A METHOD OF ASSAYING IN VIVO THE SURVIVAL OF ULTRA VIOLET-IRRADIATED TUMOUR CELLS

Abstract— The growth of ultraviolet-irradiated ascites tumour cells in adult animals which have received a total-body dose of x-radiation appears to be a suitable method to obtain data on the survival of the treated tumour cells, if the dose of total-body radiation and its time before tumour cell inoculation are chosen correctly.
In assessing tumour cell survival, the analysis of the time of appearance of the resulting solid tumours, using the linear relationship between the reciprocal of this time and the logarithm of the number of cells injected is preferred to probit or to Reed-Muench analysis of the number of tumour-takes. The 1/T method of analysis yields more precise estimates of the survival ratios and greater economy in the use of host animals without a disproportionate increase in the labour involved in obtaining the data.
An application of this method to the determination of survival-ratios of u.v.-irradiated Landschütz ascites tumour cells is described.     

Application of reversed-phase and ion-pair HPLC for investigation of nucleotide metabolism in erythrocytes,reticulocytes and tumour cells

Summary The determination of nucleotides, nucleosides, and nucleobases was carried out in cells of different metabolic complexity: in mature and immature red blood cells, in Ehrlich ascites tumour cells from different proliferation stages, and in other tumour cells. The maturation of reticulocytes to erythrocytes is accompanied by loss of organelles and energy-requiring processes as well as the switch from aerobic to anaerobic ATP production. The profile of purine nucleotides, nucleosides, bases, and pyridine dinucleotides, by reversed-phae HPLC, shows large concentration changes during the maturation of red blood cells. The concentrations of purine mono and triphosphates are two to four times greater in reticulocytes in comparison with erythrocytes; the difference in the concentrations of nucleosides and nucleobases between reticulocytes and erythrocytes is even greater. Application of ion-pair HPLC showed that the Ehrlich ascites cells loose major portions of purine mono-, diand triphosphates between the 7th and 11th day after inoculation. Fast growing solid sarcoma tumours of rats (MV 202 Ner) contain higher amounts of nucleotides than slowly growing tumours of identical cell type.     

Evaluation of the optimal LNCaP prostate tumour developmental stage to be assessed by 99mTc-HYNIC-βAla-Bombesin(7–14) in an experimental model

The lymph node carcinoma of the prostate (LNCaP) cell line is widely used in prostate cancer diagnostic studies. However, the optimal developmental stage during which to assess LNCaP prostate tumours by in vivo molecular imaging has not been established. This study aimed to assess LNCaP prostate tumours by scintigraphic imaging using technetium-99m (^99mTc)-2-hydrazinonicotinamide (HYNIC)-beta-alanine (βAla)-Bombesin_(7–14) at different stages of tumour development and to compare each developmental stage using histopathological analysis. ^99mTc-HYNIC-βAla-Bombesin_(7–14) (7.4 MBq) was injected into LNCaP prostate tumour-bearing mice after 15, 20 or 25 days of tumour inoculation. Scintigraphic images were obtained 1 and 4 h following radiopeptide injection and tumour tissues were obtained for histopathological analysis. Tumours were not observed by scintigraphic imaging after 15 days of inoculation, but after 20 and 25 days of inoculation, tumours were visualised, with a tumour-to-muscle ratio of 2.14 at 4 h after the radiopeptide injection into mice with 25-day tumours. Histopathological analysis showed that the amount of collagen fibres in the stroma was inversely proportional to the stage of tumour development, whereas the intensity of vascular proliferation was directly proportional to the tumour stage; this explains radiopeptide localisation in LNCaP cells and improved visualisation of the 25-day tumours. In conclusion, the results showed that 25-day tumours presented the most appropriate features for radiopeptide accumulation in LNCaP cells, suggesting that they are in the best stage of tumour development to be assessed by scintigraphic imaging using ^99mTc-HYNIC-βAla-Bombesin_(7–14).     

