Radiomodulation by Hoechst 33258 against radiation- induced damage in murine splenocytes.

In this study modulatory effect of Hoechst 33258 on radiation induced membrane related signaling events which ultimately leads to apoptosis has been investigated. Splenocytes from swiss albino mice were irradiated in air at room temperature in a gamma chamber (240 TBq 60Co Model 4000 A) at the dose-rate of 0.052 Gys-1. Membrane lipid peroxidation, fluidity, specific activities of antioxidant enzymes, levels of nitric oxide, glutathione and apoptosis in presence and absence of different concentrations of Hoechst 33258 has been assayed. DNA binding activity of nuclear factor kappa B and activator protein–1 was also assayed by electrophoretic mobility shift assay. Modulatory effect of Hoechst 33258 was examined at 3 and 5 Gy using different concentrations (10, 20 and 30 µM). Hoechst 33258 was found to inhibit radiation induced peroxidative damage and fluidity and lowered the level of nitric oxide and apoptosis - as evident by DNA ladder assay and FACS, indicating free radicals scavenging potential. Dot plot diagramme clearly showed that 30 µM Hoechst 33258 caused 14% and 19% decrease in apoptotic cells at 3 Gy and 5 Gy of radiation respectively (compared to irradiated control group). Further DNA binding activity of nuclear factor kappa B and activator protein–1 was also inhibited but the antioxidant potential of the cells was enhanced. These findings support that Hoechst 33258 protects the cell from undergoing apoptosis. Hoechst 33258 may have interacted and has an ability to protect splenocytes against radiation induced apoptosis through modulation of membrane-related signaling events and antioxidant status.

Chemomodulatory potential of Glycine max against murine skin and cervical papillomagenesis.

In the present study, chemopreventive potential of Glycine max (G. Max) seeds was examined against DMBA-induced skin and MCA-induced cervical papillomagenesis in Swiss albino mice. Different doses (2.5, 5, and 7.5% w/w) of G. max were provided to animals in feed. Results exhibited a significant reduction in skin as well as cervical tumor incidence and tumor multiplicity (up to 75%) at all doses of test diet as compared to the control. Relatively, 7.5% test diet was most effective in protecting the animals against carcinogenesis. Further, detoxifying enzymes and antioxidative status was also evaluated in the liver of mice to understand the role of G. max in prevention of cancer. It was observed that the test diet containing G. max significantly elevated the specific activities of glutathione-S-transferase (GST), DT-diaphorase (DTD), superoxide dismutase (SOD), catalase (CAT), and glyoxalase I (Gly I). The test diet also elevated the content of reduced glutathione whereas it decreased the level of the peroxidative damage along with the specific activity of lactate dehydrogenase. It appeared that G. max seeds provided chemoprevention against skin and cervical papillomagenesis probably by modulating the detoxifying and antioxidative enzymes. It could be inferred that intake of G. max might help in reducing the risk of cancer.

Modulation of antioxidant potential in liver of mice by kernel oil of cashew nut (Anacardium occidentale) and its lack of tumour promoting ability in DMBA induced skin papillomagenesis.

Cashew nut shell oil has been reported to possess tumour promoting property. Therefore an attempt has been made to study the modulatory effect of cashew nut (Anlacardium occidentale) kernel oil on antioxidant potential in liver of Swiss albino mice and also to see whether it has tumour promoting ability like the shell oil. The animals were treated orally with two doses (50 and 100 microl/animal/day) of kernel oil of cashew nut for 10 days. The kernel oil was found to enhance the specific activities of SOD, catalase, GST, methylglyoxalase I and levels of GSH. These results suggested that cashew nut kernel oil had an ability to increase the antioxidant status of animals. The decreased level of lipid peroxidation supported this possibility. The tumour promoting property of the kernel oil was also examined and found that cashew nut kernel oil did not exhibit any solitary carcinogenic activity.

Cancer preventive potential of Momordica charantia L. against benzo(a)pyrene induced fore-stomach tumourigenesis in murine model system.

Bitter melon ( Momordica charantia Linnaeus) fruit extract was tested against 3,4 benzo(a)pyrene [B(a)P] induced forestomach papillomagenesis in Swiss albino mice. Extract of M. charantia in two concentrations, 2.5 and 5% of standard mice feed was used for the short-term and long-term studies. A significant decrease in tumour burden was observed in short and long-term treatment. Also, total tumour incidence reduced to 83.33% with 2.5% dose and 90.90% with 5% dose in short term treatment, while in long-term treatment tumor incidence decreased to 76.92% with 2.5% dose and 69.23% with 5% dose of M. charantia. The possible mechanism involved in the cancer chemoprevention has also been discussed.

Adaptive response and split-dose effect of radiation on the survival of mice.

