Heat-induced nuclear protein binding and its relation to thermal cytotoxicity

When nuclei were isolated from exponentially growing HeLa S3 cells immediately after a treatment with hyperthermia and/or procaine-HCl, an increase in nuclear protein binding was observed. The extent of this increase, however, did not correlate with cell survival under all conditions of the various treatments. For example, an increase up to 40 per cent in nuclear protein binding as a result of procaine treatment did not lead to a decrease of survival, while a 40 percent increase of nuclear protein binding as a result of hyperthermia corresponded with over 90 per cent cell killing. In addition the extent of heat-induced enhancement of nuclear protein content was approximately equal for thermotolerant and heated control cells, or for cells heated in the presence of procaine. The rate of decay in nuclear protein binding upon post-heat incubations at 37 degrees C of the cells, however, was enhanced in tolerant cells and retarded in cells heated in the presence of procaine as compared to heated control cells. These results show that, in spite of suggestions in other reports, neither the initial rate of enhanced protein binding nor the extent of the protein bound to the nucleus seems a reliable measure for heat toxicity. The capacity of the cell to reverse this heat-induced protein binding must be considered.     

Reduced DNA break formation and cytotoxicity of the topoisomerase II drug 4'-(9'-acridinylamino)methanesulfon-m-anisidide when combined with hyperthermia in human and rodent cell lines

The interaction between hyperthermia and the anticancer drug 4'-(9'-acridinylamino)methanesulfon-m-anisidide (mAMSA) was studied in the human HeLa S3 and the rodent Ehrlich ascites tumor cell line. For both cell lines it was found that hyperthermia preceding the drug treatment reduced the extent of mAMSA induced DNA breakage as well as mAMSA cytotoxicity. Formation and resealing of mAMSA induced DNA break formation was found to be related to cytotoxicity. Hyperthermic protection for the action of mAMSA was found not to be a result of changed permeability for the drug. The data also do not support the possibility that heat has caused inactivation of the putative target enzyme of mAMSA, topoisomerase II. It is suggested that the hyperthermic protection for the mAMSA drug action is due to a hyperthermic alteration of the chromatin organization, especially at topoisomerase II target sequences that are found to be enriched in the nuclear matrix (P.N. Cockerill and W.T. Garrard. Cell, 44: 273-282, 1986). We show here that heat has caused an alteration of protein binding to the nucleus that seems related to the hyperthermic inhibition of mAMSA induced DNA break induction. It is concluded that preheating cells before treatment with mAMSA should not be used, at least not in this sequence, in cancer therapy.     

Initial events in radiation-induced atheromatosis. III. Effect on lipase activity

Treatment of large blood vessels with ionizing irradiation leads to the development of atheromatosis when the serum lipid levels are sufficiently high. In order to answer the question whether a disturbed lipid clearance in the arterial wall plays a role in the accumulation of the lipids it was of interest to examine the effect of X-irradiation on the lipase activity. Triglycerol lipase was tested in a homogenate from rabbit carotid arteries, glycerol-tri-oelate was used as a substrate and the hydrolysis assayed at pH 6.4 at 37 degrees C. Under the conditions used, an hydrolytic activity could be measured of 7.5 nmoles . mg prot-1 . hr-1. The lipase activity was also tested in the carotid arteries obtained from rabbits which had been locally irradiated with 20 Gy of X-rays. No clear radiation effect on the lipase activity was found 4, 8, 20, 24 and 72 hours after irradiation. The accumulation of lipids is thought to be caused by a higher influx of lipids from the serum as a result of endothelial damage by X-irradiation rather than a defect in the clearance capacity of the arterial wall.     

