Chemotherapy and signaling: How can targeted therapies supercharge cytotoxic agents?

In recent years, oncologists have begun to conclude that chemotherapy has reached a plateau of efficacy as a primary treatment modality, even if toxicity can be effectively controlled. Emerging specific inhibitors of signaling and metabolic pathways (i.e., targeted agents) contrast with traditional chemotherapy drugs in that the latter primarily interfere with the DNA biosynthesis and the cell replication machinery. In an attempt to improve on the efficacy, combination of targeted drugs with conventional chemotherapeutics has become a routine way of testing multiple new agents in early phase clinical trials. This review discusses the recent advances including integrative systematic biology and RNAi approaches to counteract the chemotherapy resistance and to buttress the selectivity, efficacy and personalization of anti-cancer drug therapy.

Hepatic epigenetic phenotype predetermines individual susceptibility to hepatic steatosis in mice fed a lipogenic methyl-deficient diet.

BACKGROUND/AIMS: The importance of epigenetic changes in etiology and pathogenesis of disease has been increasingly recognized. However, the role of epigenetic alterations in the genesis of hepatic steatosis and cause of individual susceptibilities to this pathological state are largely unknown. METHODS: Male inbred C57BL/6J and DBA/2J mice were fed a lipogenic methyl-deficient diet (MDD) that causes liver injury similar to human non-alcoholic steatohepatitis (NASH) for 6, 12, or 18 weeks, and the status of global and repetitive elements cytosine methylation, histone modifications, and expression of proteins responsible for those epigenetic modifications in livers was determined. RESULTS: The development of hepatic steatosis in inbred C57BL/6J and DBA/2J mice was accompanied by prominent epigenetic abnormalities. This was evidenced by pronounced loss of genomic and repetitive sequences cytosine methylation, especially at major and minor satellites, accompanied by increased levels of repeat-associated transcripts, aberrant histone modifications, and alterations in expression of the maintenance DNA methyltransferase 1 (DNMT1) and de novo DNMT3A proteins in the livers of both mouse strains. However, the DBA/2J mice, which were characterized by an initially lower degree of methylation of repetitive elements and lower extent of histone H3 lysine 9 (H3K9) and H3 lysine 27 (H3K27) trimethylation in the normal livers, as compared to those in the C57BL/6J mice, developed more prominent NASH-specific pathomorphological changes. CONCLUSIONS: These results mechanistically link epigenetic alterations to the pathogenesis of hepatic steatosis and strongly suggest that differences in the cellular epigenetic status may be a predetermining factor to individual susceptibilities to hepatic steatosis.

Role of DNA damage and alterations in cytosine DNA methylation in rat liver carcinogenesis induced by a methyl-deficient diet.

Currently, cancer is recognized as a disease provoked by both genetic and epigenetic events. However, the significance of early genetic and epigenetic alterations with respect to carcinogenic process in general and to liver carcinogenesis in particular remains unexplored. A lack of knowledge regarding how specific alterations during early preneoplasia may be mechanistically related to tumor formation creates a major gap in understanding the role of these genetic and epigenetic abnormalities in carcinogenesis. In the present study we investigated the contribution of DNA damage and epigenetic alterations to liver carcinogenesis induced by a methyl-deficient diet. Feeding Fisher 344 rats a methyl-deficient diet for 9 weeks resulted in DNA damage and aberrant DNA methylation. This was evidenced by an early up-regulation of the base excision DNA repair genes, accumulation of 8-oxodeoxyguanosine and 3'OH-end strand breaks in DNA, pronounced global loss of DNA methylation, and hypermethylation of CpG islands in the livers of methyl-deficient rats. These abnormalities were completely restored in the livers of rats exposed to methyl-deficiency for 9 weeks after removal of the methyl-deficient diet and re-feeding a methyl-sufficient diet. However, when rats were fed a methyl-deficient diet for 18 week and then given a methyl-sufficient diet, only DNA lesions were repaired. The methyl-sufficient diet failed to restore completely the altered DNA methylation status and prevent the progression of liver carcinogenesis. These results suggest that stable alterations in DNA methylation are a factor that promotes the progression of liver carcinogenesis. Additionally, the results indicate that epigenetic changes may be more reliable markers than DNA lesions of the carcinogenic process and carcinogen exposure.

