Toxicology, 61 (1990) 59-71 Elsevier Scientific Publishers Ireland Ltd.
Depression of glutathione by cold-restraint in mice Henry F. Simmons a, Robert C. James b, Raymond D. Harbison b, and S t e p h e n M. R o b e r t s °'* ~Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR, and bCenter for Environmental Toxicology, University of Florida, Gainesville, FL (U. S. A) (Received June 29th, 1989; accepted October 17th, 1989)
Summary The effects of cold-restraint as a physiological stressor on the glutathione (GSH) content of the liver and other tissues were examined in male mice. Mice of the ICR, NIH, ND/4, and B6C3F1 strains subjected to cold-restraint for 2 or 3 h experienced a loss of hepatic GSH concentrations ranging from approximately 15 to 50%. Though 3 of these strains (ICR, NIH, and B6C3F1) experienced hypothermia as result of the cold-restraint treatment, with average decreases in core body temperature ranging from 3.3 to 9.8°C, hepatic GSH levels were depressed in the ND/4 mouse in the absence of changes in core body temperature. The ability of cold-restraint as a stressor to diminsh hepatic GSH therefore could not be attributed simply to hypothermia. The decrease in hepatic GSH from cold-restraint in ND/4 mice was paralleled by a decrease in non-protein sulfhydryl (NPSH) content of the liver. In addition to its effects on liver GSH and NPSH concentrations, 1.5 h of cold-restraint stress significantly depressed plasma, heart, kidney, and lung NPSH concentrations. The extent of NPSH depression was equivalent to the GSH depression in the liver, heart, and kidney, despite the observation that the normal contribution of GSH to total NPSH content in these tissues ranged from a high of 89% (liver) to a low of 49% (heart). These results with cold-restraint in the ND/4 mouse suggest that other stressors may significantly depress cellular concentrations of GSH and other thiols, and may thereby render the affected tissues more susceptible to the toxicity of free radicals, electrophilic xenobiotic metabolites, or reactive oxygen species.
Key words: Glutathione; Cold-restraint; Stress; Non-protein sulfhydryl content
Introduction In 1888, glutathione or y-glutamyl-cysteinyl-glycine was discovered in yeast. Forty-seven years later its structure was elucidated. This tripeptide is found in virtually every living cell with the possible exception of some bacteria. Quite probably, it is the most abundant low molecular weight thiol that occurs naturally, with many tissue concentrations in the millimolar range [1]. The bulk of the Address all correspondence to: Dr. Stephen M. Roberts, Center for Environmental Toxicology, University of Florida, One Progress Blvd., Mailbox 17, Alachua, FL 32615, U.S.A. 0300-483X/90/$03.50 (~) 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
59
reduced glutathione (GSH) in most cells is found in the cytosol where it plays a role in numerous biochemical functions. These functions include the scavenging of free radicals, electrophilic metabolites and reactive oxygen species; the synthesis of deoxyribonucleotide precursors; and the reduction of various disulfide linkages [21. Because of the importance of GSH in the detoxification of a number of compounds, a significant depletion of GSH may increase the toxicity of these agents. There are several examples in the literature where a chemically-induced depletion of GSH (e.g. with diethyl maleate) has resulted in a potentiation of the toxicity of a compound (e.g. acetaminophen [3], bromobenzene [4], doxorubicin [5], and cocaine [6]). Depression of GSH by physiological means can also apparently influence toxicity as indicated by the effects of diurnal fluctuations in GSH levels on 1,1-dichloroethylene toxicity [7]. One of the more interesting, and perhaps important, non-chemically induced means of depressing GSH concentrations is through physiological stress. Studies spanning decades have shown that rats or mice subjected to various stressors have diminished hepatic levels of GSH, or of non-protein sulfhydryl (NPSH) content of which GSH is the principal component. Examples of stressors observed to cause a reduction in hepatic GSH or NPSH content include limb ligation trauma, scalding, bacterial endotoxins, tumbling trauma, hemorrhage, and insulin shock [8-11]. While changes have been observed occasionally in NPSH or GSH content of tissues other than the liver in stressed animals, there is little in the way of comprehensive information on stress-induced effects on GSH in extrahepatic tissues. In order to further examine the influence of stress on GSH, we have measured the effects of cold-restraint on tissue GSH concentrations in mice. Cold-restraint of laboratory rodents has been used for decades to study stress-related phenomena, and rats subjected to cold-restraint have been shown to develop gastric ulcers within a few hours [12,13]. Beck and Linkenheimer [8[ observed that cold exposure depressed hepatic NPSH content in mice, but cold plus restraint was found to be more effective in lowering hepatic NPSH content than cold alone in a study in rats [14]. Cold-restraint is a relatively humane stressor, and can be readily applied to varying extents to examine "dose"-response relationships. Initial experiments were directed to characterizing the effects of cold-restraint on hepatic GSH concentrations in mice, and later experiments examined the magnitude and temporal course of cold-restraint changes in NPSH and GSH concentrations in extra-hepatic tissues including spleen, stomach, lung, heart, kidney, and plasma. Materials and methods
