DNA cell cycle distribution and glutathione (GSH) content according to circadian stage in bone marrow of cancer patients.

DNA cell cycle distribution and glutathione (GSH) content in bone marrow were measured both at daytime and midnight over single 24 h periods in 15 cancer patients. Between patients the S-phase demonstrated a difference from lowest to highest value of 700%, whereas the corresponding difference for the G2/M-phase was nearly 900%. The mean GSH content measured in the bone marrow at the two timepoints was 2.24 +/- 0.21 nmol mg-1 protein, range 0.91-4.19 nmol mg-1 protein. A statistically significant higher fraction of cells in S-phase and G2/M-phase was found at daytime as compared to midnight when excluding the four patients with an abnormal circadian variation in cortisol. No significant temporal variation in total bone marrow GSH content was found, although a weak correlation between S-phase and GSH content was demonstrated (r = 0.42; P less than 0.05). This correlation was strengthened when not including the six patients with an abnormal cortisol pattern (4) and bone marrow infiltration (2) (r = 0.66; P = 0.005). Cells in S-phase demonstrated a positive correlation with cells in G2/M-phase (r = 0.64; P less than 0.0001). A negative correlation was found between GSH content and age (r = 0.53; P less than 0.005). Finally, a statistically significant positive correlation was demonstrated between cortisol and both S-phase and G2/M-phase (r = 0.57; P less than 0.001 and r = 0.38; P less than 0.05, respectively). The present study suggests a possibility of optimising cancer therapy and use of hematopoietic growth factors by determining individual average values and circadian stage dependent variation in bone marrow DNA cell cycle distribution. Furthermore, GSH content in bone marrow may predict this tissue's sensitivity to cytotoxic agents.     

鼻咽癌患者外周血血浆可的松水平和全血还原型谷胱甘肽含量的昼夜节律

背景与目的:谷胱甘肽与细胞对抗癌药物的解毒作用以及对放射性损伤的保护机制密切相关。本文通过对鼻咽癌患者和健康志愿者外周血血浆可的松水平和全血还原型谷胱甘肽含量的昼夜节律进行研究,从而为鼻咽癌患者进行肿瘤的时间治疗提供参考资料。方法:13名鼻咽癌患者(实验组)和14名健康志愿者(正常对照组)参加本项研究。每位受试者均从中午12点开始抽取外周静脉血4.5mL,每隔4h抽血一次,在24h内共抽血6次。血浆可的松水平的检测采用放射免疫法,全血还原型谷胱甘肽含量则采用高效液相色谱法进行检测。结果:鼻咽癌患者组和正常对照组的血浆可的松水平均呈现出明显的昼夜节律的特征,且节律特性相似。两组的血浆可的松水平的峰值均出现在早晨,而谷值均出现在午夜。不同时间点时,两组的全血还原型谷胱甘肽含量不同,且均具有显著性差异(重复测量的方差分析,F=5.18,P=0.02)。余弦分析表明,鼻咽癌患者组呈现出昼夜节律变化趋势(P=0.06),峰值出现在早晨(05∶02),正常对照组也呈现出明显的昼夜节律的特征,峰值出现在早晨(07∶44±01∶56)(P<0.01)。鼻咽癌患者组外周全血中还原型谷胱甘肽的节律调整均值为(19.60±1.11)nmol/mgprotein,正常对照组为(8.95±0.46)nmol/mgprotein。结论:包括晚期患者在内的鼻咽癌患者仍然具有正常的生物节律。鼻咽癌患者全血中还原型谷胱甘肽水平呈现出昼夜节律变化的趋势,与正常对照组节律特性相似。这为临床上选择恰当的时间对患者进行化疗和放疗提供了有价值的参考资料。     

Cellular glutathione and thiol measurements from surgically resected human lung tumor and normal lung tissue

