Endothelial alkaline phosphatase activity loss as an early stage in the development of radiation-induced heart disease in rats

Alkaline phosphatase activity of capillary endothelial cells in the heart of Wistar and Sprague-Dawley rats was studied sequentially after single doses of 10, 15, 20, or 25 Gy. Following irradiation capillary density and alkaline phosphatase activity were focally lost before myocardial degeneration or clinical symptoms of heart disease developed. Recovery from both changes took place after doses of 10 or 15 Gy. The decrease in capillary density and enzyme activity showed the same strain difference in latency times and in the extent of the lesions as previously described for pathological and clinical signs of heart disease.     

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation.     

Influence of protein deficiency on the inhibition of hepatic microsomal detoxication by methyl mercury in two rat strains

N- and C-oxygenation of N,N-dimethylaniline was studied in liver microsomes from 2 rat strains (Wistar, Strain R and Sprague-Dawley) subjected to protein deprivation and methyl mercury pretreatment, separately and in combination. A striking interstrain difference was observed. Strain R microsomes from 2 rat strains (Wistar, Strain R and Sprague-Dawley) subjected to tion, but little effect after methyl mercury pretreatment. With Sprague-Dawley microsomes, C-oxygenation was slightly reduced after both treatments. N-oxygenation was little affected in either strain. Methyl mercury treatment of protein deprived rats strongly inhibited C-oxygenation in microsomes from both strains, with N-oxygenation being unaffected in strain R microsomes whereas markedly reduced in microsomes from Sprague-Dawley.     

Study on the optimum number of trials and intensity of unconditioned stimulants and strain difference in the two-way shuttle-box avoidance test using rats

In order to determine the optimun conditions suitable for a number of trials and the intensity of unconditioned stimulant (US) in the two-way shuttle-box avoidance test in Sprague-Dawley strain rats, which are used most frequently in reproduction studies, conditioned avoidance response was observed under various conditions for 30 and 60 trials and the low and high US levels. Investigation was also conducted in Wistar rats under a high US level with 30 and 60 trials. Latency time of the escape response in Sprague-Dawley rats was shortened with increasing trials. Body weight gains of both strains of rats in the high US level with the 60-trial group decreased during the observation period. These findings suggest that the high US level with the 60-trial group is not suitable for the two-way shuttle-box avoidance test. The rate and latency time of the avoidance response were lower in Wistar rats than in Sprague-Dawley rats, although those of the escape response were higher. Significant changes in the following were observed, mainly from first to third sessions: the avoidance rate of all groups in strains of rats, escape rate of 60-trial group in Sprague-Dawley rats, avoidance and escape latency time of the 60-trial groups in both strains of rats and escape latency time of the 30-trial group in Sprague-Dawley strain rats.     

Radiobiology and stathmokinetics of intestinal crypts associated with patches of Peyer

The stathmokinetics and radiobiology of intestinal crypts directly adjoining the lymphoid patches of Peyer, have been compared with those of non-patch-associated crypts. Patch crypts contain an additional one to two rings of cells, the Mitotic Index for the whole crypt is higher than in non-patch crypts, and the apparent cell cycle time is insignificantly lower. Using single and split doses of gamma-rays, dose-survival curves were obtained for whole intestinal crypts, from which single-cell survival curves were derived for the clonogenic cells of the crypt. For a single-hit, multitarget, model, the extrapolation numbers of the cell survival curves for patch and non-patch crypts were the same (approximately 35) but the final D0 for cells of the patch crypts was significantly higher (2.1 versus 1.7 Gy). A linear-quadratic fit gave a similar ratio of alpha/beta (approximately 10) for the two curves. For a given level of crypt depletion, the number of clonogenic cells per crypt derived by the use of equal split doses of radiation, was the same for patch and non-patch crypts. This number is a function of the dose regime employed: the higher the level of crypt depletion, the higher the derived number of cells (range 10 to 45, for non-patch crypts).     