Chemicals
Trichioroacetic acid, perchloric acid, disodium ethylenediamine tetraacetate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and methanol (HPLC grade) were purchased from Fisher Chemical Company (Fair Lawn, NJ)
60
as ASC grade. 5,5'-Dithiobis (2-nitrobenzoic acid), reduced glutathione, sodium chloride, monochloroacetic acid, and penicillamine (DL-isomeric mixture) were purchased from Sigma Chemical Company (St. Louis, MO). Sodium hydroxide (ACS grade) was purchased from MCB Manufacturing Chemists, Inc. (Cincinnati, OH), and sodium octyl sulfate was obtained from Eastman Kodak Company (Rochester, NY). Triple-distilled mercury was purchased from Bioanalytical Systems (West Lafayette, IN).
Animals and Treatments ICR, NIH, and ND/4 male mice all weighing 20-25 g were purchased from Harlan Sprague Dawley (Indianapolis, IN). Male B6C3F1 mice, 20-30 g, were obtained from the National Center for Toxicological Research (Jefferson, AR). The animals were housed 4-5 per cage in stainless steel and polycarbonate cages with hardwood chip bedding material. Aside from restraint periods, all animals had free access to food and water. A 12-h light/dark cycle was provided in the animal quarters, and the temperature was maintained at 22_+ I°C. Relative humidity ranged from 40 to 60%. Treatment and control group sizes ranged from a minimum of 5 to a maximum of 10 animals. All treatments were initiated in the morning, usually between 0700 and 0900 h. Sacrifice times varied with experimental design, but a control group was always sacrificed at the same time of day as a treatment group to preclude an influence of diurnal variation in GSH on experimental outcome. In studies determining strain responsiveness to cold-restraint and in studies in which the cold-restraint "dose"-response relationship was evaluated, treated animals were sacrificed by asphyxiation with carbon dioxide immediately post-cold-restraint. In the multiple tissue time course studies, groups of animals and their controls were asphyxiated immediately after termination of cold-restraint (0 h), or 1, 2, 3, or 5 h later. Cold-restraint was carried out in a draft-free portion of a refrigerated room maintained at 7.5 + 1°C. The restrainers used were modified 50 ml polystyrene centrifuge tubes with screw-on caps supplied by Fisher Scientific Company (Fair Lawn, N J). The wall of each tube was perforated with 4 rows of 44 2-mm holes. A single 5 mm perforation was drilled through the tapered tip. Five additional 2-mm holes were bored through the cap. Animals readily entered the tubes without shoving, orienting their heads at the tapered ends. An approximate 2:1 ratio of tube volume to body volume was achieved in most cases. In the cold room, each loaded tube was placed in a wooden rack that insured its isolation from other tubes. Rectal temperatures were measured with a flexible, small rodent rectal probe coupled to a calibrated electronic thermometer (YSI Instruments, Yellow Springs, OH).
Assay for reduced glutathione (GSH) Tissues samples were excised, rinsed with cold saline, blotted dry, and weighed. These samples, weighing between 0.3 and 0.6 g, were homogenized in 3 ml of
61
cold 0.3 M perchloric acid with 0.1% EDTA. Immediately after homogenization, precipitated protein was pelleted at 10 000 x g for 15 min in a refrigerated (4°C) centrifuge. A 50-/xl aliquot of the supernatant was pipetted into a 0.2-/xm pore-size microfiltration tube (Bioanalytical Systems, West Lafayette, IN) along with 250/xl cold 0.1% EDTA and 10/zl penicillamine internal standard solution (penicillamine 1 mg/ml in 0.1% EDTA). The microfiltration tube was capped, vortexed, and centrifuged to accomplish the mixing and filtration of the sample solution. The filtered samples were placed on ice for analysis the same day or stored frozen at -80°C for assay within 72 h. Previous stability studies revealed no significant differences in tissue GSH concentrations among samples analyzed immediately or stored for 5 days under these conditions (unpublished observations). GSH content of tissue samples was measured by HPLC with electrochemical detection in a manner similar to that described by Allison and Shoup [15]. A Bioanalytical Systems LC-448 Liquid Chromatograph was used equipped with modifications to exclude oxygen from the system including the mobile phase reservoir. The mobile phase consisted of 0.075 M monochloroacetic acid and 3% acetonitrile, adjusted to pH 2.8 with sodium hydroxide. Sodium octyl sulfate was included at a concentration of 348 mg/l as an ion-pairing agent. Chromatographic separation was achieved with a 3/xm, 3.2 x 100 mm reverse phase (ODS) column (Bioanalytieal Systems, West Lafayette, IN). The potential of the gold/mercury electrode vs. silver/silver chloride reference was 0.150 V.