Cellular glutathione (GSH) levels were measured from 27 human lung tumor biopsies, enzymatically disaggregated, and compared with cells isolated from normal lung of the same patients. GSH levels from normal lung were similar among patients with a mean value of 11.20 +/- 0.58 (SEM) nmol GSH/mg protein (24 patients) with a range from 6.1 to 17.5 nmol GSH/mg protein. GSH levels varied considerably within and across histological tumor types with the following values: adenocarcinomas, 8.83 +/- 0.96 nmol/mg protein (8 patients); large cell carcinomas, 8.25 +/- 2.51 nmol/mg protein (3 patients); and squamous cell carcinomas, 23.25 +/- 5.99 nmol/mg protein (8 patients). The cyclic GSH reductase assay gave only average GSH values and could not distinguish possible GSH variation among subpopulations of cells isolated. Cell volume measurements and microscopic evaluation of cells isolated from both tumors and normal lung revealed heterogeneity with respect to cell types present. To determine the extent of thiol variation among tumor cell subpopulations, tumor cell suspensions were stained with the thiol-specific stain, monochlorobimane (MCB). The accuracy of MCB staining was tested by flow cytometric analysis of 12 in vitro human tumor cell lines and 3 rodent cell lines. A linear relationship was found between the bimane cellular fluorescence and the cyclic GSH reductase assay for cell lines having less than 80 nmol GSH/mg protein (R2 = 0.82). Above 80 nmol GSH/mg protein the rate of change of the bimane fluorescence intensity with respect to increasing GSH concentrations was much reduced. However, by labeling cells with MCB it was possible to distinguish between cell lines with low versus high GSH content. MCB staining of tumor samples revealed multiple populations of cells with respect to thiol levels. In particular, 2 of 8 squamous cell carcinomas had a proportion of cells with elevated fluorescence intensities (from 10 to 35% of the population) suggesting the presence of cells with greatly elevated thiol levels. These findings underscore the complexity of quantitating intracellular GSH levels from tumor biopsies. The combined use of MCB with flow cytometry and conventional GSH assays may help to delineate subpopulations of cells within tumors with different thiol levels.     

Depletion of tumour versus normal tissue glutathione by buthionine sulfoximine

We have investigated in detail the effects of buthionine sulfoximine (BSO), a selective glutathione (GSH) depleting agent, on the GSH contents of a number of normal tissues and three experimental tumours in mice. Significant variations in the rate and degree of GSH depletion and recovery were observed among the normal tissues. Following a dose of 2.5 mmol kg-1 BSO, GSH nadirs were reached by approximately 5 h for the liver and kidney, 8 h for the lung and bone marrow, 12 h for red blood cells (RBCs) and by 24 h for the heart. The degree of depletion was greatest for the kidney (80%), liver (74%) and bone marrow (83%), intermediate for the heart (54%) and lung (40%), and least for RBCs (13%). Recovery of GSH content was fastest for the liver followed in descending order by the kidney, the lung, the bone marrow, RBCs and the heart. In contrast, the rate and extent of GSH depletion and recovery showed considerably less variation among the 3 murine tumours. In the tumours GSH nadirs were reached by 10-12 h. The extent of depletion was about 55-65%. Recovery of GSH levels in the tumours required 48 h or more, a longer period than required by the liver, kidney and lung but shorter than that needed for the bone marrow, heart and RBCs. Attempts to preferentially deplete tumour GSH by exploiting the differences in recovery rates between normal tissues and tumours were only partially successful. Multiple BSO dosing at 16 h intervals allowed the liver to recover between doses, but the recovery in the kidney, lung and bone marrow was only partial and no recovery was seen in the heart. Finally, dose-depletion relationship investigations showed that, with the exception of the lungs, GSH depletion could be achieved in tumours with doses of BSO lower than those required for normal tissues.     

