Dose and time dependent apoptotic response in a human melanoma cell line exposed to accelerated boron ions at four different LET.

The aim was to investigate and compare the influence of linear energy transfer (LET), dose and time on the induction of apoptosis in a human melanoma cell line exposed to accelerated light boron ((10)B) ions and photons. Cells were exposed in vitro to doses up to 6 Gy accelerated boron ions (40, 80, 125 and 160 eV nm(-1)) and up to 12 Gy photons (0.2 eV nm(-1)). The induction of apoptosis was measured up to 9 days after irradiation using morphological characterization of apoptotic cells and bodies. In parallel, measurements of cell-cycle distribution, monitored by DNA flow cytometry, and cell survival based on the clonogenic cell survival assay, were performed. In addition, the induction and repair of DNA double-strand breaks (DSB), using pulsed-field gel electrophoresis (PFGE) were studied. Accelerated boron ions induced a significant increase in apoptosis as compared with photons at all time points studied. At 1-5 h the percentage of radiation-induced apoptotic cells increased with both dose and LET. At the later time points (24-216 h) the apoptotic response was more complex and did not increase in a strictly LET-dependent manner. The early premitotic apoptotic cells disappeared at 24 h following exposure to the highest LET (160 eV nm(-1)). A postmitotic apoptotic response was seen after release of the dose-, time- and LET-dependent G2/M accumulations. The loss of clonogenic ability was dose- and LET-dependent and the fraction of un-rejoined DSB increased with increasing LET. Despite the LET-dependent clonogenic cell killing, it was not possible to measure quantitatively a LET-dependent apoptotic response. This was due to the different time course of appearance and disappearance of apoptotic cells.

Low and high LET radiation-induced apoptosis in M059J and M059K cells.

PURPOSE: To investigate and compare the ability of DNA-dependent protein kinase (DNA-PK)-deficient and -proficient cells to undergo apoptosis after exposure to low and high linear energy transfer (LET) radiation. MATERIALS AND METHODS: A human glioma cell line M059J lacking the catalytic subunit of DNA-PK (DNA-PKcs) and its DNA-PKcs-proficient counterpart, M059K, were exposed to 1 and 4 Gy of accelerated nitrogen ions (14N, 140 eV nm(-1), 8-12 Gy min(-1)) or 60Co gamma-rays (0.2 eV nm(-1), 0.7 Gy min(-1)). The induction of apoptosis was studied up to 144 h post-irradiation using two different methods: morphological characterization of apoptotic cells after fluorescent staining and cell size distribution analysis to detect apoptotic bodies. In parallel, protein expression of DNA-PKcs and poly(ADP-ribose) polymerase (PARP) as well as DNA-PK and caspase-3 activity were investigated. RESULTS: Low and high LET radiations (4 Gy) induced a time-dependent apoptotic response in both cell lines. Low LET radiation induced a significantly elevated apoptotic response in M059J as compared with M059K cells at 144 h post-irradiation. Following high LET radiation exposure, there was no difference between the cell lines at this time. PARP cleavage was detected in M059J cells following both low and high LET irradiation, while only high LET radiation induced PARP cleavage in M059K cells. These cleavages occurred in the absence of caspase-3 activation. CONCLUSIONS: M059J and M059K cells both display radiation-induced apoptosis, which occur independently of caspase-3 activation. The apoptotic course differs between the two cell lines and is dependent on the quality of radiation.

Relative biological effectiveness of boron ions on human melanoma cells.

PURPOSE: To compare the difference in relative biological effectiveness (RBE) between (10)B ions and a (60)Co gamma-ray beam for human melanoma cells using in vitro cell survival based on a clonogenic assay. MATERIALS AND METHODS: Cells were irradiated in vitro under aerobic conditions with (60)Co and (10)B ions with different linear energy transfer (LET) (40, 80 and 160 eV nm(-1)). The dose to the cells was determined using ferrous sulphate dosimetry and an ionisation chamber. The standard linear-quadratic model and the newly proposed repairable conditionally repairable damage (RCR) model were used to calculate the RBE. RESULTS: The RBE at 10% cell survival for 40, 80 and 160 eV nm(-1) boron ions compared with (60)Co were 1.98 (1.83-2.22), 2.85 (2.64-3.11) and 3.37 (3.17-3.58), respectively, of almost independence of the model used in the calculation. CONCLUSIONS: Different cell survival models may generate different RBE, especially at low doses and high cell survival levels.