Repopulation kinetics of rat rhabdomyosarcoma tumors following single and fractionated doses of low-LET and high-LET radiation

Measurements were made of clonogenic cell survival in rat rhabdomyosarcoma tumors as a function of time following in situ irradiation with single or fractionated doses of 225-kVp X rays or with 557-MeV/u neon ions in the distal position of a 4-cm extended-peak ionization region. Single doses of 20 Gy of X rays or 7 Gy of peak neon ions reduced the initial surviving fraction to approximately 0.025 for each modality. Daily fractionated doses (four fractions in 3 days) of either peak neon ions (1.75 Gy per fraction) or X rays (6 Gy per fraction) achieved a cell survival of approximately 0.02-0.03 after the fourth dose of radiation. In the single-dose experiments, significant 5- and 10-fold decreases in the fraction of clonogenic cells were observed between the third and fourth days after irradiation with peak neon ions and X rays, respectively. After the sixth day postirradiation, the residual clonogenic cells exhibited a rapid burst of proliferation leading to doubling times for the surviving cell fractions of approximately 1.5 days. Radiation-induced growth delay was consistent with the cellular repopulation dynamics. In the fractionated-dose experiments with both radiation modalities, a large delayed decrease in cell survival was observed at 1-3 days after completion of the fractionated-dose schedule. Cellular repopulation was consistent with postirradiation tumor volume regression and regrowth for both radiation modalities. The extent of decrease in survival following the four-fraction radiation schedule was approximately two times greater in X-irradiated than in neon-ion-irradiated tumors that produced the same survival level immediately after the fourth dose. Mechanisms underlying the marked reduction in cell survival 3-4 days postirradiation are discussed, including the possible role of a toxic host cell response against the irradiated tumor cells.     

Morphological and functional changes in the rat heart after X irradiation: strain differences

The hearts of mature male rats of the Wistar and Sprague-Dawley strains were locally irradiated with single doses of 17.5 and 20.0 Gy of X rays, respectively. These two dose levels had previously been shown to result in a comparable latent period between irradiation and the death of rats of these two strains from cardiac failure. Morphological changes in the myocardium and modifications in cardiac function were assessed in the animals at 28, 70, and 100 days after irradiation. The first radiation-induced change which was observed in the myocardium was a rapid decline in capillary density and a loss of alkaline phosphatase activity by the capillary endothelial cells. The capillary density was reduced to approximately 50% of that of unirradiated control values at 28 days and to approximately 40% of the control values between 70 and 100 days after irradiation. The loss of enzyme activity was also detected at 28 days. Examination of histological sections showed an increase by 70 days in the areas with negative enzyme activity up to approximately 70% of the myocardium. The reduction in capillary density and the loss of enzyme activity occurred before any marked pathological changes were seen in the myocardium. The pathological lesions seen in the myocardium at 100 days after irradiation were qualitatively and quantitatively the same in the two strains of rat. Measurements of cardiac output in Sprague-Dawley rats showed a gradual decline in output after irradiation; however, measurements in Wistar rats showed a progressive increase in cardiac output over the same period of time. It was shown by rubidium extraction that there was an increase in the percentage of the total cardiac output distributed to the ventricular muscle of Sprague-Dawley rats, while similar measurements in Wistar rats showed no significant change. In spite of the marked strain differences observed in cardiac output and rubidium extraction, blood perfusion per gram of ventricular muscle was apparently not modified in both strains of rat after irradiation. These findings indicated that the correlation between morphological effects after irradiation and the functional expression of damage is highly complex.     

Ionizing Irradiation Not Only Inactivates Clonogenic Potential in Primary Normal Human Diploid Lens Epithelial Cells but Also Stimulates Cell Proliferation in a Subset of This Population