Effect of acute and fractionated irradiation on hippocampal neurogenesis

Ionizing radiation has become an inevitable health concern emanating from natural sources like space travel and from artificial sources like medical therapies. In general, exposure to ionizing radiation such as γ-rays is one of the methods currently used to stress specific model systems. In this study, we elucidated the long-term effect of acute and fractionated irradiation on DCX-positive cells in hippocampal neurogenesis. Groups of two-month-old C57BL/6 female mice were exposed to whole-body irradiation at acute dose (5 Gy) or fractional doses (1 Gy × 5 times and 0.5 Gy × 10 times). Six months after exposure to γ-irradiation, the hippocampus was analyzed. Doublecortin (DCX) immunohistochemistry was used to measure changes of neurogenesis in the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). The number of DCX-positive cells was significantly decreased in all acute and fractionally irradiation groups. The long-term changes in DCX-positive cells triggered by radiation exposure showed a very different pattern to the short-term changes which tended to return to the control level in previous studies. Furthermore, the number of DCX-positive cells was relatively lower in the acute irradiation group than the fractional irradiation groups (approximately 3.6-fold), suggesting the biological change on hippocampal neurogenesis was more susceptible to being damaged by acute than fractional irradiation. These results suggest that the exposure to γ-irradiation as a long-term effect can trigger biological responses resulting in the inhibition of hippocampal neurogenesis.     

THE LETHAL EFFECTS OF RADIATION ON SIX SPECIES OF PROTOZOANS

Abstract. The lethal response of six strains of protozoa to both ionizing radiation and u.v. light has been established. Values have been determined by observation of isolated animals and death was considered to be failure of irradiated individuals to produce unlimited progeny regardless of the actual time of death following irradiation. The strains were selected to give a large variety of size. Lethal doses of u.v. radiation have been determined in the same units used for ionizing radiation (the rad). The results reported here have been supplimented by values from the literature and plotted in the form of a graph of LD-50 value versus individual organism size.     

PHOTOCHEMICAL DEGRADATION OF KERATINS

Abstract— Twenty-nine samples of fur, hair. wool and feathers have been irradiated with a Sunlamp (Λ max. 310 nm) and with a 2 kW mercury vapour lamp emitting mixed U.V. and visible radiation resembling sunlight. Photodegradation is accompanied by progressive yellowing when irradiated with the Sunlamp. It is accompanied by bleaching followed by yellowing, or by bleaching throughout irradiation if the initial yellowness is high, when exposed to the 2 kW lamp. A group of nine fur and hair samples, representing four different orders of mammals, are the most sensitive to yellowing, followed by a group of ten wool and hair samples from the Artiodactyla and finally by the feather keratins which are less susceptible than the α-keratins. Amino acid analysis of the keratins before and after Sunlamp irradiation shows strong correlation of yellowing both with the initial concentration of tryptophan and with its destruction during irradiation. Similar correlations of yellowing with destruction of other amino acids are less significant. Preliminary washing with non-ionic detergent increases susceptibility to u.v. yellowing.     

Increased levels of 8-hydroxy-2'-deoxyguanosine in Drosophila larval DNA after irradiation with 364-nm laser light but not with X-rays

Exposure to UVA light causes damage to cellular components such as DNA and membrane lipids. We showed previously that UVA irradiation can induce mutations in Drosophila larvae and that the major lesions responsible for mutations were not thymidine dimers when wavelengths tested became longer. The use of a longer wavelength with UVA laser apparatus (364 nm) has made it possible to test the effects of this powerful light in biological organisms. In the present study, we irradiated third instar larvae of the urate-null Drosophila mutant strain y v ma-l, which is sensitive to oxidative stress, and compared the effects of 364 nm light irradiation with the effects of X-rays. To assay viability, some of the larvae were kept at 25 degrees C until they eclosed in order to obtain a measure of viability. The remaining larvae were used to measure the amount of 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative DNA damage. The amount of 8-OHdG increased and viability decreased in response to increased UV dose in both the y v ma-l and wild-type strains. With irradiation of 600 kJ m(-2), 8-OHdG/10(6)dG was 7.2 +/- 3.2 and 6.2 +/- 2.0 in y v ma-l and wild-type strains, respectively, whereas the respective levels were 2.2 +/- 0.6 and 2.3 +/- 0.8 without irradiation. Our results indicated that irradiation with a 364-nm laser light caused significant oxidative damage in Drosophila larval DNA; however, induction of the damage was not prohibited by urate. To the best of our knowledge, this is the first report of a study in whole animals that shows increased levels of 8-OHdG in response to 364-nm UVA. X-ray ionizing radiation is also thought to generate reactive oxygen species in irradiated cells. We found that the amount of 8-OHdG in DNA following X-ray radiation remained unchanged in both strains, though survival rates were affected. X-ray-generated oxidative damage in Drosophila cells was followed by cell death but not DNA base oxidation, and the damage was suppressed by urate. The overall results suggest significant differences in the major in vivo oxidative damage caused by 364-nm light and X-rays.     