荷人鼻咽癌裸鼠骨髓细胞DNA合成及凋亡相关基因表达的生物节律的初步研究

背景与目的：有关鼻咽癌的生物节律特征尚未见文献报道,本文拟探索荷人鼻咽癌裸鼠骨髓细胞的DNA合成及几种凋亡相关基因表达的生物节律,为临床制定鼻咽癌时辰化疗方案提供必要的参数。方法：BALB/C裸小鼠69只,置于程控的独立光照系统（12h光照,12h黑暗）同步化至少3周。然后双侧腋窝皮下接种人鼻咽低分化鳞癌细胞（CNE-2）裸小鼠移植瘤,成瘤后按6个不同时间点分批处死小鼠,收集骨髓细胞,固定,染色后流式细胞仪测DNA含量,用ANOVA法检验各期细胞在6个时间点差异的显著性,Cosinor法考察G1、S、G2/M期细胞在24h的分布是否符合余弦函数,部分骨髓细胞经裂解,蛋白定量后用Western Blot法检测不同时间点p53,p21和Bcl-2三种基因蛋白表达的变化情况。结论：G1、S、G2/M期细胞在3、7、11、15、19、23HALO（Hours After Light Onset即灯亮后第3、7、11、19、23h)的分布随时间变化有显著性差异,G1、G2/M期细胞24h变化符合余弦节律,高峰值分别位于10.8HALO和1.8HALO,骨髓细胞p53和Bcl-2蛋白表达24h强度不同,p53在11HALO最强,在7HALO最弱,Bcl-2在15HALO最强,在19HALO最弱,p21蛋白未见表达。结论：荷人鼻咽癌裸小鼠骨髓细胞DNA合成随昼夜交替呈节律变化。p53和Bcl-2蛋白表达随昼夜时间变化而规律性波动。     

Eradication of Helicobacter pylori restores glutathione S-transferase activity and glutathione levels in antral mucosa.

Glutathione S-transferases (GST) and glutathione peroxidases (GPO) are important in detoxification. GST activity in the mucosa of the gastrointestinal tract is inversely correlated with the development of gastrointestinal cancer. Helicobacter pylori (H. pylori) infection has been associated with gastric cancer. We studied GST activity and the substrate glutathione (GSH) in patients with H. pylori-associated gastritis. GST activity and isoenzyme levels, GPO activity and GSH levels were studied in antral biopsies of 38 H. pylori-positive patients, before and after eradication treatment. In 31 patients in whom H. pylori was successfully eradicated, antral GST enzyme activity before therapy was 532 (465 - 598) nmol / mg protein. min (mean and 95% confidence interval) and that after therapy was 759 (682 - 836) nmol / mg protein. min (P < 0.0001). Correspondingly, levels of GST alpha and GST-P1 were higher after eradication (P < 0.001). GSH concentration significantly increased: 21.2 (16.2 - 26.2) nmol / mg protein before and 27.1 (23.6 - 30.6) nmol / mg protein after therapy (P < 0.05). In 7 patients in whom H. pylori was not eradicated, GST activity was 671 (520 - 823) nmol / mg protein. min and 599 (348 - 850) nmol / mg protein before and after treatment respectively (P = 0.32). GSH levels were 17.4 (9.0 - 25.7) nmol / mg protein and 18.2 (9.1 - 27.3) nmol / mg protein, respectively (P = 0.84). No differences in antral GPO enzyme activity, both of selenium (Se)-dependent and total GPO, before and after successful treatment were found. Eradication of H. pylori infection increases GST activity and GSH levels in antral mucosa. Low GST activity and GSH concentration due to H. pylori infection might play a role in gastric carcinogenesis.     

Eradication of Helicobacter pylori Restores Glutathione S-Transferase Activity and Glutathione Levels in Antral Mucosa

Glutathione S-transferases (GST) and glutathione peroxidases (GPO) are important in detoxification. GST activity in the mucosa of the gastrointestinal tract is inversely correlated with the development of gastrointestinal cancer. Helicobacter pylori (H. pylori) infection has been associated with gastric cancer. We studied GST activity and the substrate glutathione (GSH) in patients with H. pylori-associated gastritis. GST activity and isoenzyme levels, GPO activity and GSH levels were studied in antral biopsies of 38 H. /pyfori-positive patients, before and after eradication treatment. In 31 patients in whom H. pylori was successfully eradicated, antral GST enzyme activity before therapy was 532 (465–598) nmol/mg protein-min (mean and 95% confidence interval) and that after therapy was 759 (682–836) nmol/mg protein-min ( P <0.0001). Correspondingly, levels of GST α and GST-P1 were higher after eradication ( P <0.001). GSH concentration significantly increased: 21.2 (16.2–26.2) nmol/mg protein before and 27.1 (23.6–30.6) nmol/mg protein after therapy ( P <0.05). In 7 patients in whom H. pylori was not eradicated, GST activity was 671 (520–823) nmol/mg protein min and 599 (348–850) nmol/mg protein before and after treatment respectively ( P =0.32). GSH levels were 17.4 (9.0–25.7) nmol/mg protein and 18.2 (9.1–27.3) nmol/mg protein, respectively ( P =0.84). No differences in antral GPO enzyme activity, both of selenium (Se)-dependent and total GPO, before and after successful treatment were found. Eradication of H. pylori infection increases GST activity and GSH levels in antral mucosa. Low GST activity and GSH concentration due to H. pylori infection might play a role in gastric carcinogenesis.     