High-LET radiation induces apoptosis in lymphoblastoid cell lines derived from atazia-telangiectasia patients.

PURPOSE: To investigate and compare the propensity of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL), derived from ataxia-telangiectasia (A-T) patients and from unaffected healthy individuals (controls), to undergo apoptosis after exposure to high-linear energy transfer (LET) radiation. MATERIALS AND METHODS: Four A-T (ARO, BMA, CSA and RJO) and two control (JAC and KKB3) LCL were exposed to doses of up to 4Gy of accelerated nitrogen ions (32-45 MeV/u, 8-12Gy/min). For comparative purposes X-ray irradiation (1.36 Gy/min) was also performed. The induction of apoptosis was studied 0-48 h after irradiation with the use of two methods: (1) monitoring of high molecular weight (HMW) DNA fragments by field inversion pulse gel electrophoresis (FIGE); and (2) morphological characterization ofapoptotic cells after fluorescent staining. In parallel, cell-cycle distribution, monitored by DNA flow cytometry, as well as measurements of p53/p21(WAF1) protein levels by Western blots, were investigated in these cells. RESULTS: High-LET radiation-induced apoptosis and G2/M-arrest in both A-T and control LCL. No significant increase in the amount of p53/p21(WAF1) proteins preceded apoptosis in control or in A-T LCL after high-LET irradiation. However, low-LET radiation did induce significant enhanced levels of p53 proteins in control but not in A-T LCL. CONCLUSIONS: LCL from both A-T homozygous and unaffected healthy individuals undergo apoptosis without accumulation of p53/p21(WAF1) proteins after exposure to high-LET radiation. In contrast, low-LET radiation induces apoptosis and significantly increases levels of p53 protein in control but not in A-T LCL.

Influence of dose-rate, post-irradiation incubation time and growth factors on interphase cell death by apoptosis and clonogenic survival of human peripheral lymphocytes.

PURPOSE: To investigate the effects of dose-rate, post-irradiation incubation time and growth factors on radiation-induced interphase cell death by apoptosis and reproductive cell death in human peripheral lymphocytes. MATERIALS AND METHODS: Lymphocytes in G0-phase were exposed in vitro to 1-3Gy 137Cs gamma-radiation at a high- (HDR, 45Gy/h) or a low dose-rate (LDR, 0.024Gy/h). HDR exposures were performed either on day 1 (HDR1) simultaneously with the start of the 3 Gy LDR exposure, or on day 6 (HDR6) when the LDR exposures ended. Apoptosis was studied at different times after irradiation by measuring (1) cellular membrane integrity, (2) morphological changes and (3) cell size reduction. Clonogenic survival was analysed for cells plated directly after irradiation (LDR, HDR6, HDR1 day 1) or after 5 days post-irradiation incubation (HDR1 day 6). RESULTS: A significant decrease in reproductive cell death was observed after 3 Gy LDR exposure as compared with the HDR1 exposure for cells plated day 6. For the lower doses applied, the dose-rate effect could not be statistically verified. A decrease in apoptosis for all three doses applied (i.e. 1, 2 and 3Gy) was observed when the LDR exposures were compared with the HDRI analysed day 6, although not of statistical significance. Radiation-induced apoptosis was efficiently counteracted by growth factors up to 24-48 h after 3 Gy HDR exposure. The prevention of radiation-induced cell death by growth factors was dependent on dose and post-irradiation time in G0. When the growth factors were added after a prolonged post-irradiation incubation in G0 (HDR 1 cells plated day 6), a significant increase in reproductive cell death was found (3 Gy) as compared with HDR protocols where the growth factors were added directly after irradiation (HDR 1 plated day 1 and HDR6). CONCLUSIONS: A dose-rate effect on radiation-induced apoptosis was indicated but not statistically verified. A significant dose-rate effect on reproductive cell death was observed. This dose-rate effect was, however, inverted when growth factors were added directly after the HDR irradiations.

Epstein-Barr virus-transformed lymphoblastoid cell lines of ataxia telangiectasia patients are defective in X-ray-induced apoptosis.