Epigenetic downregulation of the suppressor of cytokine signaling 1 (Socs1) gene is associated with the STAT3 activation and development of hepatocellular carcinoma induced by methyl-deficiency in rats.

The members of the platelet-derived growth factor (PDGF) and the transforming growth factor-beta (TGFbeta) pathways are important in the induction of liver fibrosis and cirrhosis; however, their role in the subsequent progression to hepatocellular carcinoma (HCC) remains elusive. Our study provides new insights into mechanisms of dysregulation of PDGFs, TGFbeta and signal transducer and activator of transcription (STAT) pathways in the pathogenesis of methyl-deficient rodent liver carcinogenesis, a remarkably relevant model to the development of HCC in humans. We demonstrated a progressive increase in the Pdgfs and TGFbeta expression in preneoplastic tissue and liver tumors indicating their promotional role in carcinogenesis, particularly in progression of liver fibrosis and cirrhosis. However, activation of the STAT3 occurred only in fully developed HCC and was associated with downregulation of the Socs1 gene. The inhibition of the Socs1 expression in HCC was associated with an increase in histone H3 lysine 9, H3 lysine 27, and H4 lysine 20 trimethylation at the Socs1 promoter, but not with promoter methylation. The results of our study suggest the following model of events in hepatocarcinogenesis: during early stages, overexpression of the Socs1 effectively inhibits TGFbeta- and PDGF-induced STAT3 activation, whereas, during the advanced stages of hepatocarcinogenesis, the Socs1 downregulation resulted in loss of its ability to attenuate the signal from the upregulated TGFbeta and PDGFs leading to oncogenic STAT3 activation and malignant cell transformation. This model illustrates that the Socs1 acts as classic tumor suppressor by preventing activation of the STAT3 and downregulation of Socs1 and consequent activation of STAT3 may be a crucial events leading to formation of HCC.

Induction of oxidative stress and DNA damage in rat brain by a folate/methyl-deficient diet.

The age-associated decline in cellular antioxidant defenses and resultant accumulation of DNA damage in central nervous system has been mechanistically implicated in the etiology and pathogenesis of neurodegenerative diseases. Neurons possess a high metabolic activity and are especially vulnerable to the long-term effects of continuous exposure to endogenous reactive oxygen species. It is well recognized that adequate availability of essential nutrients involved in cellular one-carbon metabolism is essential for normal brain development and function. Additionally, the synthesis of the primary low-molecular cellular antioxidant glutathione is inter-dependently linked to one-carbon metabolic pathway. Thus, any aberrant disruptions in one-carbon metabolism can result in potentially deleterious effects including cell death as a result of an imbalance in the cellular redox state. Hence, in the present study, we examined the long-term effects of a folate/methyl-deficient (FMD) diet on cellular antioxidant defenses and DNA damage in the rat brain. Feeding male Fisher 344 rats a FMD diet resulted in perturbations in the levels of one-carbon metabolites along with induction of oxidative stress and oxidative DNA damage in the brain. This was evidenced by a decrease in the reduced oxidized/glutathione ratio, imbalance of cellular antioxidant defense system; specifically, altered activity and expression of antioxidant enzymes Mn-containing superoxide dismutase (Mn-SOD), catalase, and glutathione peroxidase (GPX), increased accumulation of oxidative DNA lesions, 8-hydroxydeoxyguanosine (8-OH-dG) and DNA single-strand breaks, even in the presence of increased expression of critical DNA repair genes apurinic/apyrimidinic endonuclease 1 (Apex1) and DNA polymerase beta (Polbeta), and apoptosis in the brains of folate/methyl-deficient rats. These results indicate that chronic methyl group deficiency leads to an imbalance in cellular antioxidant defense systems, increased oxidative stress, and apoptosis. Any of these events may compromise normal central nervous system function and contribute to the development of various neurological, behavioral, and neurocognitive dysfunctions.

Mechanisms of peroxisome proliferator-induced DNA hypomethylation in rat liver.