Over the past century, ionizing radiation has been known to induce cataracts in the crystalline lens of the eye, but its mechanistic underpinnings remain incompletely understood. This study is the first to report the clonogenic survival of irradiated primary normal human lens epithelial cells and stimulation of its proliferation. Here we used two primary normal human cell strains: HLEC1 lens epithelial cells and WI-38 lung fibroblasts. Both strains were diploid, and a replicative lifespan was shorter in HLEC1 cells. The colony formation assay demonstrated that the clonogenic survival of both strains decreases similarly with increasing doses of X-rays. A difference in the survival between two strains was actually insignificant, although HLEC1 cells had the lower plating efficiency. This indicates that the same dose inactivates the same fraction of clonogenic cells in both strains. Intriguingly, irradiation enlarged the size of clonogenic colonies arising from HLEC1 cells in marked contrast to those from WI-38 cells. Such enhanced proliferation of clonogenic HLEC1 cells was significant at ≥2 Gy, and manifested as increments of ≤2.6 population doublings besides sham-irradiated controls. These results suggest that irradiation of HLEC1 cells not only inactivates clonogenic potential but also stimulates proliferation of surviving uniactivated clonogenic cells. Given that the lens is a closed system, the stimulated proliferation of lens epithelial cells may not be a homeostatic mechanism to compensate for their cell loss, but rather should be regarded as abnormal. This is because these findings are consistent with the early in vivo evidence documenting that irradiation induces excessive proliferation of rabbit lens epithelial cells and that suppression of lens epithelial cell divisions inhibits radiation cataractogenesis in frogs and rats. Thus, our in vitro model will be useful to evaluate the excessive proliferation of primary normal human lens epithelial cells that may underlie radiation cataractogenesis, warranting further investigations.     

Effects of paradoxical sleep deprivation on genital reflexes in five rat strains

This study examined the effects of cocaine on genital reflexes in paradoxical sleep-deprived (PSD) male rats of five strains since it has been demonstrated that this drug enhances genital reflexes in Wistar PSD rats. At the end of a 4-day period of PSD or at the equivalent time-point to control animals, cocaine or saline was acutely administered to the animals and penile erection (PE) and ejaculation (EJ) were quantified. Results indicated that PSD induced genital reflexes in all strains, and cocaine potentiated these behaviors in Wistar and Long-Evans rats. Wistar PSD rats injected with cocaine performed significantly more PE than all the other PSD + cocaine strains. The number of Wistar and Long-Evans PSD + cocaine ejaculating was significantly higher than the respective PSD + saline and control, whereas a tendency of increase was seen in relation to other groups. Wistar PSD + cocaine rats showed the highest EJ frequency compared to F344, Sprague-Dawley and Wistar-Kyoto strains, and the Long-Evans displayed more EJ than Sprague-Dawley and Wistar-Kyoto. Analysis of testosterone concentrations revealed that after sleep deprivation, Wistar, Long-Evans, and F344 rats showed significantly lower testosterone concentrations than control rats. In F344, Sprague-Dawley and Wistar-Kyoto controls rats, testosterone was significantly lower than in the control Wistar and Long-Evans. Progesterone concentrations were significantly higher in Wistar and Long-Evans PSD rats than in respective control groups. In the other strains, this hormone was significantly lower compared to the Wistar and Long-Evans PSD. This study demonstrates that genital reflexes are differently influenced by PSD associated to cocaine in five rat strains.     

Medium from irradiated cells induces dose-dependent mitochondrial changes and BCL2 responses in unirradiated human keratinocytes

Exposure of unirradiated human keratinocytes to irradiated cell conditioned medium (ICCM) is known to cause a cascade of events that leads to reproductive death and apoptosis. This study investigates the effect of ICCM on clonogenic survival, mitochondrial mass and BCL2 expression in unirradiated keratinocytes. Exposure to 5 mGy, 0.5 Gy and 5 Gy ICCM resulted in a significant decrease in clonogenic survival. Human keratinocytes incubated with ICCM containing an antioxidant, N-acetylcysteine, showed no significant decrease in clonogenic survival. HPV-G cells incubated with ICCM containing a caspase 9 inhibitor showed no significant decrease in clonogenic survival when the ICCM dose was < or =0.5 Gy. A significant increase in mitochondrial mass per cell was observed after exposure to 5 mGy and 0.5 Gy ICCM. A change in the distribution of the mitochondria from a diffuse cytoplasmic distribution to a more densely concentrated perinuclear distribution was also observed at these doses. No significant increase in mitochondrial mass or change in distribution of the mitochondria was found for 5 Gy ICCM. Low BCL2 expression was observed in HPV-G cells exposed to 5 mGy or 0.5 Gy ICCM, whereas a large significant increase in BCL2 expression was observed in cells exposed to 5 Gy ICCM. This study has shown that low-dose irradiation can cause cells to produce medium-borne signals that can cause mitochondrial changes and the induction of BCL2 expression in unirradiated HPV-G cells. The dose dependence of the mitochondrial changes and BCL2 expression suggests that the mechanisms may be aimed at control of response to radiation at the population level through signaling pathways.     