ENHANCED GROWTH AND EXPERIMENTAL METASTASIS OF CHEMICALLY INDUCED TUMOR IN ULTRAVIOLET IRRADIATED SYNGENEIC MICE

Abstract— Recent studies have shown that ultraviolet (UV) irradiation induces a systemic effect which enhances subsequent tumor induction by benzo[a]pyrene in a manner which is dependent on the dose of benzo[a]pyrene. The present study was designed to test whether UV-B irradiation renders mice susceptible to subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene. The sources of UV-B irradiation were banks of 6 Westinghouse FS-40 sunlamps, situated 20 cm above the mouse cages. Female BALB/cAnNHsd received five 30-min dorsal UV-B radiation treatments per week for 12 weeks, resulting in a total dose of approx. 6.4 × 10⁵ J m^-2. Two to seven days after termination of UV treatments, syngeneic regressor tumor cells (BP2) induced by benzo[a]pyrene were injected subcutaneously or intravenously into irradiated mice and unirradiated controls. By 38 days post subcutaneous implantation, 24/30 and 3/30 BP2 implants were detectable in the irradiated and unirradiated mice, respectively. Ultraviolet irradiated mice were also unable to reject lung colonies resulting from intravenous administration of BP2 cells, although they were rejected by unirradiated mice. The mean number of lung colonies per mouse was 16- to 35-fold greater in UV irradiated mice than in unirradiated controls, at 14 to 17 days post injection. Thus, UV irradiation rendered mice, with no known exposure to benzo[a]pyrene, susceptible to a subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene.     

Study by electron paramagnetic resonance and spectrophotometry of absorption of the primary processes in photochemistry of frozen aqueous solutions at 77 degree K of aromatic amino acids and polypeptides

Abstract— Neutral, acidic or basic frozen aqueous solutions of aromatic amino acids undergo photoionisation under u.v. irradiation, at 77°K. In neutral or basic solutions, photo-ejected electrons are trapped in the solvent matrix and exhibit a characteristic absorption band in the visible region. In acidic solutions electrons are trapped by protons and ESR signal spectrum of hydrogen atoms may be observed. Hydrogen atoms are also produced in low yield in neutral or basic frozen aqueous solutions, u.v. irradiated at 77°K. In basic media the ESR spectrum of 0^- radical ions is observed. Kinetic studies as a function of light intensity show that photoionisation takes place after absorption of a second photon by the phosphorescent molecule in its lowest triplet state. Recombination of trapped electrons in neutral or basic solutions may be induced using secondary excitation with visible light. In all instances we could record the absorption spectrum of photolytic products of aromatic amino acids and polypeptides which are u.v. irradiated at 77°K.     