Genomic hypomethylation is a consistent finding in both human and animal tumors and mounting experimental evidence suggests a key role for epigenetic events in tumorigenesis. Furthermore, it has been suggested that early changes in DNA methylation and histone modifications may serve as sensitive predictive markers in animal testing for carcinogenic potency of environmental agents. Alterations in metabolism of methyl donors, disturbances in activity and/or expression of DNA methyltransferases, and presence of DNA single-strand breaks could contribute to the loss of cytosine methylation during carcinogenesis; however, the precise mechanisms of genomic hypomethylation induced by chemical carcinogens remain largely unknown. This study examined the mechanism of DNA hypomethylation during hepatocarcinogenesis induced by peroxisome proliferators WY-14,643 (4-chloro-6-(2,3-xylidino)-pyrimidynylthioacetic acid) and DEHP (di-(2-ethylhexyl)phthalate), agents acting through non-genotoxic mode of action. In the liver of male Fisher 344 rats exposed to WY-14,643 (0.1% (w/w), 5 months), the level of genomic hypomethylation increased by approximately 2-fold, as compared to age-matched controls, while in the DEHP group (1.2% (w/w), 5 months) DNA methylation did not change. Global DNA hypomethylation in livers from WY-14,643 group was accompanied by the accumulation of DNA single-strand breaks, increased cell proliferation, and diminished expression of DNA methyltransferase 1, while the metabolism of methyl donors was not affected. In contrast, none of these parameters changed significantly in rats fed DEHP. Since WY-14,643 is much more potent carcinogen than DEHP, we conclude that the extent of loss of DNA methylation may be related to the carcinogenic potential of the chemical agent, and that accumulation of DNA single-strand breaks coupled to the increase in cell proliferation and altered DNA methyltransferase expression may explain genomic hypomethylation during peroxisome proliferator-induced carcinogenesis.

Genetic and epigenetic changes in rat preneoplastic liver tissue induced by 2-acetylaminofluorene.

Genotoxic carcinogens, including 2-acetylaminofluorene (2-AAF), in addition to exerting their genotoxic effects, often cause a variety of non-genotoxic alterations in cells. It is believed that these non-genotoxic effects may be indispensable events in tumorigenesis; however, there is insufficient knowledge to clarify the role of carcinogens in both the genetic and epigenetic changes in premalignant tissues and a lack of conclusive information on the link between epigenetic alterations and carcinogenic exposure. In the current study, we investigated whether or not the mechanism of 2-AAF-induced hepatocarcinogenesis consists of both genotoxic (genetic) and non-genotoxic (epigenetic) alterations. Male and female Sprague-Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 6, 12, 18 or 24 weeks. The levels of DNA adducts obtained from 2-AAF in liver and kidney tissues were assessed by high-performance liquid chromatography combined with electrospray tandem mass spectrometry (HPLC-ES-MS/MS). N-(Deoxyguanosine-8-yl)-2-aminofluorene was the major adduct detected at all time points in both tissues. Global DNA methylation in the livers and kidneys, as determined by an HpaII-based cytosine extension assay and by HPLC-ES-MS/MS, did not change over the 24-week period. In the livers of male rats, there was a progressive decrease of global and long interspersed nucleotide element-1-associated histone H4 lysine 20 trimethylation, as well as hypermethylation of the p16(INK4A) gene. These epigenetic changes were not observed in the livers of female rats or the kidneys of both sexes. Importantly, morphological evidence of formation and progression of neoplastic process was observed in the liver of male rats only. In conclusion, we have demonstrated that exposure of rats to genotoxic hepatocarcinogen 2-AAF, in addition to formation of 2-AAF-specific DNA lesions, resulted in substantial alterations in cellular epigenetic status.

Hypoxia induces oxidative stress in tissues of a goby, the rotan Perccottus glenii.

The effects of hypoxia (0.4 mg O2/L) for 2, 6 or 10 h and subsequent normoxic recovery on the levels of lipid peroxides, thiobarbituric acid reactive substances, protein carbonyls (CP), free thiols, and the activities of six antioxidant and associated enzymes were measured in the brain, liver, and skeletal muscle of the rotan Perccottus glenii. Hypoxia increased CP content in the brain (5.0-7.4-fold), liver (2.2-3.3-fold) and muscle (3.2-61-fold) relative to controls and the levels remained elevated during recovery. Lipid peroxide content rose within 2 h of hypoxia in all tissues examined with the most marked increase (8.7-fold) in the liver, but decreased again during longer hypoxic exposure except in the muscle. Levels of low-molecular mass thiols were transiently lowered after 2 h hypoxia in all tissues, but were higher compared with controls after longer hypoxic exposure and recovery. Hypoxia decreased protein thiol content in the liver and muscle that return to control levels during recovery. Experimental conditions affected enzyme activities in a different manner. Superoxide dismutase activity rose two-fold in the liver of hypoxic fish, and a similar tendency was seen in muscle glutathione-S-transferase. Activities of other enzymes were decreased or unchanged during hypoxia and elevated in some cases during normoxic recovery. Taken together, these data show that hypoxia resulted in the development of oxidative stress and a compensatory changes of antioxidant enzymes in the tissues.