Effects of single and split doses of cobalt-60 gamma rays and 14 MeV neutrons on mouse stem cell spermatogonia

The long-term effects of ionizing radiation on male gonads may be the result of damage to spermatogonial stem cells. Doses of 10 cGy to 15 Gy (60)Co gamma rays or 10 cGy to 7 Gy 14 MeV neutrons were given to NMRI mice as single or split doses separated by a 24-h interval. The ratios of haploid spermatids/2c cells and the coefficients of variation of DNA histogram peaks as measures of both the cytocidal and the clastogenic actions of radiation were analyzed by DNA flow cytometry after DAPI staining. The coefficient of variation is not only a statistical examination of the data but is also used here as a measure of residual damage to DNA (i.e. a biological dosimeter). Testicular histology was examined in parallel. At 70 days after irradiation, the relative biological effectiveness for neutrons at 50% survival of spermatogonial stem cells was 3.6 for single doses and 2.8 for split doses. The average coefficient of variation of unirradiated controls of elongated spermatids was doubled when stem cells were irradiated with single doses of approximately 14 Gy (60)Co gamma rays or 3 Gy neutrons and observed 70 days later. Split doses of (60)Co gamma rays were more effective than single doses, doubling DNA dispersion at 7 Gy. No fractionation effect was found with neutrons with coefficients of variation.     

Enhanced recovery from ionizing radiation damage in a lepidopteran insect cell line

TN-368 lepidopteran insect cells display a pronounced resistance to the lethal effects of ionizing radiation and exhibit superior DNA repair capabilities. When a TN-368 cell population entering stationary growth phase is irradiated with 137Cs gamma rays and then incubated for several hours before cell dilution and plating for colony formation, the surviving fraction is increased several-fold over cells diluted and plated immediately after irradiation. Similarly, the survival of cells plated immediately following the second of two equivalent doses separated by several hours is greater than the survival of cells plated immediately following a single dose equal to the sum of the split doses. Both processes exhibit similar biphasic repair kinetics and reach maximal levels by 6 h. The phenomena appear initially to be analogous to confluent-holding and split-dose recovery as described for mammalian cells. However, the survival levels obtained for doses of 61-306 Gy after allowing for these recovery processes to occur are quite high and greatly exceed survival levels for all but relatively low doses less than 50 Gy. For example, while the survival of cells irradiated with 150 Gy is near 0.15, the survival of cells receiving 306 Gy in two equivalent split doses is approximately 0.77. Even if damage induced by the first of the split doses was completely repaired, it might be expected that the survival would be near the level of the second dose alone, or near 0.15. Instead the survival is approximately five times greater, suggesting that the first split dose stimulated a repair system not present in unirradiated cells. The situation for confluent-holding recovery is similar to that for split-dose recovery.     

Delayed reproductive death and ROS levels in the progeny of irradiated melanoma cells