Although the importance of radiation-induced adaptive response has been recognized in human health, risk assessment and clinical application, the phenomenon has not been understood well in terms of survival of animals. To examine this aspect Swiss albino mice were irradiated with different doses (2-10 Gy) at 0.015 Gy/s dose rate and observed on a regular basis for 30 days. Since almost 50% lethality was seen with 8 Gy, it was selected as the challenging dose for further studies. Irradiation of mice with conditioning doses (0.25 or 0.5 Gy) and subsequent exposure to 8 Gy caused significant increase in the survival of mice compared to irradiated control. The splitting of challenging dose did not influence the efficiency of conditioning doses (0.25 Gy and 0.5 Gy) to induce an adaptive response. However conditioning doses given in fractions (0.25 Gy + 0.25 Gy) or (0.5 Gy + 0.5 Gy) were able to modulate the response of challenging dose of 8 Gy. These results clearly showed the occurrence of adaptive response in terms of survival of animals. The conditioning dose given in small fractions seemed to be more effective. The findings have been discussed from a mechanistic point of view. The possible biological implications, potential medical benefits, uncertainties and controversies related to adaptive response have also been addressed

Post-irradiation free radical generation: evidence from the conversion of xanthine dehydrogenase into xanthine oxidase.

The xanthine oxidoreductase (XOR) system which consists of xanthine dehydrogenase (XDH) and xathine oxidase (XO), is one of the major sources of free radicals in biological systems. The XOR system is pre-dominantly present as XDH in normal tissues and converts into the free radical generating XO-form in the damaged tissue. Therefore, the XO-form of the XOR system is expected to be mainly found in radiolytically damaged tissues. In such an event, XO may catalyze the generation of free radicals and potentiate radiation effects in the post-irradiation period. Recent findings on the effect of ionizing radiation on the XOR system in the liver of mice, peroxidative damage and lactate dehydrogenase support this possibility. From these results it has been hypothesized that free radical generating systems could be activated in the radiolytically damaged cell and in turn contribute to the cause and complications of late effects and their persistence in post-irradiation period. This aspect may have great significance in the understanding of radiation-induced damages. It may also have serious implication in various fields like radiation therapy, health physics, carcinogenesis, space travelling radiation exposures and post nuclear accident care. Further, it is suggested that efforts need to be made to search more system(s) which could be activated particularly at lower doses of radiation to generate free radicals in the post-exposure period.

Radiation induced peroxidative damage: mechanism and significance.

An interest has been generated in free radicals after the discovery of superoxide dismutase. These free radicals cause a number of diseases and are involved in the detrimental effect of ionizing radiation. Efforts have been made to understand their role in damage and death of the cell using lipid peroxidation process. Lipid peroxidation is an important effect of radiation on membranes, which apart from DNA, are critical targets of radiation action. This paper addresses the basic mechanism of radiation induced lipid peroxidation. Various factors, which determine the mode and magnitude of lipid peroxidation, are also discussed. Lipid peroxidation is shown to have importance in understanding the modifications of radiation effects. Efforts are made to show similarities between radiolytic and non-radiolytic lipid peroxidation. Recent findings related to the close link between radiation-induced lipid peroxidation and apoptosis are likely to open new avenues for future research and to develop new approaches for radiomodification of biological effects.

Effects of vanadate, insulin and fenugreek (Trigonella foenum graecum) on creatine kinase levels in tissues of diabetic rat.

The in vivo effects of insulin, and other insulino mimetic agents like vanadate and fenugreek (T. foenum graecum) were followed on the changes in the activities of creatine kinase in heart, skeletal muscle and liver of experimental diabetic rats. As compared to control rats, creatine kinase activities were found to decrease significantly in the tissues during experimental diabetes. All the antidiabetic compounds used namely, insulin, vanadate and Fenugreek seed powder normalised the decreased activities to almost control values. The effects of insulin and vanadate were comparable in restoring normoglycemia and the creatine kinase activities.

Paradoxical influence of Ca(2+) on lipid peroxidation.

Lipid peroxidation and Ca2+ are suggested to be linked as a mediator of cell damage and death. Lipid peroxidation is a free radical phenomenon. It is highly destructive process and induces plethora of alterations in structure and function of cellular membranes which could lead to cell injury. Ca2+, a regulator of variety of physiological and biochemical functions, was shown to enhance as well as inhibit lipid peroxidation. The explanations provided for the protection as well as the potentiation of lipid peroxidation by Ca2+ were, to some extent, speculative in nature and some of the basic facts seem to be overlooked. Moreover, Ca2+ has a closed-shell electronic state and lacks electronic transitions and hence it may not participate directly in a free radical reaction to modulate lipid peroxidation process. Perhaps this could be the reason for conflicting reports on this aspect. In the present review, the interactions between Ca2+ and lipid peroxidation are critically examined.

Influence of Ca2+ on kinetics and thermodynamics of the NADPH-dependent microsomal lipid peroxidation.