Interaction of hyperthermia and radiation in tolerant and nontolerant HeLa S3 cells: role of DNA polymerase inactivation

The activities of DNA polymerase alpha and beta were measured in tolerant and nontolerant HeLa S3 suspension cells. The heat-inactivation of the enzymes and their recovery when cells were incubated at 37 degrees C after the heat challenge was compared to the synergistic action of heat and radiation and its disappearance at the level of cell survival. Thermotolerant cells were radiosensitized by heat similarly to nontolerant cells, but the sensitization decreased more rapidly in the tolerant cells when time at 37 degrees C was allowed between the two treatments. For polymerase activities the extent of inactivation, as well as the kinetics of recovery, were similar in tolerant and nontolerant cells. The results show that the activities of DNA polymerase alpha and beta do not always correlate with the extent of heat radiosensitization. It is concluded that heat inactivation of these enzymes may not be taken as a general cause for the synergistic effect of hyperthermia and radiation. As an alternative mechanism, changes in nuclear protein binding due to cellular heating are suggested, since these correlate well with effects observed for radiosensitization under different experimental conditions, including the use of thermotolerant cells.     

Isolation, anticholinesterase properties, and acute toxicity in mice of the four stereoisomers of the nerve agent soman

The four stereoisomers of the nerve agent pinacolyl methylphosphonofluoridate (soman), designated as C(+)P(+), C(+)P(-), C(-)P(+), and C(-)P(-), have different toxicologic properties due to stereospecific interactions in living organisms. We report the isolation of these stereoisomers with more than 99% optical purity. This result was realized by means of (i) complete optical resolution of pinacolyl alcohol, (ii) synthesis of C(+)- and C(-)-soman from the (+)- and (-)-enantiomers of the alcohol, (iii) optimalization of conditions for stereospecific inhibition of alpha-chymotrypsin with the P(-)-isomers of C(+)- and C(-)-soman, followed by isolation of the C(+)P(+)- and C(-)P(+)-isomers, (iv) isolation of the C(+)P(-)- and C(-)P(-)-isomers after incubation of C(+)- and C(-)-soman, respectively, in rabbit plasma, which hydrolyzes stereospecifically the P(+)-isomers. The bimolecular rate constants for inhibition of electric eel acetylcholinesterase (AChE) at pH 7.7, 25 degrees C, are at least 3.6 X 10(4) larger for the P(-)- than for the P(+)-isomers. The enzyme inhibited with C(+)P(-)-soman is much more effectively reactivated with the oximes HI-6, HGG-42, and obidoxime than AChE inhibited with C(-)P(-)-soman. The LD50 values (sc, mice) are in accordance with the P(-)/P(+) ratio of inhibition rates of AChE, i.e. 99, 38, greater than 5000, greater than 2000, 214, 133, and 156 micrograms/kg for C(+)P(-)-, C(-)P(-)-, C(+)P(+)-, C(-)P(+)-, C(+)-, C(-)-soman, and "soman", respectively. The relative LD50 values of the C(-)P(-)- and C(+)P(-)-isomers do not correspond with the small differences in their rates of inhibition of AChE, indicating that such small rate ratios may be overruled by other stereospecific effects, e.g., in vivo rates of detoxification.     

An adaptation of the Lashley cup for use in rat saliva collection

A miniaturized Lashley cup for collecting rat parotid and submandibular/sublingual saliva is described. The small dimensions of the cup enabled its proper positioning on the orifices of both salivary ducts. The method avoids surgical intervention, causes no tissue damage, allows simultaneous collection of both types of saliva and its appropriate for long-term studies of salivary composition and secretion in the rat.     

Gender and ethnic differences in urban and rural first-contact schizophrenia outpatients in Trinidad

This study investigated gender and ethnic differences in the rate of first contact outpatients with schizophrenia in the setting of a more-urban region (MUR) and a less-urban region (LUR) in Trinidad. In a prospective study, 134 first-contact patients with a diagnosis of schizophrenia were selected from two ecologically different regions. RESULTS: Of this population, 56.7% were of African origin and 32.1% were of Indian descent. Gender differences were significant, with males accounting for 66.4% (n=89) of patients with schizophrenia (chi2 = 14.45, d.f. = 1, p = 0.0001). Further analysis by age categories revealed a significant male predominance at ages 20-24 (p = 0.0001) and 25-29 (p = 0.002). Young African males (15-19 y, p = 0.049) predominated in MUR compared with LUR. The results showed a marked presence of Afro-Trinidadian males in both outpatient clinics (p < 0.05). We conclude that gender and ethnicity are important variables in the presentation of schizophrenia in Trinidad, whereas neither rural nor urban environments appeared to influence the expression of schizophrenia.