Influence of CW microwave radiation on in vitro release of enzymes from retinol-treated hepatic lysosomes

Hepatic lysosomes were exposed in vitro to microwave radiation (2450 MHz) either prior to or simultaneously with treatment with retinol (vitamin A), and the release of the lysosomal enzymes, β-glucuronidase, acid phosphatase, and cathepsin D, determined. A 60-min microwave exposure (10 or 100 mW/g) of retinol-treated lysosomes had no effect on the amount of release of β-glucuroni-dase, cathepsin D, or acid phosphatase. In addition, 10 and 100 mW/g irradiation of lysosome fractions for 40 min prior to a 20-min retinol and microwave treatment, had no influence on the release of these enzymes. Finally, the effect of microwave radiation on the loss of latency of acid phosphatase and β-glucuronidase from retinol-treated lysosomes was determined. Microwave radiation had no influence on the rate of appearance of these enzymes in the suspending medium. The results indicate that microwave radiation had no effect on the retinol-induced lysosomal enzyme release.     

Microwave irradiation and in vitro release of enzymes from hepatic lysosomes

Summary An exposure system for examining in vitro effects of microwave irradiation on cellular and subcellar components has been developed. The system was used to test the effect of 2.45-GHz CW microwaves on the release of two lysosomal enzymes. At a specific absorption rate (SAR) of 10, 50 or 100 mW/g (90 min) no effects were noted at 37° C (pH of 7.3) on lysosomal fragility as determined by the release of the lysosomal enzymes cathepsin D and-glucuronidase. When the medium was adjusted to pH 5.0, microwave irradiation of the lysosomal suspension had no effect on the acid-induced enhancement of release of lysosomal enzymes. The data indicate that microwave irradiation had no labilizing effect on the lysosomal membrane, although other microwave-membrane interactions not associated with enzyme release may occur.     

Influence of 2.45-GHz CW microwave radiation on spontaneously beating rat atria

The chronotropic and inotropic effects of 2.45-GHz continuous wave (CW) microwave radiation were investigated in the isolated spontaneously beating rat atria. Isolated atria were placed in specially designed tubes inserted into a waveguide exposure system. The atria were then irradiated for a period of 30 min, followed by a 30-min recovery period. The control atria were prepared simultaneously and sham exposed. Experiments were conducted at two temperatures, 22 and 37 °C, and two specific absorption rates, 2 mW/g and 10 mW/g. At both temperatures the rate of atrial contraction was not altered by a 30-min exposure at either 2 or 10 mW/g. The average rate (beats per min) was approximately 100 for both the control and exposed atria at 22 °C and 215 beats per min for both the control and exposed atria at 37 °C. In addition, no inotropic effects on the spontaneously beating atria were noted at any exposure level. These data suggest that 2.45-GHz CW microwave radiation at these intensities has no overt effect on these variables in isolated rat atria.     

Influence of in vitro microwave radiation on the fertilizing capacity of turkey sperm

Turkey sperm were exposed to 2.45-GHz microwave radiation in a temperature-controlled wave-guide apparatus. Temperature was maintained at either 25 or 40.5 degrees C. The sperm were exposed for 30 min at a specific absorption rate (SAR) of 10 or 50 mW/g. Following irradiation, the sperm were used to inseminate virgin turkey hens artificially. During the 9 weeks following the single insemination, the following were assessed: mean number of eggs, percentage of fertile eggs, rate of decrease in egg fertility, percentage of hatched eggs, and percentage of early and late deaths. These data demonstrate that, for the conditions used in these experiments, microwave radiation has no effect on the fertilizing capacity of turkey sperm.     

Tissue producing the serum factor stimulating the release of cathepsin D from lysosomes in vitro

Pancreatectomy as well as thyroparathyroidectomy resulted in the quick disappearance of a serum factor (stimulating cathepsin D release from lysosomes in vitro) from the rat or mouse blood. Extirpation of other organs such as duodenum, stomach, spleen, kidney, submaxillary gland, testis, adrenal gland or hypophysis, showed no effect on the serum factor level. Glucagon (but not insulin or thyroxine) given to the pancreatectomized animals restored the serum factor level in a dose-dependent manner. The serum factor-like activity was detected only in the parathyroids (but not thyroid), and the release of activity from parathyroid-slices was stimulated by glucagon, suggesting that the parathyroid may produce and/or secrete the serum factor under the influence of glucagon.     

Influence of 2.45 GHz microwave radiation on enzyme activity

Summary The in vitro activity of acetylcholinesterase and creatine phosphokinase was determined during in vitro exposure to 2.45 GHz microwave radiation. The enzyme activities were examined during exposure to microwave radiation at specific absorption rates (SAR) of 1, 10, 50, and 100 mW/g. These specific absorption rates had no effect on the activity of either enzyme when the temperature of the control and exposed samples were similar. These data demonstrate that the activity of these two enzymes is not affected by microwave radiation at the SARs and frequency employed in this study.     