Heterogeneity of glutathione content in human ovarian cancer

Intracellular glutathione (GSH) has been shown to be one of the major factors modulating tumor response to a variety of commonly used anti-neoplastic agents. In this study the GSH contents of human ovarian tumors from primary biopsies, nude mouse xenografts, and in vitro cell cultures were compared. Pronounced intratumor cell-to-cell heterogeneity in GSH content was observed in primary patient biopsies when assessed using flow cytometry. For example, in an ascites biopsy from a newly diagnosed patient, a 5.6-fold difference in GSH concentration existed between the cell subpopulations with the 5% highest and 5% lowest GSH contents. Similar intratumor heterogeneity in GSH content was also evident in nude mouse xenografts. In addition, for a particular tumor line, the intertumor variations of GSH content among individual whole tumors were much less than the intratumor variation among slices from an individual tumor. Nude mouse xenografts of human ovarian cancer had GSH contents that were on average only slightly lower (30%) than those found in primary biopsies. In contrast, tumor cells grown as in vitro cultures, particularly those in exponential growth phase, had GSH contents considerably greater (1.3- to 3.5-fold) than those found in situ. Plateau phase cultures, however, had lower GSH contents and were more comparable to those observed in tumors in vivo. Overall, it may be concluded that in situations where GSH plays an important part in determining tumor response to a particular treatment, nude mouse xenografts may represent the most appropriate experimental model system.     

Inhibition of the protective effect of cyclophosphamide by pre-treatment with buthionine sulfoximine

Low dose cyclophosphamide (CTX) is protective against a subsequent challenge with a lethal dose of the same drug administered 5 days later. At the time of maximal protection, elevation of glutathione (GSH) and glutathione transferase (GST) levels are detectable in the bone marrow of pre-treated animals. Elevation of GSH levels in the bone marrow was inhibited with the use of D,L-buthionine-S,R-sulfoximine (BSO), and this resulted in loss of the protective effect of CTX pre-treatment. In contrast, the overshoot in GST levels observed in these animals was not affected by BSO therapy. Bone marrow GSH levels in animals treated with BSO alone were minimally depleted (68% of control); whereas, animals pre-treated with CTX followed by BSO exhibited a greater reduction in GSH levels (47% of control). These results suggest that GSH is important in the protective effect afforded by low dose CTX pre-treatment and that the elevation of GSH levels observed is the result of a rebound synthetic process. In CTX pre-treated animals, BSO treatment resulted in greater than predicted depletion in GSH levels, and, therefore, caution is recommended with the potential use of combinations of BSO and cytotoxic drugs in the presence of a regenerating bone marrow.     

Habitual consumption of fruits and vegetables: associations with human rectal glutathione S-transferase

The glutathione (GSH)/glutathione S-transferase (GST) system is an important detoxification system in the gastrointestinal tract. A high activity of this system may benefit cancer prevention. The aim of the study was to assess whether habitual consumption of fruits and vegetables, especially citrus fruits and brassica and allium vegetables, is positively associated with parameters reflecting the activity of the GSH/GST enzyme system in human rectal mucosa. GST enzyme activity, GST isoenzyme levels of GST-alpha (A1-1, A1-2 and A2-2), -mu (M1-1) and -pi (P1-1), and GSH levels were measured in rectal biopsies from 94 subjects. Diet, lifestyle, GSTM1 and GSTT1 null polymorphisms were assessed. Mean GST enzyme activity was 237 nmol/min/mg protein (SD = 79). Consumption of citrus fruits was positively associated with GST enzyme activity [difference between high and low consumption: 28.9 (95% confidence interval (CI) = 9.3-48.6) nmol/min/mg protein], but was not associated with the other parameters. A positive association with brassica vegetables was found among carriers of the GSTM1-plus genotype [difference between high and low consumption: 22.6 (95% CI = 0.2-45.0) nmol/min/mg protein], but not among GSTM1-null individuals (-25.8 nmol/min/mg protein, 95% CI = -63.3-11.8). This is in line with a positive association between consumption of brassica vegetables and GSTM isoenzyme level [difference between high and low consumption: 67.5%, 95% CI = (6.8-162.7)]. Consumption of allium vegetables was not associated with GST enzyme activity, but negatively with GSTP1-1 levels [difference between high and low consumption: -23.3%, 95% CI = (-35.5; -8.6)]. Associations were similar among those with the GSTT1-plus and GSTT1-null genotype. In conclusion, variations in habitual consumption of fruits, particularly citrus fruits, and of vegetables, in particular brassica vegetables, among those with the GSTM1-plus genotype, may contribute to variations in human rectal GST enzyme activity.     