Oxidative stress and antioxidant defenses in goldfish liver in response to short-term exposure to arsenite.

Arsenic is an environmental pollutant capable of causing oxidative stress, disturbance of metabolism, and cancer development. The present study was undertaken to investigate the effects of exposure to sodium arsenite on the glutathione pool, lipid peroxidation, protein carbonyl levels, global DNA methylation, and activities of six antioxidant enzymes in goldfish liver. In a preliminary experiment, 7-day exposure to 200 microM sodium arsenite, but not 10 or 100 microM, disturbed the glutathione status. A detailed investigation of oxidative stress development and antioxidant responses was further examined during different periods of exposure to 200 microM sodium arsenite. This treatment increased lipid peroxide levels after 1 and 4 days of exposure but did not affect thiobarbituric acid reactive substances and protein carbonyls. Oxidized glutathione and the oxidative stress index rose after 4 days, but de novo glutathione synthesis decreased both parameters after 7 days. Activities of the main antioxidant enzymes-superoxide dismutase, catalase, and glutathione peroxidase, were elevated after longer periods of exposure, indicating an enhanced antioxidant response. Arsenite exposure led to DNA hypomethylation, which is an early marker of disturbed epigenetic regulations. The findings suggest that goldfish livers cope with arsenic-induced oxidative stress mainly through adaptive changes in the glutathione pool and antioxidant enzymes.

Gene expression profiling reveals underlying molecular mechanisms of the early stages of tamoxifen-induced rat hepatocarcinogenesis.

Tamoxifen is a widely used anti-estrogenic drug for chemotherapy and, more recently, for the chemoprevention of breast cancer. Despite the indisputable benefits of tamoxifen in preventing the occurrence and re-occurrence of breast cancer, the use of tamoxifen has been shown to induce non-alcoholic steatohepatitis, which is a life-threatening fatty liver disease with a risk of progression to cirrhosis and hepatocellular carcinoma. In recent years, the high-throughput microarray technology for large-scale analysis of gene expression has become a powerful tool for increasing the understanding of the molecular mechanisms of carcinogenesis and for identifying new biomarkers with diagnostic and predictive values. In the present study, we used the high-throughput microarray technology to determine the gene expression profiles in the liver during early stages of tamoxifen-induced rat hepatocarcinogenesis. Female Fisher 344 rats were fed a 420 ppm tamoxifen containing diet for 12 or 24 weeks, and gene expression profiles were determined in liver of control and tamoxifen-exposed rats. The results indicate that early stages of tamoxifen-induced liver carcinogenesis are characterized by alterations in several major cellular pathways, specifically those involved in the tamoxifen metabolism, lipid metabolism, cell cycle signaling, and apoptosis/cell proliferation control. One of the most prominent changes during early stages of tamoxifen-induced hepatocarcinogenesis is dysregulation of signaling pathways in cell cycle progression from the G(1) to S phase, evidenced by the progressive and sustained increase in expression of the Pdgfc, Calb3, Ets1, and Ccnd1 genes accompanied by the elevated level of the PI3K, p-PI3K, Akt1/2, Akt3, and cyclin B, D1, and D3 proteins. The early appearance of these alterations suggests their importance in the mechanism of neoplastic cell transformation induced by tamoxifen.

Diethyldithiocarbamate injection induces transient oxidative stress in goldfish tissues.