PURPOSE: To investigate and compare the propensity of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) obtained from unaffected healthy individuals and ataxia telangectasia (A-T) patients to undergo apoptosis after X-ray exposure. MATERIAL AND METHODS: The LCL were exposed to 1-4 Gy X-rays at a dose-rate of 1.36 Gy/min. At various post-irradiation times (0, 24, 48 and 72 h) the induction of apoptosis was analysed by: (1) monitoring the formation of high molecular weight (HMW) DNA fragments by field inversion pulse gel electrophoresis (FIGE); and (2) morphological characterization of apoptotic cells after fluorescence staining. In parallel, cell-cycle distribution, monitored by DNA flow cytometry, was investigated in these cells. RESULTS: The LCL obtained from the A-T homozygotes were resistant to undergoing radiation-induced apoptosis during the observation time used. On the contrary, LCL from unaffected healthy controls displayed significant radiation-induced chromatin fragmentation seen at 48 h and 72 h after irradiation. In these cells, radiation-induced G -arrest (24h post-irradiation) preceded chromatin cleavage. In A-T LCL, the defective G1-arrest was not followed by apoptosis. CONCLUSIONS: In spite of a defective cell-cycle control, EBV-transformed LCL of A-T patients compared with unaffected healthy controls do not undergo X-ray-induced apoptosis, at least during their first post-irradiation cell cycle.

RBE for the induction of apoptosis in human peripheral lymphocytes exposed in vitro to high-LET radiation generated by accelerated nitrogen ions.

PURPOSE: To investigate the relative biological effectiveness (RBE) of accelerated nitrogen ions (32-45 MeV/u) compared with 137Cs gamma-rays for the induction of apoptosis in G0 lymphocytes. MATERIALS AND METHODS: Human peripheral G0 lymphocytes were exposed in vitro to doses up to 3 Gy. RBE for the induction of apoptosis was studied at different times after irradiation (0, 24, 48 and 72 h) with the use of three different methods: (1) morphological characterization of apoptotic cells after fluorescence staining; (2) cell size distribution analysis of the cell population to detect apoptotic bodies; and (3) electrophoretic analysis of DNA to detect 'DNA-ladders'. RESULTS: RBE values in the range of 1.3-3.0 were obtained from the linear components of the dose response curves. The variation in RBE was primarily dependent on post-irradiation time, where the highest RBE values were obtained after 48 h. The three different techniques used for analysis of apoptosis gave similar results. The significantly increased RBE was also seen as an earlier appearance of DNA-ladders, as well as a more rapid disappearance of the total number of viable cells. CONCLUSIONS: These results show that nitrogen ions of high linear energy transfer (LET) produce RBE > 1.0 and induce a faster apoptotic response as compared with gamma-photons of low-LET radiation.

Chromosomal instability in human lymphocytes after low dose rate gamma-irradiation and delayed mitogen stimulation.

PURPOSE: To study the possibility that radiation induced chromosomal instability in human lymphocytes is promoted by a conflict between mitogen-induced growth stimulation and radiation-induced genotoxic stress. MATERIALS AND METHODS: Peripheral blood lymphocytes were exposed to low LET-irradiation at: (1) low-dose rate (LDR, 1-3 Gy, 0.024 Gy h[-1]) in order to minimize genotoxic stress; (2) high dose rate (HDR, 1-3 Gy, 45 Gy h[-1]) followed by immediate mitogen stimulation; and (3) HDR followed by a recovery period of 5 days before mitogen stimulation. Subsequent analyses included cell viability and clonogenic cell survival, chromosome aberrations at the first post-irradiation mitosis, and karyotype analysis of long term cultured cells, 11-57 days after mitogen stimulation. RESULTS: Dose (1-3 Gy) and dose rate (LDR and HDR) effects on the frequency of dicentric chromosomes at the first post-irradiation mitosis were in agreement with published data, with a pronounced dose rate effect of 2 and 3 Gy exposures. G-handed karyotypes after 11 days of growth in vitro showed increased frequencies of chromosome breaks and rearrangements in all irradiated cell cultures. Clones with complex karyotype abnormalities and increased frequencies of de novo aberrations developed in the irradiated cultures during extended growth for 22-57 days. These results show that: (1) LDR-irradiation induces chromosomal instability in primary human lymphocytes; (2) mitogen stimulation rescues HDR-irradiated cells from death at the expense of an increased level of chromosome aberrations; and (3) HDR-irradiated cells that are allowed 5 days of recovery before mitogen stimulation develop chromosomal instability during subsequent long-term proliferation. CONCLUSIONS: Neither the acute genotoxic stress of HDR-irradiation compared with LDR-irradiation, nor the hypothesized conflict between mitogen-induced growth stimulation and irradiation-induced growth arrest, seem to be critical conditions for the development of chromosomal instability in primary human T lymphocytes. Post-irradiation incubation allowing apoptotic processes to remove damaged cells does not prevent the subsequent development of chromosomal instability during long-term cell proliferation.