The effect of hOGG1 and glutathione peroxidase I genotypes and 3p chromosomal loss on 8-hydroxydeoxyguanosine levels in lung cancer

Polymorphic genes for the peroxide scavenger glutathione peroxidase I (GPX1) and 8-hydroxydeoxyguanosine (8-OHdG) DNA glycosylase/apurinic (AP) lyase (hOGG1) map to loci on chromosome 3p which are subject to frequent loss of heterozygosity (LOH) in lung tumours. Levels of the pro-mutagenic, oxidative DNA lesion 8-OHdG, were measured in 37 paired normal and tumorous lung specimens using HPLC with electrochemical detection. Lung tumours were also analysed for 3p LOH by fluorescent PCR with Genescan analysis. No significant difference was observed between 8-OHdG levels in tumour [7.7 +/- 6.7 (mean +/- SE) 8-OHdG/10(6) 2'-deoxyguanosine (dG)] and normal (8.1 +/- 8.8 8-OHdG/10(6) dG) lung tissue. Adduct levels in normal lung tissue DNA were not associated with constitutive hOGG1 genotype although there was a trend towards lower 8-OHdG levels in individuals possessing the ALA6 GPX1 polymorphism. Lung tumours exhibiting 3p LOH (40%) contained higher levels of 8-OHdG adducts (10.9 +/- 2.6 8-OHdG/10(6) dG) (P = 0.05) and lower GPX1 enzyme activity [45.5 nmol glutathione (GSH)/min/mg] (P = 0.09) when compared with tumours without LOH at these sites (5.55 +/- 0.87 8-OHdG/10(6) dG and 63.6 nmol GSH/min/mg, respectively). In conclusion, tumours with 3p LOH at loci associated with hOGG1 and GPX1 appear to have compromised oxidative defence mechanisms as measured by reduced GPX1 enzyme activity and elevated 8-OHdG levels and this may affect the prognosis of lung cancer patients.     

谷胱甘肽还原酶循环法测定血浆谷胱甘肽含量在肿瘤化疗中的意义

肿瘤细胞内谷胱甘肽增加与含铂类、烷化剂及含醌类等抗癌药物的耐药性有关.本文用谷胱甘肽还原酶循环法(Glutathione Reductase Recycling Assay)测定40例正常人,20例化疗前恶性肿瘤病人及20例以顺铂、环磷酰胺及阿霉素为主的化疗病人的血浆谷胱甘肽含量.化疗后病人血浆谷胱甘肽含量比正常人及化疗前者明显增高(P<0.01),而化疗前病人与正常人无显著差异(P>0.05).提示血浆谷胱甘肽增高与长期运用顺铂、环磷酰胺及阿霉素等药有关.5例临床产生耐药者血浆谷胱甘肽明显高于化疗组平均值(P<0.01),故可能与临床耐药有一定相关性.     

Modulation by glutathione of DNA strand breaks induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its aldehyde metabolites in rat hepatocytes.

