In vivo radioprotective effect of Moringa oleifera leaves.

Radioprotective property of Moringa oleifera leaves was investigated in healthy adult Swiss albino mice. Animals were injected (ip) with 150 mg/kg body weight of 50% methanolic extract (ME) of M. oleifera leaves, as a single dose, or in 5 daily fractions of 30 mg/kg each, and exposed to whole body gamma irradiation (RT, 4 Gy) 1 hr later. Five animals from each group were sacrificed at 1, 2 and 7 days after treatment. Bone marrow protection was studied by scoring aberrations in metaphase chromosomes and micronucleus induction in polychromatic erythrocytes and normochromatic erythrocytes. Pretreatment with a single dose of 150 mg/kg ME significantly reduced the percent aberrant cells to 2/3rd that of RT alone group on day 1 and brought the values to normal range by day 7 post-irradiation. A similar effect was also seen for the micronucleated cells. Fractionated administration of ME (30 mg/kg x 5) gave a higher protection than that given by the same dose administered as a single treatment. ME also inhibited the Fenton reaction-generated free radical activity in vitro in a concentration dependent manner. These results demonstrate that pretreatment with the methanolic leaf extract of M. oleifera confers significant radiation protection to the bone marrow chromosomes in mice and this may lead to the higher 30 day survival after lethal whole body irradiation.

Modification of radiation induced damage in mouse intestine by WR-2721.

Intestinal protection in mice against radiation injury by WR-2721 (300 mg/kg body wt, i.p., 30 min before irradiation) was studied after whole body gamma irradiation (0.5, 1.5, 3.0, 4.5, or 6.0 Gy). Crypt survival and induction of apoptosis, and abnormal mitoses in crypt cells in the jejunum were studied on day 1, 3 and 7 after irradiation. Irradiation produced a significant decrease in crypt survival, whereas apoptosis and abnormal mitoses showed a significant increase from sham-treated control animals. Maximum changes in all the parameters were observed on day 1 after irradiation and the effect increased linearly with radiation dose. There was recovery at later intervals, which was inversely related to radiation dose. WR-2721 pre-treatment resulted in a significant increase in the number of surviving crypts, whereas the number of apoptotic cells in the crypts showed a significant decrease from respective irradiated controls on day 1 after exposure. The recovery was also faster in WR-2721 pre- treated animals. It is concluded that WR-2721 protects against gastrointestinal death by reducing radiation induced cell death, thereby maintaining a higher number of stem cells in the proliferating compartment.

Radiosensitization of a mouse melanoma by withaferin A: in vivo studies.

The radiosensitizing effect of a plant withanolide, withaferin A, on the B16F1 mouse melanoma was studied in vivo. Treatment of 100 mm3 tumours with 10 to 60 mg/kg withaferin A intraperitoneally produced a dose dependent increase in growth delay and volume doubling time. Injection of 30-50 mg/kg withaferin A, followed by 30 Gy local gamma irradiation, significantly enhanced the tumour response. No systemic or local adverse reactions were noted in these groups. The drug was most effective when injected intraperitoneally 1 h before irradiation. However, neither the individual agents nor their combination could produce any complete response (tumour cure). Melanoma is a relatively radioresistant tumour. The present results indicate that the radiation response of this tumour can be significantly enhanced by pretreatment with withaferin A.

Evaluation of anti-tumor efficacy of injectable Centchroman in mice bearing Ehrlich ascites carcinoma.

Anti-tumor efficacy of Centchroman formulated as niosomes and gel implant was evaluated in Swiss albino mice bearing Ehrlich ascites carcinoma at 10 mg/kg body weight dose given subcutaneously. Median day of death, percentage increase in host life span and changes in body weight were studied. Centchroman significantly (P < 0.05) increased the median day of death both in free and formulated systems. Also, injectable formulations exhibited a significant (P < 0.05) increase in host life span compared to free drug, hence, enhanced anti-tumor efficacy against Ehrlich ascites carcinoma.