Assay for non-protein sulfhydryl (NPSH) content NPSH content was measured by the method of Ellman [16]. Tissues were processed in the fashion described above for the measurement of GSH, with samples removed and homogenized under cold conditions with 0.3 M perchloric acid. Following cold (4°C) centrifugation of the tissue homogenate, protein-free supernatant was diluted with cold 0.1% EDTA. For heart tissue, 400 /xl of deproteinized supernatant was added to 100 txl of 0.1% EDTA prior to assay. For kidney, spleen, lung, and stomach, 200 /xl of supernatant was added to 100 txl 0.1% EDTA. The preparation of plasma for NPSH measurement differed from that of other tissues somewhat. Immediately following carbon dioxide asphyxiation, blood was collected from mice via cardiac puncture into a heparinized syringe. The blood was centrifuged at 13 000 x g for 3 min to recover the plasma. A 250-/xl aliquot of plasma was quickly pipetted into 500/xl cold 0.3 M perchloric acid in 0.1% EDTA and allowed to stand in ice for approximately 5 min. Following a 5-rain spin at 13 000 x g, the supernatant was removed for measurement of NPSH content.
Statistical analyses For most experiments in this study, each treatment group had a distinct, matching control group. For these experiments, the treatment and control groups were compared with an unpaired Student's t-test. In those experiments in which
62
more than one treatment group was compared with a single control group sacrificed at the same time, statistical comparisons were made using the Dunnett's test. In all instances, statistical significance was considered to be P ~
Depression of glutathione by cold-restraint in mice Henry F. Simmons a, Robert C. James b, Raymond D. Harbison b, and S t e p h e n M. R o b e r t s °'* ~Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR, and bCenter for Environmental Toxicology, University of Florida, Gainesville, FL (U. S. A) (Received June 29th, 1989; accepted October 17th, 1989)
Summary The effects of cold-restraint as a physiological stressor on the glutathione (GSH) content of the liver and other tissues were examined in male mice. Mice of the ICR, NIH, ND/4, and B6C3F1 strains subjected to cold-restraint for 2 or 3 h experienced a loss of hepatic GSH concentrations ranging from approximately 15 to 50%. Though 3 of these strains (ICR, NIH, and B6C3F1) experienced hypothermia as result of the cold-restraint treatment, with average decreases in core body temperature ranging from 3.3 to 9.8°C, hepatic GSH levels were depressed in the ND/4 mouse in the absence of changes in core body temperature. The ability of cold-restraint as a stressor to diminsh hepatic GSH therefore could not be attributed simply to hypothermia. The decrease in hepatic GSH from cold-restraint in ND/4 mice was paralleled by a decrease in non-protein sulfhydryl (NPSH) content of the liver. In addition to its effects on liver GSH and NPSH concentrations, 1.5 h of cold-restraint stress significantly depressed plasma, heart, kidney, and lung NPSH concentrations. The extent of NPSH depression was equivalent to the GSH depression in the liver, heart, and kidney, despite the observation that the normal contribution of GSH to total NPSH content in these tissues ranged from a high of 89% (liver) to a low of 49% (heart). These results with cold-restraint in the ND/4 mouse suggest that other stressors may significantly depress cellular concentrations of GSH and other thiols, and may thereby render the affected tissues more susceptible to the toxicity of free radicals, electrophilic xenobiotic metabolites, or reactive oxygen species.