Activation of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) produced methylating species and two aldehydes: formaldehyde and 4-oxo-4-(3-pyridyl)-butanal (OPB). We investigated the modulation by glutathione of single-strand breaks (SSB) generated by N-methyl-N-nitrosourea (MNU) and the two aldehydes. Hepatocytes were simultaneously exposed to 0.2 mM MNU and to 0-2.00 mM formaldehyde or OPB for 4 h. Both aldehydes induced SSB in a dose-dependent manner. Formaldehyde and OPB exerted a synergistic effect on the formation of DNA SSB by MNU. It is postulated that both aldehydes interfere with DNA repair processes and thus increase the genotoxic effect of DNA methylating species. We investigated whether glutathione (GSH) could protect DNA from NNK-derived intermediates. Formaldehyde (2 mM) and OPB (2 mM) decreased intracellular GSH contents to 60 and 86% of control respectively. DL-Buthionine-[S,R]-sulfoximine (BSO) treatment reduced the GSH contents of hepatocytes to 19% of control but did not reduce the content of cytochrome P450 nor the metabolism of NNK. The frequency of DNA SSB induced by NNK, formaldehyde or OPB was significantly higher in GSH-depleted hepatocytes. GSH repletion with GSH monoethyl ester returned NNK-induced SSB to its initial frequency. OPB but not NNK nor formaldehyde induced double-strand breaks. We conclude that OPB and formaldehyde inhibit the repair of DNA damage induced by methylating species and that GSH reduces the level of DNA damage induced by NNK-derived reactive metabolites.     

Pharmacokinetics of buthionine sulfoximine (NSC 326231) and its effect on melphalan-induced toxicity in mice

Intravenous doses of buthionine sulfoximine (BSO, NSC 326231), an inhibitor of glutathione synthesis, were eliminated rapidly from mouse plasma in a biexponential manner. The initial phase of the plasma concentration versus time curve had a half-life of 4.9 min and accounted for 94% of the total area under the curve. The half-life of the terminal phase of the curve was 36.7 min and the area accounted for only 6% of the total area under the curve. Plasma clearance of BSO was 28.1 ml/min/kg and the steady state volume of distribution was 280 ml/kg. The oral bioavailability of BSO, based on plasma BSO levels, was extremely low. However, comparable glutathione depletion was apparent after i.v. and p.o. doses of BSO, suggesting a rapid tissue uptake and/or metabolism of BSO. Therefore, due to the rapid elimination of BSO from mouse plasma, plasma drug levels do not directly correlate with BSO-induced tissue glutathione depletion. Administration of multiple i.v. doses of BSO to male and female mice resulted in a marked 88% depletion of liver glutathione at doses of 400-1600 mg/kg/dose. Toxicity of i.v. administered BSO was limited to a transient depression of peripheral WBC levels in female mice given six doses of 1600 mg/kg. Multiple i.v. doses of BSO of up to 800 mg/kg/dose (every 4 h for a total of six doses) did not alter the toxicity of i.v. administered melphalan. However, multiple doses of 1600 mg/kg/dose of BSO did potentiate histopathological evidence of melphalan-induced bone marrow toxicity in 30% of the mice and, additionally, the combination of BSO and melphalan produced renal tubular necrosis in 80% of the male mice. The potentiation of melphalan induced toxicity did not appear to be related to GSH depletion, since: quantitatively similar amount of GSH depletion occurred at lower dose of BSO without any increase in melphalan toxicity.     

Diminished bone marrow responsiveness to erythropoietin in myelophthisic anemia.

The purpose of this study was to examine a mechanism which might explain the diminished erythropoiesis that occurs when tumor cells invade the bone marrow (myelophthisic anemia). To this end we compared 21 patients with neoplastic bone marrow invasion with seven normal subjects. When erythropoietin was added to bone marrow cell cultures from the normal subjects, the baseline rate of heme synthesis for a specific number of marrow erythroid cells increased 65%. In contrast, in patients with greater than 60% of the marrow replaced by tumor, the marrow cell culture response to erythropoietin was 14% above baseline. A significant inverse correlation was noted between the degree of marrow infiltration with tumor and the stimulation of heme synthesis with erythropoietin. It is proposed that marrow invasion with tumor may result in decreased marrow response to erythropoietin, and hence, diminished red cell production.     