The effect of Ca2+ on kinetics and thermodynamics of lipid peroxidation in microsomes prepared from liver of male Swiss albino mice (7-8 weeks old) was studied. Ca2+ was found to increase the Vmax in temperature dependent manner. Michaelis-Menten constant (Km) also increased with temperature. However, the linearity and extent of change in Km remained unaffected in presence of Ca2+, and was suggestive of non-competitive and mixed type of activation. The activation constant (Ka) obtained by the replotting of slopes of the Lineweaver-Burk plots against the reciprocal of Ca2+ concentration showed linear variation with temperature. The linear pattern of Arrhenius plots indicated non-involvement of parallel reactions of other intermediate species in the lipid peroxidation. Thermodynamic parameters delta H degree, delta S degree and delta G degree, associated with lipid peroxidation process were studied. The positive value of delta H degree is suggestive of the endothermic nature of the process. It appears that the NADPH induced lipid peroxidation is an entropy driven process.

Influence of Ca2+ on microsomal lipid peroxidation.

Lipid peroxidation in microsomes prepared from liver of mice was initiated by NADPH, ascorbic acid and ferrous ions. The presence of Ca2+ modulated the lipid peroxidation in all these three systems. The mode and magnitude depend on the system and concentration of cofactors used for initiation of lipid peroxidation. In ascorbate system, Ca2+ enhanced the lipid peroxidation up to 30 microM concentration of ascorbic acid and beyond 30 microM concentration it inhibited. Ca2+ increased NADPH-dependent lipid peroxidation at all concentrations. Depending on concentration of Fe2+, lipid peroxidation was either decreased or increased in presence of Ca2+. It suggested that the in vitro findings may be cautiously extrapolated to the animal systems. In absence of cofactors, Ca2+ enhanced lipid peroxidation. EGTA inhibited Ca2+-enhanced lipid peroxidation. However in presence of ionophore A23187, Ca2+ potentiated lipid peroxidation. Since Ca2+ has a closed-shell electronic state and lacks electronic transitions, it may not participate directly in lipid peroxidation process. The effect of Ca2+ on lipid peroxidation may be through some biochemical processes or its interactions with membranes leading to various changes in their characteristics.

Divalent cations and radiation induced lipid peroxidation.

Response of Co2+, Cu2+, Fe2+, Ni2+ and Mn2+ against radiation induced lipid peroxidation has been examined. Liposomes prepared from L-alpha-lecithin were irradiated with or without divalent cations, Co2+ and Cu2+ inhibited lipid peroxidation in concentration dependent manner. On the other hand Fe2+, Ni2+ and Mn2+ enhanced the peroxidation. The enhancement of peroxidation due to Ni2+ was biphasic in nature. Higher concentration of Mn2+ decreased the peroxidation. Except Cu2+ no other cation studied could change the non-linear pattern of radiation induced lipid peroxidation. The pro- or antioxidant effect of divalent cations may be due to their reactivity towards oxygen and oxygen free radicals, and at least in part to their hard/soft acid character.

Diosgenin--a growth stimulator of mammary gland of ovariectomized mouse.

Estrogenic action of diosgenin on the mammary epithelium of ovariectomized (OVX) mouse has been reported. Diosgenin when administered (sc) at the dose levels of 20 and 40 mg/kg body weight for a period of 15 days stimulated the growth of mammary epithelium. This was indicated by the increase in DNA content, increase in number of ducts and appearance of terminal endbuds. There was a significant increase in the mammary development scores in the presence of diosgenin. Concomitant treatment of estrogen and diosgenin showed augmentation of estrogenic effect of diosgenin especially at the higher dose level (40 mg/kg body wt). Diosgenin showed a lack of progesterogenic action as was apparent from the absence of alveolar development even in the presence of exogenous estrogen.

Non-linear pattern of radiation induced lipid peroxidation is not affected by vitamin E, Fe2+ ions and molecular oxygen.

Results of the present study on liposomes have clearly shown that non-linear pattern of radiation-induced lipid peroxidation was not changed even in the presence of vitamin E, Fe2+ ions or molecular oxygen. These results are important from biological point of view as lipid peroxidation is used as a measure of membrane damage.

Modification of radiation induced lipid peroxidation by calmodulin antagonists.

It has been shown that calmodulin antagonists provide radio-protection in euoxic and sensitization in hypoxic conditions. This differential protection in euoxic conditions might have arisen from the interaction of calmodulin antagonists with oxygen free radicals. This possibility has been tested in the present communication. Radiation induced lipid peroxidation process in liposomes has been used for this purpose. Liposomes prepared from L-alpha-lecithin were irradiated with or without calmodulin antagonists. Calmodulin antagonists inhibited lipid peroxidation significantly. The inhibition was found to increase with increase in concentration of the drugs. These observations suggest that calmodulin antagonists have a capacity to scavenge oxygen free radicals involved in initiation and/or propagation of lipid peroxidation process. This may be the reason for their differential radioprotection in euoxic conditions in biological systems.