The cell death and survival of proliferating (clonogenic) cells were investigated in two human melanoma cell lines to assess the optimal conditions for preparation of apoptotic bodies from melanoma cells. After 50 J/m2 UVB+UVC the maximal levels of apoptotic cells assayed by Trypan blue staining, nucleosomal DNA fragmentation, MTT, and TUNEL tests were observed within 2-3 d of radiation. In 100 Gy gamma-irradiated cultures these apoptosis indicators were delayed for up to 3 weeks. In addition, clonogenic cells were observed only in exponentially growing cultures irradiated with UV at high cell density but not in gamma-irradiated cultures. The response of melanoma cultures after high UV radiation doses contrasted to the response in lethally gamma-irradiated cultures. UV-irradiated melanoma cultures were recovered within two weeks. Most of the clonogenic cells in the recovered colonies contained micronuclei. ROS levels determined by DCF fluorescence and a modified MTT test were also normalized obviously due to the extensive antioxidant defense system of melanoma cells. UV radiation of tumor cells might be the preferential method for preparation of apoptotic bodies. The presence of clonogenic cells in the suspension of apoptotic bodies from melanoma cells used for pulsing of dendritic cells with tumor antigens might compromise this protocol for preparation of cell vaccines.     

The clonogen content of murine intestinal crypts: dependence on radiation dose used in its determination.

The number of colony-forming (clonogenic) cells in each of the crypts in mouse small intestine was deduced using a two-dose irradiation technique. The number was 7.5 +/- 0.8 cells using two equal doses each less than 9 Gy and 38 +/- 7 cells using 9 Gy or more per dose. The significant dose dependence could not be accounted for by considerations of intra- or intercrypt variability, or by the factor introduced to correct the sampling frequency for the influence of crypt size. The results suggest that more colony-forming cells may be recruited when the injury is more severe.     

Strain differences in the radiosensitivity of mouse spermatogonia

The radiosensitivity of spermatogonia was found to be greater by up to a factor of 2 in C3H mice than in B6D2F1 mice, whether assessed for the highly sensitive spermatogonia (types A2 to In) or the much more resistant clonogenic spermatogonia which repopulate tubules. The latter were similarly resistant in the B6D2F1 hybrid and in the DBA2 parent, but were much more sensitive in the C57BL parent strain. A difference in sensitivity by up to a factor of 2 results in a variation by a factor of 10 or more in the level of survival of clonogenic cells after high doses. This variation is also observed when comparing data in the literature from different authors using various strains of mice. Using the radiosensitizer misonidazole, it was shown that hypoxia did not play a major role in the lesser sensitivity demonstrated in B6D2F1 mice. The variation in sensitivity is similar to the range reported in the literature for reciprocal translocations.     

Comparison of intestine and bone marrow radiosensitivity of the BALB/c and the C57BL/6 mouse strains and their B6CF1 offspring

The radiosensitivity as measured by LD50/6 or LD50/30 of the F1 hybrid B6CF1 (C57BL/6 X BALB/c) is similar to that of C57BL/6 mice but markedly different from BALB/c. The LD50/6 for BALB/c mice was about 8.8 Gy compared to 16.4 Gy for the B6CF1. The difference in LD50/6 between the parent strains or between BALB/c and the F1 hybrid could not be explained by any differences in crypt cell number, cell cycle time, or transit time. Likewise, the observed differences in the LD50/6 do not appear to result from marked differences in the radiosensitivity of marrow stem cells (CFU-S) since the D0's for the three genotypes of mice were similar. Also, there were no apparent differences in the red blood cell contents of several enzymes associated with antioxidant defenses. The microcolony assay was used to determine the D0 for the crypt clonogenic cells and the D0 values for 60Co gamma rays were about 0.8 Gy for BALB/c mice and 1.4 Gy for B6CF1 mice. However, the D0 values for JANUS fission neutrons were similar; 0.6 Gy for the BALB/c mice and 0.5 for the B6CF1 mice. A comparison of clonogenic cell kinetics, using prolonged colcemid block to distinguish between slowly and rapidly cycling cells suggest that, normally, the stem cells are slowly cycling in both the BALB/c and the B6CF1 hybrid. However, the stem cells of the B6CF1 appear to go into rapid cell cycle more rapidly than those of the BALB/c following irradiation or prolonged colcemid treatment. The more rapid recovery in intestinal epihelial cell production in the B6CF1 hybrid after irradiation may provide an increased mucosal barrier and may, in part, explain the difference in the response to radiation compared to that in the BALB/c.     