Key words: Glutathione; Cold-restraint; Stress; Non-protein sulfhydryl content
Introduction In 1888, glutathione or y-glutamyl-cysteinyl-glycine was discovered in yeast. Forty-seven years later its structure was elucidated. This tripeptide is found in virtually every living cell with the possible exception of some bacteria. Quite probably, it is the most abundant low molecular weight thiol that occurs naturally, with many tissue concentrations in the millimolar range [1]. The bulk of the Address all correspondence to: Dr. Stephen M. Roberts, Center for Environmental Toxicology, University of Florida, One Progress Blvd., Mailbox 17, Alachua, FL 32615, U.S.A. 0300-483X/90/$03.50 (~) 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
59
reduced glutathione (GSH) in most cells is found in the cytosol where it plays a role in numerous biochemical functions. These functions include the scavenging of free radicals, electrophilic metabolites and reactive oxygen species; the synthesis of deoxyribonucleotide precursors; and the reduction of various disulfide linkages [21. Because of the importance of GSH in the detoxification of a number of compounds, a significant depletion of GSH may increase the toxicity of these agents. There are several examples in the literature where a chemically-induced depletion of GSH (e.g. with diethyl maleate) has resulted in a potentiation of the toxicity of a compound (e.g. acetaminophen [3], bromobenzene [4], doxorubicin [5], and cocaine [6]). Depression of GSH by physiological means can also apparently influence toxicity as indicated by the effects of diurnal fluctuations in GSH levels on 1,1-dichloroethylene toxicity [7]. One of the more interesting, and perhaps important, non-chemically induced means of depressing GSH concentrations is through physiological stress. Studies spanning decades have shown that rats or mice subjected to various stressors have diminished hepatic levels of GSH, or of non-protein sulfhydryl (NPSH) content of which GSH is the principal component. Examples of stressors observed to cause a reduction in hepatic GSH or NPSH content include limb ligation trauma, scalding, bacterial endotoxins, tumbling trauma, hemorrhage, and insulin shock [8-11]. While changes have been observed occasionally in NPSH or GSH content of tissues other than the liver in stressed animals, there is little in the way of comprehensive information on stress-induced effects on GSH in extrahepatic tissues. In order to further examine the influence of stress on GSH, we have measured the effects of cold-restraint on tissue GSH concentrations in mice. Cold-restraint of laboratory rodents has been used for decades to study stress-related phenomena, and rats subjected to cold-restraint have been shown to develop gastric ulcers within a few hours [12,13]. Beck and Linkenheimer [8[ observed that cold exposure depressed hepatic NPSH content in mice, but cold plus restraint was found to be more effective in lowering hepatic NPSH content than cold alone in a study in rats [14]. Cold-restraint is a relatively humane stressor, and can be readily applied to varying extents to examine "dose"-response relationships. Initial experiments were directed to characterizing the effects of cold-restraint on hepatic GSH concentrations in mice, and later experiments examined the magnitude and temporal course of cold-restraint changes in NPSH and GSH concentrations in extra-hepatic tissues including spleen, stomach, lung, heart, kidney, and plasma. Materials and methods
Chemicals
Trichioroacetic acid, perchloric acid, disodium ethylenediamine tetraacetate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and methanol (HPLC grade) were purchased from Fisher Chemical Company (Fair Lawn, NJ)
60
as ASC grade. 5,5'-Dithiobis (2-nitrobenzoic acid), reduced glutathione, sodium chloride, monochloroacetic acid, and penicillamine (DL-isomeric mixture) were purchased from Sigma Chemical Company (St. Louis, MO). Sodium hydroxide (ACS grade) was purchased from MCB Manufacturing Chemists, Inc. (Cincinnati, OH), and sodium octyl sulfate was obtained from Eastman Kodak Company (Rochester, NY). Triple-distilled mercury was purchased from Bioanalytical Systems (West Lafayette, IN).