The effects of intraperitoneal injection of diethyldithiocarbamate (DDC) on free radical processes were examined in brain, liver and kidney of goldfish (Carassius auratus). Levels of oxidatively modified lipids and proteins as well as the activities of antioxidant and associated enzymes were measured. Intraperitoneal injection of DDC at a concentration of 0.01 mg/g wet mass decreased SOD activities by about 30-50% after 48 and 168 h compared to corresponding sham-injected values. This treatment resulted in transient oxidative stress. Lipid peroxide content increased after DDC injection at all time points in the kidney, after 48 h in the liver and was elevated in most experimental groups in the brain. Thiobarbituric-acid reactive substances (end products of lipid peroxidation) rose within the first 48 h after injection, but returned to initial levels after 168 h. Two other indices of oxidative stress were also transiently modified: protein carbonyl levels in the brain and kidney increased 24h post-injection, and the low-molecular mass thiol content was reduced over the same period in all tissues examined. Activities of catalase, glutathione peroxidase, glutathione-S-transferase, glutathione reductase, and glucose-6-phosphate dehydrogenase showed differential responses to DDC treatment that rebounded by 168 h post-injection. Glutathione peroxidase activities were reduced by 60, 45 and 65% in the brain, liver and kidney, respectively, after 24h but rebounded thereafter. After 48 h post-injection with DDC significant decreases were also seen in liver and kidney catalase, GST activities in all three tissues, and kidney GR and G6PDH activities. In some cases, catalase, GST, GR and G6PDH activities transiently increased after 24 h. It was concluded that DDC injection depleted SOD and simultaneously stimulated lipid peroxidation, but did not require compensatory enhancement of other enzymatic defenses. Different actions of the superoxide anion in cellular metabolism and possible consequences of the impairment of superoxide dismutase are discussed.

Coordinated response of goldfish antioxidant defenses to environmental stress.

Different kinds of oxidative stress cause responses of antioxidant defenses which often act in concert. In previous works, some relationships have been found between oxidative stress markers and antioxidant enzyme activities in goldfish treated with different levels of oxygen or heat shock. This study aimed to check whether or not there are general patterns of relationships between antioxidant enzyme activities and oxidative stress indices in goldfish tissues, regardless of the stressor. For this, goldfish were treated with different concentrations of iron sulphate, 20 or 500 microM, as well as limestone water for 7 days. Both iron ions and limestone water led to a pH shift. Therefore, complex effects of iron ions and/or a pH shift on levels of oxidative stress indices and antioxidant enzyme activities in goldfish liver and kidney were investigated. Experimental conditions resulted in increased protein carbonyl content by 1.5-1.9-fold. Externally added iron ions did not change lipid peroxide levels in the liver but decreased them in the kidney, while levels of thiobarbituric acid reactive substances (TBARS) were elevated by 1.4-2.5-fold. Limestone water raised levels of both lipid peroxidation products in the liver. The treatment affected activities of superoxide dismutase and catalase only slightly, but activities of glutathione-associated enzymes, glutathione-S-transferase (GST), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH) were lowered in many cases. G6PDH activities correlated inversely with protein carbonyl levels (R2 = 0.77-0.97), suggesting possible inactivation of the enzymes due to their carbonylation. Levels of lipid peroxidation products had a positive correlation to activities of catalase in the liver and GR in the kidney (R2 = 0.83), indicating possible up-regulation of the enzymes by these products. A negative link between TBARS levels and GST activities may reflect the involvement of GST in the detoxification of lipid peroxide products. The main conclusions are: (i) experimental conditions resulted in increased levels of protein carbonyls and end products of lipid peroxidation (TBARS); (ii) under oxidative stress, some enzymes can be inactivated due to oxidation; (iii) lipid peroxidation products seem to be involved in up-regulation of some antioxidant enzymes.

Effects of different environmental oxygen levels on free radical processes in fish.

Changes in oxygen levels occur frequently in aquatic environments; therefore, water organisms, including fishes, evolve a wide spectrum of adaptations to both anoxia/hypoxia and hyperoxia. The review describes oxidative damage to cellular constituents by reactive oxygen species, alterations in glutathione status, and response of antioxidant enzymes to variable oxygen availability in fish. Anoxia- and hypoxia-tolerant species demonstrate an anticipatory increase of some antioxidant enzymes during low-oxygen state in order to enhance their antioxidant potential for dealing with possible oxidative stress upon return to normoxia. Under hyperoxic conditions, it seems that the glutathione system plays an important adaptive role. Most stressful conditions lead to a quick increase in lipid peroxidation products that, in turn, are detoxified rapidly by respective low- and high-molecular weight antioxidants. A scheme on possible ways of regulating antioxidant enzymes by different oxygen levels is proposed.