Lack of effect of melatonin on sexual maturation in female rats

Daily subcutaneous injections of 100 micrograms melatonin given to prepubertal female rats housed in 14L:10D or 12L:12D failed to delay puberty as evidenced by the age at which vaginal opening occurred; neither the Sprague-Dawley nor the Wistar strain rats were responsive to melatonin treatment. Reproductive organ weights (ovaries and uteri) at vaginal opening were unaffected by such treatment. Administration of melatonin through the drinking water in doses of 100, 500 or 1000 micrograms/day did not alter the timing of puberty or the reproductive organ weights in rats of the Sprague-Dawley or Long-Evans strains (housed in 12L:12D). Our experimental methods are identical to a previous report and we have no explanation for our failure to reproduce the earlier results.     

Effect of radiation on fecundity and fertility of codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) from South Africa

Codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) is the key pest of pome fruit in South Africa, and it’s control in apple and pear orchards relies on the application of insecticides and pheromone‐mediated mating disruption. Development of resistance to insecticides and placement of restrictions on the use of certain insecticides has made control of codling moth in South Africa increasingly problematic. The use of the sterile insect technique (SIT) as a control tactic for codling moth is under investigation as a potential addition to the current control strategy. We investigated the radiosensitivity of a laboratory strain of codling moth that was established from moths collected from commercial and organic orchards in the Western Cape, South Africa. Fecundity and fertility of this strain following radiation were consistent with values for the codling moth strain in the Canadian rearing facility in British Columbia. For both strains, the female codling moth was considerably more radiosensitive than the male. At a radiation dose of 100 Gy or higher, treated females were 100% sterile. The fertility of the South African strain was higher (86.3%) than for the Canadian strain (71.9%). This difference in fertility between the two strains was maintained when the dose of radiation was 100 Gy. However, the level of fertility was very similar between the two strains for doses ≥150 Gy. Therefore, based upon previously published work and the data from this study, an operational dose of 150 Gy is recommended for future codling moth SIT programmes in South Africa.     

Synthesis of DNA in dividing and quiescent cells following gamma-irradiation

Changes in clonogenic capacity of gamma-irradiated minimally transformed 3T3 cells (Do = 1.3 Gy, D37 = 5 Gy, and Dq = 5.3 Gy) were determined and conditions for obtaining dormant population of these cells were chosen to be adequate for registering the radiation-induced unscheduled DNA synthesis. The comparison between the postirradiation changes in DNA synthesis in dividing and resting 3T3 cells suggests the participation of DNA-polymerase alpha in providing the unscheduled DNA synthesis in resting cells after gamma-irradiation with doses of 5 to 50 Gy.     

Lack of optimal activation of natural killer levels by interleukin-2 in rat spleen cells: evidence for suppression

The effect of human recombinant IL-2 on the levels of natural killer (NK) activity in spleen cells derived from BALB/c mice and different strains of rats was studied. Enhancement of murine NK activity in response to IL-2 was readily demonstrable. Levels of NK activity in control as well as IL-2-treated spleen cells from Wistar, Sprague-Dawley, Fischer, and Long-Evans rats increased initially and peaked on Day 1 or 2 of culture. No significant differences between the control and the IL-2-treated cultures was found in this duration. The subsequent fall in NK activity was slower in IL-2-treated cultures of Fischer and Long-Evans rat spleen cells resulting in a significant difference between the NK levels in control and IL-2-treated cells on Day 5 and Day 7 of the culture. In the case of Wistar and Sprague-Dawley rats, the boosting effect of IL-2 on NK levels was either poor or nonexistent. Even though NK activation was poor, spleen cells of all strains of rats did proliferate in response to IL-2, indicating that the IL-2 preparation used was biologically active on rat spleen cells. Rat spleen cells cultured alone or with IL-2 released a factor(s) in the culture supernatant which could suppress the IL-2-induced NK activation in mouse spleen cells. Indomethacin could block the release of suppressor factor by cultured rat spleen cells. Moreover the NK levels in rat spleen cells could be augmented by IL-2 in the presence of indomethacin. These results indicate that the subdued IL-2-induced NK activation in bulk cultures of rat spleen cells could be due to spontaneous release of prostaglandins by the cultured cells.