Prevention of doxorubicin myocardial toxicity in mice by reduced glutathione

The effect of reduced glutathione (GSH) on acute myocardial toxicity due to doxorubicin (DXR) was investigated. At 20 mg/kg i.p. DXR was 100% lethal to BALB/c X DBA/2 F1 mice. At 500 or 1000 mg/kg i.p. GSH significantly decreased the lethality (P less than 0.01). Electron micrographs of the myocardium from DXR-treated mice showed narrowing of myofibrils, edematous cytoplasm, and mitochondrial swelling which were detectable at day 2, was strongest at day 14, but had disappeared by day 56. Light microscopic examination revealed that intercellular spaces between myocardial cells were widened at day 56, indicating shrinking of myocardial cells. These changes were significantly decreased by treatment with GSH (500 mg/kg i.p.). Treatment with DXR (14 mg/kg) significantly decreased myocardial non-protein sulfhydryl content (P less than 0.02) and administration of GSH (500 mg/kg) prevented the drop of non-protein sulfhydryl levels due to DXR treatment. Thus it was considered that administration of exogenous GSH contributes to prevention of the acute myocardial toxicity of DXR by increasing extracellular GSH levels and intracellular GSH synthesis, which detoxifies DXR-oxygen metabolites. The administration of GSH did not interfere with the antineoplastic effect of DXR against L1210 mouse leukemia.     

Altered mouse bone marrow glutathione and glutathione transferase levels in response to cytotoxins

The mouse bone marrow has been used as a model for the investigation of the response of cells to cytotoxins and carcinogens. The effects of cyclophosphamide, 1-beta-D-arabinofuranosylcytosine, and X-irradiation on the levels of glutathione and glutathione transferases have been studied. A high dose of cyclophosphamide (500 mg/kg) caused a significant depletion of glutathione levels in marrow, liver, and blood. A lower dose, 75 mg/kg, caused a similar depletion but only in marrow and liver. In this case, 5 to 7 days following treatment, the glutathione content of surviving cells was 1.8- to 3-fold higher than in controls. Glutathione transferase activity was also increased at this time (2- to 3-fold). 1-beta-D-Arabinofuranosylcytosine and X-irradiation also caused a depletion of marrow glutathione and glutathione transferase levels followed increased cellular levels (approximately 2-fold) 3 to 4 days later. Animals given cyclophosphamide (75 mg/kg) survived an otherwise lethal dose of this compound administered 5 to 7 days later. The time course of this effect closely paralleled the higher glutathione and glutathione transferase levels, suggesting a correlation between these effects.     

Interleukin 1 alpha stimulates hemopoiesis but not tumor cell proliferation and protects mice from lethal total body irradiation

Interleukin 1 alpha (IL-1) is a polypeptide/glycoprotein growth factor with multiple functions including the modulation of hematopoietic cell proliferation and differentiation. In vivo studies were performed with C57BL/6J mice injected with 0, 0.2, or 2.0 micrograms of IL-1 24 hr before or after lethal total body irradiation (TBI) (9.5 Gy). More mice in the groups administered IL-1 before TBI survived (90% of the 2.0 micrograms group) than those treated 2 or 24 hr after TBI, which was still slightly superior to the uninjected group, which all died within 15 days (p = .0001). Proliferation of bone marrow granulocyte/macrophage colonies following split dose TBI was also greatest for mouse groups treated with IL-1 prior to TBI. These experiments support data from other investigators that IL-1 stimulation of BM is related to IL-1 timing with respect to TBI. Stimulation of hemopoiesis was also assessed in terms of changes in peripheral blood and BM cell numbers and cell cycle kinetics using an electronic particle counter and flow cytometric techniques. Mice injected with 2 micrograms of IL-1 showed an initial decline (at 3-6 hr) and then a selective proliferation (24-48 hr) of early and more committed progenitor cells to 125% and 200% of control values, respectively. Peripheral blood counts rose accordingly. Cells in S and G2/M phases increased over 10 hr and then declined in number. It thus appeared that some synchronization of cell cycling occurred, which might place cells in a more radioresistant phase of the cell cycle. The glutathione (GSH) content and synthesis in BM cells were measured by isocratic paired-ion high performance liquid chromatography and 35S-labelled cysteine incorporation into the GSH tripeptide. An increase in cellular GSH content and synthesis was demonstrated following IL-1 which lasted 24 hr, suggesting a possible mechanism for the radioprotection by IL-1. To determine the potential for achieving a favorable therapeutic ratio, KHT tumor-bearing mice were injected with 2.0 micrograms IL-1. No change in tumor diameters or weights or tumor cell clonogenicity between IL-1 treated or untreated animals was observed. These experiments strongly support a role for IL-1 in stimulating bone marrow to overcome the myelosuppressive effects of irradiation.     