Delayed chromosomal instability in human T-lymphocyte clones exposed to ionizing radiation.

Recent studies have demonstrated that cells which survive alpha-particle and X-ray exposure may show chromosomal instability, i.e. they continue to develop chromosomal aberrations at an increased frequency for many division cycles after the exposure. To characterize this delayed response, we carried out repeated karyotype analyses of X-irradiated T-lymphocytes during clonal expansion in vitro. Human peripheral blood lymphocytes were obtained from a healthy donor and exposed to 3-Gy X-irradiation. Cell survival, estimated by a cell cloning assay, was 5%. Non-irradiated, control cells were studied in parallel. Monoclonal cell lines were established using the T-cell cloning procedure. G-band karyotype analyses were carried out at several intervals during expansion of the clones for up to 2 months. The irradiated clones did not differ from the control clones with regard to growth rate or cytometric DNA profile. Non-irradiated cell clones showed a normal karyotype, with < 10% of sporadic, non-clonal chromosome and chromatid breaks. In the irradiated clones, the karyotypes showed different (sub)clonal chromosome rearrangements, which developed successively during the cultivation time. In addition to these karyotypic abnormalities, > 20% of the cells in these clones had sporadic, non-clonal chromosome aberrations, and there was a tendency of increasing frequency of such aberrations by length of cultivation. Thus, two types of radiation-induced chromosomal instability were observed; (sub)clonal karyotypic abnormalities and sporadic, non-clonal chromosome aberrations. The frequency and kinetics by which these alterations occur in the progeny of X-irradiated T-cells suggest that they arise through different pathways, and argue against their causation by mutation or persistent DNA damage.

Pitfalls in the histochemical demonstration of alpha-glucan phosphorylase activity in glycogen-depleted skeletal muscle fibres.

In the present communication, an investigation is described into the reliability of histochemical methods for the demonstration of alpha-glucan phosphorylase activity in glycogen-depleted skeletal muscle fibres. Human skeletal muscles with glycogen-depleted fibres from patients with diseases of the neuromuscular system and from subjects who had suffered from malignant hyperthermia were used for the study. The location of phosphorylase activity and glycogen was demonstrated with histochemical techniques. Biochemical techniques were used to assay the activity of phosphorylase and the content of glycogen. Biochemical determinations of phosphorylase activity did frequently not reveal significant differences between glycogen-depleted and non-depleted skeletal muscle fibres. In contrast, all histochemical methods investigated, showed little or no phosphorylase activity in the glycogen depleted fibres, indicating that none of the existing histochemical methods revealed reliable staining results in these fibres. Owing to the invalid staining results of the histochemical methods for glycogen-depleted muscle fibres, it is necessary that for metabolic studies a biochemical assay for phosphorylase activity is also to be performed.

The pentose phosphate pathway in skeletal muscle under patho-physiological conditions. A combined histochemical and biochemical study.