Animals and Treatments ICR, NIH, and ND/4 male mice all weighing 20-25 g were purchased from Harlan Sprague Dawley (Indianapolis, IN). Male B6C3F1 mice, 20-30 g, were obtained from the National Center for Toxicological Research (Jefferson, AR). The animals were housed 4-5 per cage in stainless steel and polycarbonate cages with hardwood chip bedding material. Aside from restraint periods, all animals had free access to food and water. A 12-h light/dark cycle was provided in the animal quarters, and the temperature was maintained at 22_+ I°C. Relative humidity ranged from 40 to 60%. Treatment and control group sizes ranged from a minimum of 5 to a maximum of 10 animals. All treatments were initiated in the morning, usually between 0700 and 0900 h. Sacrifice times varied with experimental design, but a control group was always sacrificed at the same time of day as a treatment group to preclude an influence of diurnal variation in GSH on experimental outcome. In studies determining strain responsiveness to cold-restraint and in studies in which the cold-restraint "dose"-response relationship was evaluated, treated animals were sacrificed by asphyxiation with carbon dioxide immediately post-cold-restraint. In the multiple tissue time course studies, groups of animals and their controls were asphyxiated immediately after termination of cold-restraint (0 h), or 1, 2, 3, or 5 h later. Cold-restraint was carried out in a draft-free portion of a refrigerated room maintained at 7.5 + 1°C. The restrainers used were modified 50 ml polystyrene centrifuge tubes with screw-on caps supplied by Fisher Scientific Company (Fair Lawn, N J). The wall of each tube was perforated with 4 rows of 44 2-mm holes. A single 5 mm perforation was drilled through the tapered tip. Five additional 2-mm holes were bored through the cap. Animals readily entered the tubes without shoving, orienting their heads at the tapered ends. An approximate 2:1 ratio of tube volume to body volume was achieved in most cases. In the cold room, each loaded tube was placed in a wooden rack that insured its isolation from other tubes. Rectal temperatures were measured with a flexible, small rodent rectal probe coupled to a calibrated electronic thermometer (YSI Instruments, Yellow Springs, OH).
Assay for reduced glutathione (GSH) Tissues samples were excised, rinsed with cold saline, blotted dry, and weighed. These samples, weighing between 0.3 and 0.6 g, were homogenized in 3 ml of
61
cold 0.3 M perchloric acid with 0.1% EDTA. Immediately after homogenization, precipitated protein was pelleted at 10 000 x g for 15 min in a refrigerated (4°C) centrifuge. A 50-/xl aliquot of the supernatant was pipetted into a 0.2-/xm pore-size microfiltration tube (Bioanalytical Systems, West Lafayette, IN) along with 250/xl cold 0.1% EDTA and 10/zl penicillamine internal standard solution (penicillamine 1 mg/ml in 0.1% EDTA). The microfiltration tube was capped, vortexed, and centrifuged to accomplish the mixing and filtration of the sample solution. The filtered samples were placed on ice for analysis the same day or stored frozen at -80°C for assay within 72 h. Previous stability studies revealed no significant differences in tissue GSH concentrations among samples analyzed immediately or stored for 5 days under these conditions (unpublished observations). GSH content of tissue samples was measured by HPLC with electrochemical detection in a manner similar to that described by Allison and Shoup [15]. A Bioanalytical Systems LC-448 Liquid Chromatograph was used equipped with modifications to exclude oxygen from the system including the mobile phase reservoir. The mobile phase consisted of 0.075 M monochloroacetic acid and 3% acetonitrile, adjusted to pH 2.8 with sodium hydroxide. Sodium octyl sulfate was included at a concentration of 348 mg/l as an ion-pairing agent. Chromatographic separation was achieved with a 3/xm, 3.2 x 100 mm reverse phase (ODS) column (Bioanalytieal Systems, West Lafayette, IN). The potential of the gold/mercury electrode vs. silver/silver chloride reference was 0.150 V.
Assay for non-protein sulfhydryl (NPSH) content NPSH content was measured by the method of Ellman [16]. Tissues were processed in the fashion described above for the measurement of GSH, with samples removed and homogenized under cold conditions with 0.3 M perchloric acid. Following cold (4°C) centrifugation of the tissue homogenate, protein-free supernatant was diluted with cold 0.1% EDTA. For heart tissue, 400 /xl of deproteinized supernatant was added to 100 txl of 0.1% EDTA prior to assay. For kidney, spleen, lung, and stomach, 200 /xl of supernatant was added to 100 txl 0.1% EDTA. The preparation of plasma for NPSH measurement differed from that of other tissues somewhat. Immediately following carbon dioxide asphyxiation, blood was collected from mice via cardiac puncture into a heparinized syringe. The blood was centrifuged at 13 000 x g for 3 min to recover the plasma. A 250-/xl aliquot of plasma was quickly pipetted into 500/xl cold 0.3 M perchloric acid in 0.1% EDTA and allowed to stand in ice for approximately 5 min. Following a 5-rain spin at 13 000 x g, the supernatant was removed for measurement of NPSH content.
Statistical analyses For most experiments in this study, each treatment group had a distinct, matching control group. For these experiments, the treatment and control groups were compared with an unpaired Student's t-test. In those experiments in which
62
more than one treatment group was compared with a single control group sacrificed at the same time, statistical comparisons were made using the Dunnett's test. In all instances, statistical significance was considered to be P ~
