

Parameter   % offsprings
            in various
            treatment
            groups
observed

            P. hexandrum


Pinna        83
detachment  100
            100
Incisor       0
eruption     50
            100
Eye           0
opening      50
            100

Values marked with * or ** were compared with control and the
significance level was * p < 0.05, ** p < 0.01.
Table 3

Radiprotective effect of P. hexandrum on the acquisition of reflexes on
specific postnatal days in offsprings which had received various
treatments in utero on 17th day of pregnancy.

Parameters               Postnatal  % offsprings in various
                                    treatment groups
observed                 day

                                    Control         Irradiation
                                                    + Irradiation

Surface righting         5th         78.57          15.38 **
                         6th         92.85          30.76 **
                         7th        100             38.46 **
Negative geotaxis        6th         42.85           0 **
                         7th         50             23.07 *
                         8th         92.85          46.15 *
                         9th        100             61.53 *
Visual placiang ability  17th        69             36 **
                         18th       100             41 **
                         19th       100             75 *

Parameters               % offsprings in various
                         treatment groups
observed

                         P. hexandrum  P. hexandrum


Surface righting          44.4 *, (a)   66.6
                          66 *, (a)     88.3
                          88           100
Negative geotaxis         33 (a)        50
                          44 (a)        50
                          88 (a)        92.85
                         100 (a)       100
Visual placiang ability   66 (aa)       66
                          88 (aa)      100
                         100           100

Values marked with * were compared with control and the significance
level was * p<0.05.

** p<0.01. Values marked with a were compared with corresponding
irradiated group and the significance level was (a)p<0.05.

(aa)p<0.01.
Table 4

Radioprotective effect of P. hexandrum on reflex suspension time (s) on
specific postnatal days in offsprings which had received various
treatments in utero on 17th day of pregnancy.

Postnatal day  % offsprings in various treatment groups

               Control             Irradiation


 7th              3 [+ or -] 0.2   1.38 [+ or -] 0.08 **
 8th              3 [+ or -] 0.2   0.57 [+ or -] 0.12 **
 9th           4.71 [+ or -] 0.26  0.95 [+ or -] 0.18 **
10th              7 [+ or -] 0.7   1.07 [+ or -] 0.76 **

Postnatal day  % offsprings in various treatment groups

               P. hexandrum               P. hexandrum
               + Irradiation

 7th           2.6 [+ or -] 45 (a)           3 [+ or -] 0.22
 8th             3 [+ or -] 21 (aa)       2.66 [+ or -] 0.54
 9th             3 [+ or -] 27 *, (aa)     4.4 [+ or -] 0.8
10th           5.3 [+ or -] 0.45 *, (aa)   6.5 [+ or -] 0.66

Values marked with * were compared with control and the significance
level was * p<0.05.

** p<0.01. Values marked with a wer compared with corresponding
irradiated group and the significance level was (a)p<0.05

(aa)p<0.01.

Acknowledgement

The authors are thankful to Dr. Rajesh Arora and Mr. Salin for their help during the course of this work and to the Director, INMAS, for his support in this study.

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* Address

H. C. Goel, Department of Radiation Biology, Institute of Nuclear Medicine and Allied Sciences, Lucknow Marg, Delhi 110 054, India

Tel: ++91-11-3970081

Fax: ++91-11-3919509

e-mail: [e-mail]. in

Phytomedicine 9: 447-454, 2002

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