Antitumour effect of Diospyros cordifolia bark on Ehrlich ascites carcinoma-bearing Swiss albino mice

Diospyros cordifolia Roxb. (Ebenaceae), commonly known as Indian ebony, is used traditionally for several medicinal purposes. In this study, the methanol extract of D. cordifolia bark (MEDC) was evaluated for its antitumour effect against Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice. Twenty-four hours after intraperitoneal inoculation of tumour (EAC) cells in mice, MEDC was administered intraperitoneally at 25 and 50?mg kg?1 bodyweight for 9 consecutive days. On the 10th day, half of the mice were sacrificed to determine the tumour volume, viable and non-viable tumour cell counts, and rest were kept alive for the assessment of median survival time and increase in life span. Haematological profiles were also determined. MEDC exhibited a marked decrease in tumour growth parameters and increased the survival rate of EAC-bearing animals. MEDC normalised the haematological parameters as compared with the EAC control mice. Therefore, this study demonstrated that D. cordifolia bark possessed remarkable antitumour efficacy.     

DISTRIBUTIONS OF FREE RADICALS AMONG AMINO ACIDS FROM LYOPHILIZED ULTRAVIOLET-IRRADIATED PROTEINS

Abstract— The effects of u.v.-irradiation at 254 nm upon lyophilized ribonuclease, lysozyme, insulin, and chymotrypsinogen have been investigated by electron spin resonance (ESR). enzymatic assay, and labeling of free radical sites with tritiated hydrogen sulfide (HST). The ESR signal of the irradiated protein diminishes on exposure to HST, and tritium becomes covalently bound to carbon. The distribution of tritium among the amino acids of each protein. studied as an indicator of the carbon free radical distribution, differs markedly from those observed previously to result from exposure to gamma radiation, electrical discharge. or hydrogen atoms. However, the earlier observation that the tritium distribution is influenced by protein conformation holds true as well for u.v.-irradiation. Moreover, the distributions of tritium among the amino acids of u.v.-irradiated proteins indicate a broad scattering of free radicals. Tyrosine and phenylalanine, residues that absorb light energy in the region of the wavelength employed, are not particularly important as radical carriers. Thus, for ribonuclease, these residues incorporated 3.8 and 1.5 per cent of the total tritium, but they absorb 51 and 12 per cent of the light, respectively. These results, together with the observed low recoveries of methionine, an amino acid that does not absorb at 254 nm, add weight to the concept that a migration of energy ensues after the initial absorption of light energy and that photolytic damage may thus be due to destruction of amino acids other than those initially absorbing the u.v.-radiation.     

Ultraviolet action spectra in perspective: with special reference to mutation

Abstract— Problems of determining action spectra are considered as well as various types of action spectra for U.V. action upon cell activities. U.V. is an effective mutagenic agent producing point mutations and chromosomal changes. U.V. is readily absorbed by superficial layers of cells in tissues; therefore, special experimental procedures are necessary for induction of mutations in animals or plants. U.V. is, however, suitable for mutagenesis in microorganisms because their cells are small, permitting the radiation to reach the nuclei. Action spectrum studies reveal that u.v. mutagenesis results from absorption of the radiation by nucleic acid. The most prominent alteration in DNA following absorption of u.v. is dimerization of pyrimidines, chiefly thymine. Such a change not only retards DNA replication but results in errors (mutations). U.V. mutagenesis therefore depends upon the conditions before, during and after irradiation. Thus immediate post-treatment with visible and long u.v. light splits pyrimidine dimers, thereby reversing impending u.v. mutagenesis. For cells kept in the dark, conditions which prevent DNA replication by interfering with the metabolism of the cell provide time for dark repair of the DNA lesion and so for reversal of the impending mutation.     

CHANGES IN U.V. SURVIVAL CURVES OF ESCHERICHIA COLl B/r CONCOMITANT WITH CHANGES IN GROWTH CONDITIONS

Abstract— For E. coli B/r u.v.-irradiated while in logarithmic growth, the nature of the dose-response curve was strongly dependent on both pre- and post-irradiation conditions of growth. Survival curves for cells grown in nutrient medium, or minimal medium with glucose, and plated immediately after irradiation, demonstrated an initial insensitive or 'shoulder' region provided the plating medium was such that no derepression was required of operons controlling inducible enzyme systems. If, however, such derepression was called for, survival curves were of exponential form. Delay in plating resulted in the return of the survival curve to the shouldered form even when 'shift-down' media were used.
Of those cells grown before u.v.-irradiation in minimal media and plated thereafter with the same sugar as carbon source, only those grown with glucose (or lactose) demonstrated the shouldered survival curve.     