Temperature increase results in oxidative stress in goldfish tissues. 1. Indices of oxidative stress.

Levels of lipid peroxides (LOOH), thiobarbituric-acid reactive substances (TBARS), protein carbonyls and low- and high-molecular weight thiols were measured in brain, liver, kidney, and white muscle of goldfish, Carassius auratus L., over 1-12 h of high temperature (35 degrees C) exposure followed by 4 or 24 h of lower (21 degrees C) temperature recovery. LOOH and TBARS contents increased during heat shock exposure with a maximal rise of 20-fold for liver TBARS, but both mainly reversed at recovery. Protein carbonyl content was unaffected by heat shock but rose in brain, liver, and kidney during recovery. Low-molecular weight thiol concentrations unexpectedly increased up to approximately 4-fold in brain, kidney and muscle under heat shock and remained high during recovery. Protein thiol contents also rose in liver and muscle during high temperature exposure by 2- and 3-fold, respectively, and decreased to control values or below in all tissues at late recovery. Low- and high-molecular weight thiol levels inversely correlated in liver (R2=0.87) suggesting that the former was used to reduce the latter over the experiment. It is concluded that the redox balance in goldfish tissues is strictly maintained probably contributing to the high tolerance of this species to heat shock.

Temperature increase results in oxidative stress in goldfish tissues. 2. Antioxidant and associated enzymes.

Activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), and glucose-6-phophate dehydrogenase (G6PDH) were measured in four tissues of goldfish, Carassius auratus L., over 1-12 h of high temperature (35 degrees C) exposure followed by 4 or 24 h of lower temperature (21 degrees C) recovery. SOD activity was strongly affected by heat shock, increasing 4-fold in brain, liver, and kidney, but was mainly reversed at recovery. In some tissues, activities of SOD, catalase, GPx, and G6PDH decreased significantly after 1 h heat shock exposure suggesting that thermal inactivation possibly occurred, but were renewed at further exposure. In many cases, 4 h of return to the initial temperature decreased enzyme activities. High correlation coefficients between SOD activities and levels of lipid peroxidation products suggest that these products might be involved in up-regulation of antioxidant defense. Several enzymes (SOD, GST, GR) responded to stress in coordinated manner.

Adaptive response of antioxidant enzymes to catalase inhibition by aminotriazole in goldfish liver and kidney.

This study was undertaken to clarify the physiological role of catalase in the maintenance of pro/antioxidant balance in goldfish tissues by inhibiting the enzyme in vivo with 3-amino 1,2,4-triazole. Intraperitoneal injection of aminotriazole (0.5 mg/g wet mass) caused a decrease in liver catalase activity by 83% after 24 h that was sustained after 168 h post-injection. In kidney catalase activity was reduced by approximately 50% and 70% at the two time points, respectively. Levels of protein carbonyls were unchanged in liver but rose by 2-fold in kidney after 168 h. Levels of thiobarbituric acid-reactive substances were elevated in both tissues after 24 h but were reversed by 168 h. Glutathione peroxidase and glutathione-S-transferase activities increased in kidney after aminotriazole treatment whereas activities of glutathione peroxidase and glutathione reductase in liver decreased after 24 h but rebounded by 168 h. Liver glucose-6-phosphate dehydrogenase activity was reduced at both time points. Activities of these three enzymes in liver correlated inversely with the levels of lipid damage products (R2=0.65-0.81) suggesting that they may have been oxidatively inactivated. Glutathione-S-transferase activity also correlated inversely with catalase (R2=0.86). Hence, the response to catalase depletion involves compensatory changes in the activities of enzymes of glutathione metabolism.

Catalase inhibition by amino triazole induces oxidative stress in goldfish brain.