Intracellular uptake and cytotoxic effect in vitro of doxorubicin and epirubicin in human leukemic and normal hematopoietic cells

Summary Leukemic cells from patients presenting with acute nonlymphoblastic leukemia and normal hematopoietic bone marrow cells from healthy donors for allogeneic bone marrow transplantation were incubated for 3 h with doxorubicin and epirubicin at different concentrations. The intracellular uptake at the end of the incubation was determined by photofluorometry in leukemic cells from 15 patients and in normal cells from 9 donors for bone marrow transplantation. Cytotoxicity in vitro against granulocyte/macrophage colony-forming units (CFU-GM) was determined in normal cells from 7 donors, and in vitro toxicity against leukemic cells was determined by a clonogenic technique in cells from 6 patients and by vital dye staining (DiSC) following 4 days' culture in cells from 15 patients. Epirubicin was significantly less toxic than doxorubicin to normal hematopoetic cells (72%±20% survival of cells for epirubicin vs 45%±13% for doxorubicin at a concentration of 0.2 m;P0.005). As analyzed by the DiSC assay, 0.2 m epirubicin was slightly more toxic to leukemic cells than was the same concentration of doxorubicin (47% vs 61% survival,P0.01), but the clonogenic assay revealed no difference in toxicity to leukemic cells. At a concentration of 0.2 m, the mean intracellular uptake of epirubicin in leukemic cells was 0.43±0.26 nmol/mg protein as compared with 0.33±0.14 nmol/mg protein for doxorubicin (not significant). In normal cells, the uptake of epirubicin at a concentration of 0.2 m was 0.47±0.25 nmol/mg protein as compared with 0.31±0.21 nmol/mg protein for doxorubicin (not significant). The reduced myelotoxicity observed in vitro together with the retained toxicity to leukemic cells indicates that the therapeutic index of epirubicin is better than that of doxorubicin.     

Glutathione S-Transferases in Small Intestinal Mucosa of Patients with Coeliac Disease

Patients with villous atrophy due to coeliac disease have an increased risk of developing small intestinal malignancies. Intestinal glutathione (GSH) and glutathione S‐transferases (GST) are involved in the protection against carcinogenesis. The aim of this study was to evaluate GSH content and GST enzyme activity in small intestinal mucosa of untreated coeliacs compared to controls. We evaluated GSH content and GST enzyme activity, including the levels of GST classes α, μ, π, θ in small intestinal biopsies of untreated coeliacs (flat mucosa, Marsh IHC, n=12) compared to normal subjects (n=23). Next, we evaluated GSH and GST's in coeliacs in remission (Marsh 0‐1, n=11), coeliacs with persisting villous atrophy while on a gluten‐free diet (partial villous atrophy, Marsh IIIA (n=5); subtotal villous atrophy, Marsh IIIB (n=6) and patients with infiltrative/crypt‐hyperplastic Marsh II lesions (n=4). Total GST enzyme activity and content of GSTa are markedly suppressed in Marsh IIIC lesions compared to controls (resp. 220±79 vs. 4641189 nmol/mg protein‐min (P<0.001) and 2.79±2.46 vs. 6.47±2.29 μg/mg protein (P<0.001). In coeliacs in remission these levels normalized. Total GST enzyme activity and GSTα levels are proportionately lowered according to the degree of mucosal pathology in Marsh II, IIIA and IIIB. (Spearman's σ correlation coefficient for total GST, ‐0.596, P<0.001; GSTα, ‐0.620, P<0.001). GSTμ, π and θ and GSH levels are not significantly different in the selected study groups of mucosal pathology compared to controls. Total GST enzyme activity and content of GSTα in small intestinal mucosa are significantly lower in untreated coeliac disease compared to controls. In Marsh II, IIIA and IIIB, GST enzyme activity and GSTα content are proportionally lower according to the degree of mucosal pathology. Normal values are seen in coeliacs in remission. This correlation between coeliac disease and a suppressed GSH/GST detoxification system may explain in part the carcinogenic risk in untreated coeliac disease.