Pork fat peroxidation by gamma-irradiation

In this paper, pork fat peroxidation by γ-irradiation and the possible effects of oxygen, UV-irradiation and storage after the γ-irradiation have been investigated. It has been found that the level of peroxides in irradiated pork increases linearly with the increasing absorbed dose. The chemical yield of peroxides formed in the irradiated fat is about 4.2 and independent on the sample temperature or absorbed dose rate, but dependent on storage time of sample before γ-irradiation. The irradiated pork exhibits some unusual features as following: 1) the peroxide content in irradiated pork is higher than that in unirradiated one; 2) the peroxide content in irradiated pork increases gradually on storage and is essentially constant in unirradiated one, which is very useful for the detection of irradiated pork; 3) the further peroxidation in irradiated pork is much more susceptible to UV radiation than that in unirradiated pork.     

Starch and amylose degradation by 60Co γ-irradiation

The effects of γ-irradiation (0.02–4.0 Mrad absorbed dose and 1.2 Mrad/hr dose rate) on depolymerization and selected physical properties of corn starch and amylose (both dry solid and solution forms) were investigated under conditions used to make graft polymers. Radiation introduces an alkali-sensitive structure, most likely β-alkoxycarbonyl, having a G value of 2.8. A dimethyl sulfoxide (DMSO)-acetic acid-water solvent for intrinsic viscosity was developed that degraded irradiated amylose less than did aqueous 90% DMSO. The G (scission) value of 1.3 for solid amylose irradiated at 0°C under nitrogen is lower than most literature values for either amylose or other polysaccharides. The protection of amylose against irradiation degradation in water by additions of DMSO is noteworthy. The G (scission) for amylose irradiated in 99.8% DMSO is 2.3; whereas, in water it is 30.     

A Highly Luminescent Tetrahydrocurcumin IrIII Complex with Remarkable Photoactivated Anticancer Activity

Curcumin has chemopreventative properties against a variety of tumours, but has poor bioavailability. Here, two new bis-cyclometallated iridium(III) complexes have been prepared, featuring the natural product curcumin (CUR) or its reduced form, tetrahydrocurcumin (THC), as bidentate, anionic O O-binding ligands. The iridium THC complex is highly luminescent in deoxygenated solution and efficiently generates singlet oxygen under aerated conditions, whereas in the CUR analogue, other non-radiative decay pathways are competitive. The complexes are rapidly taken up by a variety of human tumour cell lines from solutions of micromolar concentration. They show negligible cytotoxicity in the absence of irradiation. When briefly irradiated with visible light, Ir-THC becomes highly phototoxic, inducing rapid apoptosis within 2 h. The results show the high potential of such complexes as sensitizers in photodynamic therapy (PDT).     

GROWTH DELAY STUDIES OF THE RESPONSE OF V-79 MULTICELL SPHEROIDS EXPOSED TO DHE and RED LIGHT

Abstract Multicell tumour spheroids (MTS) of V-79 Chinese hamster cells have been used to study the role of a number of treatment and microenvironmental parameters in the modification of tumour response to Photodynamic Therapy (PDT) using visible light in combination with the photosensitizing compound dihematoporphyrin ether (DHE). The kinetics of DHE uptake into MTS, determined by fluorimetry of extracted porphyrins, indicate that after extended incubation (i.e. 24 h) the mean cellular DHE content in larger (˜300 μ.m and 400 u.m) MTS is significantly less than that for smaller (˜200 μm) MTS, consistent with a hypothesis that DHE uptake into the internal regions of spheroids is diffusion-limited. The response of spheroids to PDT, as assessed by the endpoint of growth delay, indicates that the kinetics of spheroid volume alteration and cell loss, as well as the potential for regfrrwth, are markedly dependent on both the drug and light exposure levels used. The oxygen dependence of this response has been investigated after light irradiation of spheroid cultures equilibrated with either 21% O₂ (i.e. air) or 0% 0₂ (i.e. N₂). While treatment in air results in significant growth delay, the growth kinetics of DHE-treated spheroids irradiated under N₂ were essentially unchanged from those of untreated spheroids. These observations clearly demonstrate an important role for oxygen, at the time of irradiation, in determining the response of spheroids to PDT.     