Release of enzymes from lysosomes by irradiation and the relation of lipid peroxide formation to enzyme release

1. Acid phosphatase, cathepsin and beta-glucuronidase are released from rat-liver lysosomes by irradiation in vitro. Enzyme release is detectable after a dose of 1krad and increases with dose up to 100krads. 2. Maximum radiation effects were observed when the lysosomes were kept for 20hr. at 4 degrees or 20 degrees after irradiation. 3. An atmosphere of nitrogen considerably decreases enzyme release from lysosomes. 4. Enzyme release is enhanced by ascorbic acid and decreased by vitamin E. 5. Irradiation causes formation of lipid peroxides in lysosomes, and enzyme release increases with lipid peroxide formation. 6. It is suggested that lipid peroxide formation leads to rupture of the lysosome membrane and allows release of the contained hydrolytic enzymes.     

In vitro effects of microwave radiation on rat liver mitochondria

Liver mitochondria were exposed in vitro at 30°C to microwave radiation (2.45 GHz) during the following states of respiraton: resting, state 1; substrate dependent, state 2; ADP stimulated, state 3; and ADP depleted, state 4. At 10 or 100 mW/g, with succinate as substrate, no effect of exposure was observed on states 1–4 or the respiratory control index (state 3/state 4) of either tightly or loosely coupled mitochondria. When glutamate was used as substrate, no effects were observed at 10 mW/g. However, in the loosely coupled mitochondria the 100 mW/g exposure produced an increase in states 2 and 4 and a decrease in the respiratory control index. The results suggest that the function of loosely coupled mitochondria can be affected at high power levels of microwave radiation.     

Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

Characterization of lysosomes and lysosomal enzymes from Chediak-Higashi-syndrome cultured fibroblasts.

Chediak-Higashi-syndrome cultured skin fibroblasts were used to study the possible involvement of lysosomal enzymes and lysosomal dysfunction in this disorder. Our evidence indicated that Chediak-Higashi fibroblasts displayed a significant decrease in the specific activity of the acidic alpha-D-mannosidase (pH 4.2) compared with normal controls. Additional studies revealed a small, but significant, decrease in the rate of degradation of 125I-labelled beta-D-glucosidase that had been endocytosed into Chediak-Higashi cells.     

Stimulation of the release of lysosomal and nonlysosomal granular enzymes from macrophages treated with monensin

Mouse peritoneal macrophages that had been treated with a monovalent carboxylic ionophore, monensin, selectively secreted lysosomal and nonlysosomal granular enzymes into the medium. When macrophages were incubated with 1 to 10 microM monensin, the release of beta-glucuronidase, beta-hexosaminidase and beta-galactosidase was stimulated time and does dependently. Neither the beta-glucosidase nor acid phosphatase, enzymes bound to the lysosomal membranes, however, were released by monensin. Neutral alpha-glucosidase, shown recently to be localized in nonlysosomal granules of macrophages (15), was released by monensin at concentrations lower than those required for lysosomal enzyme release. Increased release of lysosomal enzymes also took place in a manner similar to that seen with monensin-treated macrophages after treatment of macrophages with weak bases, chloroquine and ammonium chloride. Neutral alpha-glucosidase, however, was not released when chloroquine was present in concentrations that stimulated the release of lysosomal enzymes. The UDP-galactosyltransferase activity of the Golgi apparatus in the macrophages markedly decreased after treatment with low concentration of monensin.     

Influence of fixation and buffer treatment on the release of enzymes from the plasma membrane.

1. Pretreatment of frozon cryostat sections with formaldehyde or calcium ions inhibits diffusion of the plasma membrane enzymes 5' nucleotidase, ATP-ase and alkaline phosphatase during incubation. 2. Treatment of fixed sections with different kinds of buffer at 37 degrees C induces diffusion of enzyme activity from the plasma membrane to other sites of the section and into the incubation medium. This buffer influence depends on temperature: at 4 degrees C only a slight diffusion occurs. Addition of phospholipase C, digitonin or taurocholate to the buffer opposes the buffer effect. 3. Pretreatment of frozen cryostat sections with a mixture of equal parts of chloroform and acetone give a good fixation of the plasma membrane enzymes 5'-nucleotidase, ATP-ase, alkaline phosphate and leucyl-beta-naphthylamidase. During this treatment the different kinds of lipids present in the membrane are ex-racted equally. After this fixation buffer treatment does not cause a visible diffusion of enzyme activity in the section. Only a slight diffusion (1 till 7 percent) into the buffer solution takes place. 4. The mentioned treatments open up possibilities to get insight into the membrane anchorage of plasma membrane enzymes.     

Induction of micronuclei in human lymphocytes exposed in vitro to microwave radiation

Increasing applications of electromagnetic fields are of great concern with regard to public health. Several in vitro studies have been conducted to detect effects of microwave exposure on the genetic material leading to negative or questionable results. The micronucleus (MN) assay which is proved to be a useful tool for the detection of radiation exposure-induced cytogenetic damage was used in the present study to investigate the genotoxic effect of microwaves in human peripheral blood lymphocytes in vitro exposed in G(0) to electromagnetic fields with different frequencies (2.45 and 7.7GHz) and power density (10, 20 and 30mW/cm(2)) for three times (15, 30 and 60min). The results showed for both radiation frequencies an induction of micronuclei as compared to the control cultures at a power density of 30mW/cm(2) and after an exposure of 30 and 60min. Our study would indicate that microwaves are able to cause cytogenetic damage in human lymphocytes mainly for both high power density and long exposure time.