Over the last 30 years, research into the neuromuscular apparatus, has expanded greatly. Multidisciplinary investigations have rapidly advanced our understanding both of diseases and of the basic neuromuscular mechanisms. The mode of pathological reaction of the neuromuscular apparatus is now quite well understood. The most notable aspect of the reaction of the injured neuromuscular apparatus is the remarkably stereotyped character of the resulting pathological changes as demonstrated by a wide variety of harmful causes, producing surprisingly similar effects. The findings of our combined histochemical and biochemical investigations presented in this monograph, are in complete harmony with the stereotyped character of the pathological changes. For example, it is particularly striking that many affected muscle fibres of patients with muscular dystrophies, congenital myopathies, inflammatory myopathies, metabolic myopathies, endocrine myopathies, or with diseases of the lower motor neuron, display an enhanced activity of both oxidative enzymes of the pentose phosphate pathway. Likewise, we found that experimental animals with disordered skeletal muscles, provoked by different types of agents or treatments, reveal the same marked rise in activity of GPDH and PGDH in the muscle fibres, with a positive correlation between the activity of both enzymes. Other findings of our investigations point to a positive correlation between the activity of GPDH and PGDH on the one hand and that of the non-oxidative enzymes of the pentose phosphate pathway, the enzymes TA, TK, RPI and RPE on the other hand. The rise in activity of PGDH and, in particular, of GPDH is regulated by two different mechanisms. The first represents a rapid control mechanism based on the stimulation of both oxidative enzymes of the pentose phosphate pathway by NADP+ and on their inhibition by NADPH. The other mechanism represents a long-term effect directed at the synthesis of the enzymes. It is this type of mechanism which is responsible for the rise in activity of GPDH and PGDH we observed. The findings obtained with the applied enzyme histochemical techniques clearly demonstrated that the rise in activity of both enzymes is not homogeneously distributed in the disordered skeletal muscles of man and experimental animals. For that reason, in order to obtain reliable quantitative information about enzyme activities in the muscle fibres themselves, the application of biochemical assays on a micro-scale was indispensable. The biochemical assay of enzyme activities was performed on histologically and histochemically selected dissected muscle specimens.(ABSTRACT TRUNCATED AT 400 WORDS)

The histochemical localization of reduced glutathione in skeletal muscle under different pathophysiological conditions.

The localization of reduced glutathione in skeletal muscle fibres of patients with inherited or acquired neuromuscular diseases and of subjects with no apparent disease of the neuromuscular system was studied histochemically. In healthy human skeletal muscle fibres, the level of reduced glutathione is higher in aerobic type I fibres than in anaerobic type II fibres. This finding suggests that glutathione in these healthy fibres is held in the reduced state chiefly by the activity of the decarboxylating and NADPH regenerating enzyme NADP(+)-dependent isocitrate dehydrogenase. In diseased muscle fibres, there is generally a positive relationship between the activity of the NADPH producing enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the level of reduced glutathione. This positive relationship suggests that glutathione in these diseased fibres is held in the reduced state chiefly by the activity of both enzymes of the pentose phosphate pathway.

Histochemical features of ragged-red fibres in diseased skeletal muscles.

In the present communication, the activity of 24 oxidoreductases, transferases, isomerases and hydrolases was examined histochemically in ragged-red fibres of human skeletal muscle specimens. The biopsy material was obtained from patients with neuromuscular diseases caused by an abnormally metabolic functioning of the muscle mitochondria. The granular accumulations of the ragged-red fibres were characterized by an impressive activity of all mitochondrial and most non-mitochondrial enzymes examined, whether participating in the aerobic or in the anaerobic pathways. With the exception of mitochondrial Mg2(+)-stimulated ATPase, acid phosphatase and AMP-aminohydrolase, there was no activity of the other hydrolytic enzymes studied in these regions. The strong activity of mitochondrial ATPase points to the presence of loosely coupled and/or uncoupled mitochondria. Ragged-red fibres that exhibited a diffuse and corpuscular activity of acid phosphatase, were always undergoing necrotic changes. Adsorption studies with diluted enzyme solutions demonstrated that the granular accumulations display a specific, moderate affinity for glycolytic enzymes.

[Histochemical and biochemical changes in skeletal muscles of rhabdomyolysis-sensitive racehorses following exertion. III: Elevated activity of various antioxidant enzymes]. / Histochemische und biochemische Anderungen in Skeletmuskeln von rhabdomyolyse-empfindlichen Trabrennpferden nach Grenzbelastung. III: Vermehrte Aktivität einiger antoxidanter Enzyme.