Modulation of glutathione and antioxidant enzymes by Ocimum sanctum and its role in protection against radiation injury.

Aqueous extract (OE) of the leaves of Ocimum sanctum, the Indian holy basil, has been found to protect mouse against radiation lethality and chromosome damage and to possess significant antioxidant activity in vitro. Therefore a study was conducted to see if OE protects against radiation induced lipid peroxidation in liver and to determine the role, if any, of the inherent antioxidant system in radioprotection by OE. Adult Swiss mice were injected intraperitoneally (i.p.) with 10 mg/kg of OE for 5 consecutive days and exposed to 4.5 Gy of gamma radiation 30 min after the last injection. Glutathione (GSH) and the antioxidant enzymes glutathione transferase (GST), reductase (GSRx), peroxidase (GSPx) and superoxide dismutase (SOD), as well as lipid peroxide (LPx) activity were estimated in the liver at 15 min, 30 min, 1, 2, 4 and 8 hr post-treatment. LPx was also studied after treatment with a single dose of 50 mg/kg of OE with/without irradiation. OE itself increased the GSH and enzymes significantly above normal levels whereas radiation significantly reduced all the values. The maximum decline was at 30-60 min for GSH and related enzymes and at 2 hr for SOD. Pretreatment with the extract checked the radiation induced depletion of GSH and all the enzymes and maintained their levels within or above the control range. Radiation significantly increased the lipid peroxidation rate, reaching a maximum value at 2 hr after exposure (approximately 3.5 times that of control). OE pretreatment significantly (P < 0.0001) reduced the lipid peroxidation and accelerated recovery to normal levels. The results indicate that Ocimum extract protects against radiation induced lipid peroxidation and that GSH and the antioxidant enzymes appear to have an important role in the protection.

Effect of plumbagin on the radiation induced cytogenetic and cell cycle changes in mouse Ehrlich ascites carcinoma in vivo.

The effect of plumbagin, a naphthoquinone from the roots of the Indian medicinal plant Plumbago rosea, and Cobalt-60 gamma radiation was studied on Ehrlich ascites carcinoma in vivo, taking cytogenetic damage and cell cycle changes as experimental endpoints. Plumbagin (5 mg/kg body wt, P1) administered intraperitoneally produced a significant increase in the percentage of S-phase as well as G2-M cells with a corresponding decrease in the G1 phase at different post-treatment times. Radiation (7.5 Gy, RT) alone produced the classical G2 block at 1 hr, which persisted with a continuous increase throughout the post-treatment observation period. The combination treatment produced a similar effect as that of RT on G2-M cells, but its effect on the G1 phase was more pronounced than the latter. While P1 treatment produced a small increase in the percentage of labeled S-phase cells, combination treatment significantly reduced the labeled S-phase cells with a corresponding increase in the unlabeled fraction. Drug or radiation alone significantly increased micronuclei induction at various post-treatment times and the combination of the two further enhanced this effect additively. The mechanism of interaction of P1 with radiation in bringing about this effect is not clear.

Withania somnifera Dunal (Ashwagandha): potential plant source of a promising drug for cancer chemotherapy and radiosensitization.

Study of antitumor and radiosensitizing properties of W. somnifera (Ashwagandha), a well known medicinal plant, have yielded encouraging results. The alcoholic extract of the dried roots of the plant as well as the active component withaferin A isolated from the extract showed significant antitumor and radiosensitizing effects in experimental tumors in vivo, without any noticeable systemic toxicity. Withaferin A gave a sensitizer enhancement ratio of 1.5 for in vitro cell killing of V79 Chinese hamster cells at a non toxic concentration of approximately 2 microM. The mechanism of action of this compound is not known. The studies so far indicate that W. somnifera could prove to be a good natural source of a potent and relatively safe radiosensitizer/chemotherapeutic agent. Further studies are needed to explore the clinical potential of this plant for cancer therapy.

Effect of gamma radiation on fetal haemopoiesis of mouse.

