Zinc treatment affects superoxide dismutase activity in growth retardation

Children with growth dysfunction present complex diagnostic challenges. The purpose of this study was to determine the effects of oral zinc treatment on red cell copper/zinc superoxide dismutase (Cu/Zn-SOD) activity and copper and zinc concentrations in children with “growth retardation.” Twenty-nine patients, average age of 11 yr, whose percentile was under 3% of the National Center of Health Statistics parameters were selected. For the control group, 10 children whose average age was 10 yr were included. Red cell Cu/Zn-SOD activity was determined by spectrophotometer. Red cell copper and zinc concentrations were measured by atomic absorption spectrophotometer. Red cell Cu/Zn-SOD activity was higher than the control group before zinc treatment (p<0.001). There was a decrease in the Cu/Zn-SOD activity after zinc treatment, but the mean value of the Cu/Zn-SOD activity of patients was still higher than the control values (p<0.001). After zinc treatment, there was an increase in red cell zinc concentration (p<0.01) and a decrease in copper concentration (p<0.001), which were statistically significant. The results of this study suggested that Cu/Zn-SOD activity was increased significantly during growth retardation and zinc treatment appeared to ameliorate the enzyme activity. There were also insignificant alterations in red cell copper and zinc concentrations. Presented at the 23rd Congress of Endocrinology and Metabolic Diseases of Turkey Joint Meeting with the European Federation of Endocrine Societies, Bilkent Hotel, Ankara, 7–9 September, 2000.     

Corpuscular oxidative stress in desert sheep naturally deficient in copper

Oxidative stress arises when there is an imbalance between radical-generating and radical-scavenging activity; it may therefore cause an increase in oxidation products and cell damage. This study aimed to determine antioxidant status, lipid peroxidation, and their relation to anemia of grazing sheep deficient in copper (Cu). For this purpose, 39 male lambs of native (Balady) breed, aged 6–7 months and reared in El-Dakhla oasis (in the western Egyptian desert), were divided according to plasma Cu (pCu) concentration into three groups, marginally deficient (MD, pCu = 4–8 μmol/l, n = 12), functionally deficient (FD, pCu < 3 μmol/l, n = 12) and control (pCu > 9 μmol/l, n = 15). Jugular blood was sampled for determination of red blood cell count (RBC), packed cell volume (PCV), hemoglobin concentration (Hb), plasma ceruloplasmin activity (pCp), antioxidant activities of erythrocytic superoxide dismutase (eSOD), catalase (eCAT), glutathione peroxidase (eGSH-Px), and levels of erythrocytic malondialdehyde (eMDA, as a biomarker of lipid peroxidation). The Cu-deficient lambs were characterized by microcytic hypochronic anemia accompanied by decreased pCp, eSOD, eCAT and eGSH-Px activities and increased eMDA level when compared to the controls. The indices of anemia, pCp and eSOD were lower and eMDA was higher in FD compared to MD lambs. The enhanced eMDA was strongly correlated (P < 0.01) with the inhibited activity of pCu (r = −0.79), pCp (r = −0.65) and eSOD (r = −0.71) and to a lesser extent (P < 0.05) with eGSH-Px (r = −0.38) and eCAT (r = −0.41). In addition, eMDA was negatively correlated (P < 0.01) with RBC (r = −0.75), PCV (r = −0.69) and Hb (r = −0.72). This study suggests that Cu-deficient lambs incur an erythrocytic oxidative damage secondary to impaired oxidant defenses, which may be one of the mechanisms underlying Cu deficiency-induced anemia in grazing sheep.     