The effects of in vivo inhibition of catalase by 3-amino 1,2,4-triazole (AMT) on the levels of damage products resulting from reactive oxygen species attack on proteins and lipids as well as on the activities of five antioxidant and associated enzymes were studied in the brain of goldfish, Carassius auratus. Intraperitoneal injection of AMT at a concentration of 0.1 mg/g wet weight caused a gradual decrease in brain catalase activity over 72 h, whereas higher AMT concentrations (0.5 or 1.0 mg/g) reduced catalase activity by about two-thirds within 5-10 h. AMT effects on antioxidant enzyme activities and oxidative stress markers were studied in detail using fish treated with 0.5 mg/g AMT for 24 or 168 h. The levels of thiobarbituric acid-reactive substances (a lipid damage product) increased 6.5-fold by 24 h after AMT injection but fell again after 168 h. The content of carbonylproteins (CP) also rose within 24 h (by approximately 2-fold) and remained 1.5-fold higher compared with respective sham-injected fish after 168 h. CP levels correlated inversely with catalase activity (R(2) = 0.83) suggesting that catalase may protect proteins in vivo against oxidative modification. The activities of both glutathione peroxidase and glutathione-S-transferase increased by approximately 50% and 80%, respectively, in brain of AMT-treated fish and this might represent a compensatory response to lowered catalase activity. Possible functions of catalase in the maintenance of prooxidant/antioxidant balance in goldfish brain are discussed.

Hyperoxia results in transient oxidative stress and an adaptive response by antioxidant enzymes in goldfish tissues.

The effects of hyperoxia on the status of antioxidant defenses and markers of oxidative damage were evaluated in goldfish tissues. The levels of lipid peroxides, thiobarbituric acid reactive substances, carbonyl proteins and the activities of some antioxidant enzymes were measured in brain, liver, kidney and skeletal muscle of goldfish, Carassius auratus L., over a time course of 3-12 h of hyperoxia exposure followed by 12 or 36 h of normoxic recovery. Exposure to high oxygen resulted in an accumulation of protein carbonyls in tissues throughout hyperoxia and recovery whereas lipid peroxides and thiobarbituric acid reactive substances accumulated transiently under short-term hyperoxia stress (3-6 h) but were then strongly reduced. This suggests that hyperoxia stimulated an enhancement of defenses against lipid peroxidation or mechanisms for enhancing the catabolism of peroxidation products. The activities of principal antioxidant enzymes, superoxide dismutase and catalase, were not altered under hyperoxia but catalase increased during normoxic recovery; activities may rise in anticipation of further hyperoxic excursions. In most tissues, the activities of glutathione-utilizing enzymes (glutathione peroxidase, glutathione-S-transferase, glutathione reductase) as well as glucose-6-phosphate dehydrogenase, were not affected under hyperoxia but increased sharply during normoxic recovery. Correlations between some enzyme activities and oxidative stress markers were found, for example, an inverse correlation was seen between levels of thiobarbituric acid reactive substances and glutathione-S-transferase activity in liver and catalase and glucose-6-phosphate dehydrogenase in kidney. The results suggest that liver glutathione-S-transferase plays an important role in detoxifying end products of lipid peroxidation accumulated under hyperoxia stress.

Hypoxia and recovery perturb free radical processes and antioxidant potential in common carp (Cyprinus carpio) tissues.

The effects of hypoxia exposure and subsequent normoxic recovery on the levels of lipid peroxides (LOOH), thiobarbituric acid reactive substances (TBARS), carbonylproteins, total glutathione levels, and the activities of six antioxidant enzymes were measured in brain, liver, kidney and skeletal muscle of the common carp Cyprinus carpio. Hypoxia exposure (25% of normal oxygen level) for 5h generally decreased the levels of oxidative damage products, but in liver TBARS content were elevated. Hypoxia stimulated increases in the activities of catalase (by 1.7-fold) and glutathione peroxidase (GPx) (by 1.3-fold) in brain supporting the idea that anticipatory preparation takes place in order to deal with the oxidative stress that will occur during reoxygenation. In liver, only GPx activity was reduced under hypoxia and reoxygenation while other enzymes were unaffected. Kidney showed decreased activity of GPx under aerobic recovery but superoxide dismutase (SOD) and catalase responded with sharp increases in activities. Skeletal muscle showed minor changes with a reduction in GPx activity under hypoxia exposure and an increase in SOD activity under recovery. Responses by antioxidant defenses in carp organs appear to include preparatory increases during hypoxia by some antioxidant enzymes in brain but a more direct response to oxidative insult during recovery appears to trigger enzyme responses in kidney and skeletal muscle.