In this communication, the results of a histochemical and biochemical enzyme study on gluteus medius muscle of horses, sensitive to exertional myopathy, during attacks of rhabdomyolysis are presented. For the biochemical study the biopsy specimens investigated were selected by means of histological and enzyme histochemical staining methods. Dissected specimens were used which contained groups of muscle fibres with a high or low activity of glucose-6-phosphate dehydrogenase. The activity of glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, glutathione reductase, glutathione peroxidase, superoxide dismutase, and catalase was measured microbiochemically in these dissected specimens. A rise in activity of glucose-6-phosphate dehydrogenase in pathologically changed muscle fibres was always found to be coupled with a significant rise in activity of phosphogluconate dehydrogenase, glutathione reductase, and glutathione peroxidase. In these muscle fibres, the activity of superoxide dismutase and catalase was not significantly increased. On the basis of the combined histochemical and biochemical findings it is concluded that the application of the histochemical method for the demonstration of glucose-6-phosphate dehydrogenase activity can be highly recommended for the study of antioxidant enzymes in skeletal muscles with neuromuscular defects.

Elevated activity of several antioxidant enzymes in neuromuscular diseases. A histochemical and biochemical study.

In the present study the activity of glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase was measured in dissected specimens from muscle biopsies of patients with various neuromuscular diseases and from controls. The biopsy specimens investigated were selected by means of histological and enzyme histochemical staining methods. Specimens were used which contained muscle fibres with a high or low activity of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase and which were free from inflammatory infiltrates. A rise in activity of glucose-6-phosphate dehydrogenase in pathologically changed muscle fibres was always found to be coupled with a significant rise in activity of phosphogluconate dehydrogenase, glutathione peroxidase and glutathione reductase. In these muscle fibres the activity of superoxide dismutase and catalase was not significantly changed. On the basis of the histochemical and biochemical findings it is concluded that the application of the histochemical method for the demonstration of glucose-6-phosphate dehydrogenase activity can be highly recommended for the study of antioxidant enzymes in skeletal muscles with neuromuscular diseases.

[Histochemical changes in skeletal muscles of racehorses susceptible to rhabdomyolysis after exertion. I. Early myopathological changes]. / Histochemische Anderungen in Skeletmuskeln von rhabdomyolyse-empfindlichen Trabrennpferden nach Grenzbelastung. I. Frühzeitige myopathologische Anderungen.

In this communication, the results of an enzyme histochemical study on m. gluteus medius of horses, sensitive to exertional myopathy, during attacks of rhabdomyolysis are presented. The activity and location of about 25 enzymes were examined. In the present report, the early metabolic changes are discussed. Within 6 min after an attack, some large rounded fibres (approximately 2%) were seen, which showed an intense red staining in the haematoxylin and eosin sections. These hypercontracted fibres showed an increase in activity of mitochondrial adenosine triphosphatase, indicating the presence of uncoupling and/or loose coupling of the mitochondria. This finding may point to a deficient production of ATP in the m. gluteus medius of horses sensitive for exertional myopathy and this deficiency may lead to pathological alterations in the skeletal muscles. The pathological fibres revealed a changed activity of other mitochondrial enzymes as well.

[Histochemical changes in skeletal muscles of racehorses susceptible to rhabdomyolysis after exertion. II. Later myopathological and regeneration phenomena]. / Histochemische Anderungen in Skeletmuskeln von rhabdomyolse-empfindlichen Trabrennpferden nach Grenzbelastung. II. Spätere myopathologische und Regenerationserscheinungen.

Needle biopsies from m. gluteus medius of 22 horses which had suffered from repeated attacks of exertional myopathy were studied at various times after an attack, to determine if metabolic alterations can be demonstrated by enzyme histochemistry. Morphological changes and activity of 25 enzymes were studied. Immediately after onset of an attack, some large rounded fibres with a defect of the oxidative phosphorylation were seen. After some hours these fibres lost their glycolytic enzyme activity, followed by disappearance of mitochondrial enzyme activity with accumulation of Ca2+-containing substances. After 16 h inflammatory cells were found in and around necrotic fibres with a strong activity of acid phosphatase and of the 2 oxidative enzymes of the pentose phosphate pathway. The 4th d after onset of the myopathy regenerating fibres could be observed with a strong activity of both NADPH-producing enzymes of the pentose phosphate pathway. The activity of the decarboxylating enzymes NADP+-malate dehydrogenase and NADP+-isocitrate dehydrogenase was increased in these fibres as well. After some month the studied skeletal muscles were completely normal again. Metabolic interpretations based on the histochemical findings are discussed and compared with those given in literature.