Antitumour activity of Terminalia arjuna leaf against Ehrlich ascites carcinoma in mice

Terminalia arjuna Roxb. (Combretaceae), commonly known as 'Arjuna', is a large tree occurring throughout the Indian peninsula. This study was undertaken to evaluate the methanol extract of T. arjuna leaf (META) for antitumour activity against Ehrlich ascites carcinoma (EAC) in Swiss albino mice. Twenty-four hours after intraperitonial inoculation of tumour (EAC) cells in mice, META was administered at 100 and 200?mg?kg(-1) body weights for 9 consecutive days. On day 10, half of the mice were sacrificed and the rest kept alive for an assessment of the increase in life span. The antitumour effect of META was assessed by evaluating tumour volume, tumour weight, viable and non-viable tumour cell counts, median survival time and increase in life span of EAC-bearing hosts. Haematological profiles were estimated. META showed a significant (p<0.001) decrease in tumour volume, tumour weight and viable cell count, and also increased the life span of EAC-bearing mice. Haematological profiles were significantly (p<0.001) restored to normal levels in META-treated mice compared to the EAC control. Therefore, from this study, it can be concluded that T. arjuna leaf exhibited remarkable antitumour activity against EAC in Swiss mice.     

PHOTOLYSIS AND RADIOLYSIS OF NITROMETHANE AND NITROETHANE

Abstract— Prolonged photolysis of nitromethane and nitroethane in liquid and vapour phases, with simultaneous photolysis of the nitrogen dioxide formed, was studied in the absence and presence of cupric oxide. A high-pressure quartz immersion lamp providing the full range of visible and u.v. spectrum, with a high output of radiation at 366 and 313 mμ, was employed as source of light. The products of photolysis were detected by microanalytical methods, infrared-absorption spectrum analysis and gas chromatography. Photolysis of liquid nitro-alkanes resulted in formation of many compounds; saturated hydrocarbons, and in the case of nitroethane, ethylene, were submitted to detailed examination. Photolysis in the vapour phase was carried out in a specially designed glass apparatus. The main products were nitrogen dioxide, aldehydes and polyaldehydes; hydrocarbons were not estimated.
Liquid nitromethane and nitroethane were also irradiated with γ-rays from a ⁶⁰Co source. The radiolysis products were similiar to those obtained on photolysis. The mechanism of photolytic decomposition of nitroalkanes is proposed and discussed.     

Mutagenic effects of near ultraviolet and visible radiant energy on continuous cultures of escherichia coli

Abstract— –The induction of mutation to phage T5 resistance by near ultraviolet (u.v.) and visible light was studied in chemostat cultures of Escherichia coli strains B/r and B/r/1, trp. The visible light mutation rate to phage T5 resistance was independent of growth rate over the range studied. This result is consistent with a photochemical mechanism of mutagenesis. Changeovers, in which a faster growing subpopulation takes over the culture, usually causing the mutant frequency to decline sharply, occur more frequently in chemostat cultures irradiated with visible light than in cultures treated with far u.v. or caffeine. A preliminary action spectrum was obtained with aerated chemostats that revealed effective wavelengths to be between 330 nm and 500 nm. Wavelengths longer than 500 nm were not effective. Wavelengths longer than 340 nm were not mutagenic in anaerobic chemostats. This oxygen requirement for mutagenesis between 340 nm and 500 nm is consistent with a photodynamic mechanism of action. In aerated cultures, wavelengths between 400 nm and 500 nm were as effective as wavelengths between 330 nm and 400 nm. A number of naturally occurring compounds, including riboflavin and vitamin K, are consistent with the data as candidates for the chromophore responsible for near u.v. and visible light mutagenesis.