Effect of a single acute exposure to gamma-rays during the late fetal period on the fetal haemopoietic tissue of mouse was studied. Pregnant Swiss albino mice were exposed to 1 Gy of gamma-rays on day 17 post coitus (late fetal period) and 24 hr after exposure the fetuses were observed for changes in the liver weight, cytogenetic damage in liver cells by micronucleus (MN) induction and stem cell survival by spleen colony (CFU-S) assay. Irradiation resulted in a significant (P < 0.01) decrease in fetal liver weight. A significant (P < 0.001) increase in MN count was observed after exposure, while CFU-S displayed a significant (P < 0.001) reduction in the cell survival as compared to the sham-treated control. These results demonstrate the high susceptibility of mouse fetal haemopoietic system to radiation.

Long-term effect of prenatal exposure to low level of gamma radiation on neurophysiology of mouse.

Abdominal region of pregnant Swiss mice were exposed to 0.25, 0.35 or 0.50 Gy of gamma radiation on days 11.5, 12.5, 14.5 or 17.5 post coitus (pc). Changes in locomotory activity and learning performance, and hippocampal biogenic amines (noradrenaline, NA; dopamine, DA; 5-hydroxytryptomine, 5-HT; and 5-HTs metabolite 5-hydroxyindolacetic acid, 5-HIAA) were studied at 12 (adult) and 18 months (old) of age. Significant change in locomotory activity and learning performance was observed after exposure to 0.50 Gy at late organogenesis day (11.5 pc), when tested at 12 months of age, but not observed much change at 18 months. Biogenic amines did not show any significant change after any exposure dose at any of the gestation days. It was inferred from the results that gamma irradiation (0.50 Gy) at the late organogenesis (day 11.5 pc) can impair the brain functions in adults when normal faculties are functional.

Prognostic assays in multiple myeloma: correlated with flow cytometry.

DNA ploidy of plasma cells in bone marrow has been indicated to play a role in treatment response of multiple myeloma. Therefore, a prospective study was done to test this correlation. Univariate DNA flow cytometry was done on 13 proved multiple myeloma patients. Patients aged below 50 years showed hypodiploidy, irrespective of 'S' phase population, where as all patients above 50 years had diploidy or hyperdiploidy, except for one patient. Early stage patients (I & II) with less than 25% plasma cells in bone marrow were all aneuploids. Patients belonging to advanced clinical stage with more than 60% plasma cells in bone marrow with aneuploidy, especially hyperdiploidy (DI > 1.15), carried a poor prognosis. It was difficult to correlate the 'S' phase fraction with other parameters from the present data. Further study with BrdU labelling to determine the proliferative status of the 'S' phase cells is needed.

Changes in mouse behavior induced by fetal exposure to diagnostic ultrasound.

Pregnant Swiss albino mice were exposed to diagnostic ultrasound (3.5 MHz, 65mw, ISPTP = 1 W/cm2, ISATA = 240 W/cm2) for 10 min on day 14, 16 or 17 of gestation to assess any changes in physiological reflexes (pinna detachment, eye opening and fur development) and postnatal mortality. Changes in locomotor activity by open field test and dark/bright arena test and learning and memory by hole board test were also recorded. No change was observed in physiological reflexes and postnatal mortality. However there were significant alterations in behavior in all the three exposed groups. These results demonstrate that ultrasound exposure during the late fetal period can impair brain function in adult mouse.

Radiosensitizing effect of plumbagin on mouse melanoma cells grown in vitro.

Mouse melanoma cells were treated with plumbagin, a naphthoquinone, from the plant Plumbago rosea at 0.5 microgram/ml (PI) for 60 min either alone or followed by 2 Gy gamma radiation (RT). Response to the different treatments was assessed by following the cell growth up to 5 days post treatment. PI alone produced a significant decrease in the cell count on days 3 and 4, whereas RT treatment significantly enhanced the growth inhibitory effect when compared to RT or PI alone. These findings suggests the radiosensitizing effect of PI on mouse melanoma cells in vitro, supporting the earlier in vivo findings.

Toxicity and antitumor activity of niosomal bleomycin in tumor bearing mice.

Niosomal encapsulation of bleomycin enhanced the antitumor activity against Ehrilich ascites and sarcoma-180 models. The niosomal/free drug mean survival time values of 1.28 was achieved in Ehrlich ascites infected animals. Niosomal bleomycin considerably increased the 40 days survival rate in mice. Tumor volume doubling time of S-180 tumor was also increased significantly in niosomal treated animals. Histopathological studies of lung and jejunum of Niosomal bleomycin treated mice, confirmed the less toxic potential of encapsulated bleomycin. The suppression of peripheral and bone marrow WBC counts upon niosomal bleomycin treatment was not substantial.

Multimodality treatment using AK-2123, hydralazine, radiation & hyperthermia on a transplantable mouse tumour.

The in vivo response of a transplantable mouse tumour, Sarcoma 180 to AK-2123 (AK), local irradiation (RT) and local hyperthermia, as influenced by a vasoactive drug, hydralazine (HDZ), was assessed on the basis of tumour cure (complete response CR), volume doubling time (VDT), regrowth delay (RD) and animal survival up to 120 days. A single ip injection of 200 mg/kg b.wt. AK produced more than 15 per cent CR. Combination of any two agents resulted in a better response than the single agent treatments. AK in combination with 43 degrees C, 30 min (HT) was more effective than HT combination with 10 Gy. The presence of 5 mg/kg HDZ, injected immediately after 5 Gy, in combination with AK increased the therapeutic effect over that produced by AK+10Gy. Combination of all the three agents (AK+10Gy+HT) produced 100 per cent CR and prolonged disease free animal survival. A similar response could be obtained by the presence of HDZ with a lower radiation dose of 5 Gy in combination with AK and HT (AK+5Gy+HDZ+HT). This multimodality treatment offers the possibility of further reduction in the doses of individual agents, and in the possible side effects on normal tissues without compromising the tumour cure effect.

Response of mouse sarcoma-180 to cis-platin in combination with radiation & hyperthermia.

The in vivo response of a transplantable mouse tumour, sarcoma-180, to cis-platin (cDDP, 1.0, 2.5 or 5.0 mg/kg b.wt), local hyperthermia (HT, 42 degrees C, 30' or 60' and 43 degrees C, 30') and irradiation (RT, 10 Gy) was assessed on the basis of tumour cure (complete response, CR), volume doubling time (VDT) and regrowth delay (RD) as well as animal survival up to 120 days. Each agent was given as a single modality or in different combination regimens. A single injection of cDDP produced a dose dependent increase in all the parameters. Of all the single treatments, HT at 43 degrees C, 30' gave the maximum tumour cure. Combination of any of the two treatments resulted in a better response than all the single treatments. The chemosensitizing effect of heat was more pronounced than its radiosensitizing effect. Combination of all the three modalities, cDDP (2.5 mg/kg) + RT + HT (43 degrees C, 30') resulted in 100 per cent CR, without any local recurrence. This treatment also resulted in a significant increase in 120 day tumour free survival compared to all single modality treatments and bimodality treatments, except cDDP + 43 degrees C, 30'. This study indicates a potential advantage of the trimodality approach over single and bimodality treatments in the local control of solid tumours.

Radioprotective effect of leaf extract of Indian medicinal plant Ocimum sanctum.

Water or aqueous ethanol extract of O. sanctum was given ip, either as a single dose or multiple doses, before a whole-body exposure to 11 Gy(LD100/30) of 60Co gamma radiation in albino mice. The water extract was more effective and less toxic than the aqueous ethanol extract. An optimum ip dose of 50 mg/kg (< 1/100 LD50) of the water extract, at 10 mg/kg/day for 5 consecutive days, gave the maximum survival. Increasing the dose per treatment or the number of treatments did not increase protection. Intraperitoneal administration gave the best protection (70% survival). Other routes (im, iv and po) were less effective and produced 37-47% survival. The optimum dose (ip) gave a dose modifying factor of 1.28. Since the extract may contain a number of chemical compounds, it is not possible to attribute the observed protection to any particular compound at present.

In vivo tumor inhibitory and radiosensitizing effects of an Indian medicinal plant, Plumbago rosea on experimental mouse tumors.

Tumor growth inhibitory and radiosensitizing effects of the alcoholic root extract of P. rosea was studied on experimental mouse tumors, S-180 solid tumor and Ehrlich ascites carcinoma in vivo. Intraperitoneal injection of 50 mg/kg of Plumbago extract (PE) for 10 days starting from 24 hr after intradermal inoculation of S-180 cells in BALB/c mice produced about 16% complete response (CR). The CR% increased with increase in drug dose, to 50% at 100 mg/kg for 10 days. As 100 mg/kg produced toxic side effects, lower doses were used with other treatment modalities, radiation (RT) and hyperthermia (HT). Treatment of 50 mm3 tumor with PE (75 mg/kg) for 10 days with local RT (10 Gy) and/or HT (43 degrees C, 30 min) subadditively increased the CR% and tumor free survival. The combination also significantly reduced the growth rates of uncured tumors. The PE significantly reduced the tumor glutathione content and this effect was markedly enhanced by the combination of the three modalities. PE alone was not very effective in preventing the growth of Ehrlich ascites carcinoma in Swiss mice, though it increased mean survival time and ILS% of the mice. But with radiation it produced a synergistic effect in increasing the tumor inhibition and 120 day animal survival from 10% to 50%. The results demonstrate that though PE may have only a weak antitumor effect, it may be a good candidate for use with radiation to enhance the tumor killing effect.

Modulation of glutathione depletion and lipid peroxidation by WR-77913 an 2-mercaptopropionylglycine in cyclophosphamide chemotherapy.

Cyclophosphamide induced a depletion in liver glutathione (GSH) and high rate of lipid peroxidation. GSH depletion was evident from 20 min onwards and the maximum depletion was observed at 3 hr post treatment. Lipid peroxidation was significant only after the maximum depletion of GSH. Pretreatment with either MPG or WR-77913 individually, or in combination could prevent the depletion of GSH and induction of lipid peroxidation after cyclophosphamide treatment.

Antitumor and radiosensitizing effects of Withania somnifera (Ashwagandha) on a transplantable mouse tumor, Sarcoma-180.

Antitumor and radiosensitizing effects of alcoholic root extract of W. somnifera and their modification by heat were studied in vivo on Sarcoma-180 grown on the dorsum of adult BALB/c mouse. Ashwagandha (AT) was injected (ip) at a dose of 500 mg/kg body wt for 10 consecutive days into mouse bearing tumor of 50 +/- 5 mm3, with or without a local treatment of 10 Gy gamma radiation (RT) or hyperthermia at 43 degrees C for 30 min (HT) or both on 5th day of AT. The response was assessed on the basis of tumor regression, growth delay, animal survival and changes in the tumor GSH content. Ashwagandha, RT and HT individually produced 18, 38 and 45% complete response (CR) respectively, but RT gave the best long term survival. Ashwagandha increased the effect of radiation on tumor regression as well as growth delay, but AT + HT gave a better tumor cure. However, both these combinations gave almost identical long term survival, which was not much higher than that produced by RT alone. The combination of Ashwagandha for 10 days with one local exposure to RT followed by HT significantly increased the tumor cure, growth delay of partially responding tumors and animal survival. This combination also significantly and synergistically depleted the tumor GSH level, with no recovery even at 3 hr after treatment. It is concluded that Ashwagandha, in addition to having a tumor inhibitory effect, also acts as a radiosensitizer and heat enhances these effects. The severe depletion in the tumor GSH content by the combination treatment must have enhanced the tumor response, as the inherent protection by the thiol will be highly reduced.