Superoxide dismutase activity and zinc and copper concentrations in growth retardation

The aim of this study was to determine the effects of oral zinc treatment on red cell copper/zinc-superoxide dismutase (Cu/Zn-SOD) activity and zinc and copper concentrations in growth retardation. Thirty-five patients, average age of 11 yr, were selected. The control group consisted of 10 healthy children whose average age was 10 yr. Superoxide dismutase activity was determined by spectrophotometer. Copper and zinc concentrations were measured by an atomic absorption spectrophotometer. The activity of Cu/Zn-SOD before zinc treatment was higher than the controls (p<0.001). There was a decrease in Cu/Zn-SOD activity after zinc treatment (p<0.001) and the values after treatment were still higher than the controls (p<0.001). Plasma zinc concentrations before zinc treatment were lower than controls (p<0.01). After treatment, there was an increase in plasma zinc concentrations compared to controls and the patients' values before zinc treatment, respectively (p<0.001, p<0.001). After zinc treatment, plasma copper concentrations were decreased significantly (p<0.01). An increase in red cell zinc concentration (p<0.01) and a decrease in copper concentration (p<0.001), which were statistically significant, were seen after zinc treatment. The results suggested that there were significant alterations in Cu/Zn-SOD activity and zinc and copper concentrations during growth retardation. With zinc treatment, these parameters appeared to approach normal values.     

Trace element concentrations and superoxide dismutase and catalase activities in benign and malignant larynx tumors

The plasma and erythrocyte levels of zinc, copper, and magnesium and the activities of red-cell copper-zinc superoxide dismutase (Cu/Zn-SOD) and catalase (CAT) were determined in patients with benign and malignant tumors of the larynx. Blood samples from patients and healthy controls were drawn using heparinized tubes. The erythrocyte Cu/Zn-SOD and CAT activities were determined spectrophotometrically and the zinc, copper, and magnesium concentrations were determined in erythrocyte and plasma by atomic absorption spectrometry. Variance analysis was employed in the statistical evaluation of the findings. There was a significant increase in red-cell Cu/Zn-SOD activity in the subjects with malignant and benign tumors compared to controls (p<0.001). The CAT activity increased only in the benign tumor group (p<0.01). The plasma zinc concentrations were significantly lower in the malignant tumor group (p<0.05) and significantly higher in the benign tumor group (p<0.01). The erythrocyte copper concentrations were significantly lower in both benign and malignant tumor groups (p<0.001). The plasma copper and magnesium and the erythrocyte magnesium concentrations did not show significant differences relative to controls (p>0.05). The increases in the activities of SOD and CAT activities and the changes in trace elements concentrations can indicate the presence of increased reactive oxygen species that might play a part in the pathogenesis larynx tumors. Presented at the IX Asian-Pacific Congress of Clinical Biochemistry, March 9–14, 2002, New Delhi, India.     

Heterologous expression of a novel psychrophilic Cu/Zn superoxide dismutase from Deschampsia antarctica

Superoxide dismutase (SOD) catalyzes the conversion of the superoxide radical (O₂⁻) into oxygen and hydrogen peroxide. Deschampsia antarctica is a plant that grows in Antarctica and survives to extreme low temperature and high UV radiation, thus it is an ideal model to study novel antioxidants. A cDNA Cu/Zn-SOD gene from D. antarctica was cloned into a pET vector and expressed in Escherichia coli BL21-SI. 112 mg/L of recombinant Cu/Zn-SOD was attained in batch cultures in bioreactor. Using Ni-affinity gel chromatography, the recombinant Cu/Zn-SOD was recovered with a purity of 90% and a specific enzyme activity of 749 at 25 °C. However, zymogram test showed that the enzyme has more activity at 4 °C. This D. antarctica SOD could be used to reduce the oxidation of refrigerated and frozen foods.     

Cu,Zn-superoxide dismutase is differently regulated by cadmium and lead in roots of soybean seedlings

The cadmium (Cd²⁺) and lead (Pb²⁺)-induced changes in Cu,Zn-SOD gene expression on the level of mRNA accumulation and enzyme activity were analyzed in roots of soybean (Glycine max) seedlings. The Cd²⁺ caused the induction of copper–zinc superoxide dismutase (Cu,Zn-SOD) mRNA accumulation, at each analyzed metal concentration (5–25 mg/l), whereas in Pb²⁺-treated roots this effect was observed only at the medium metal concentrations (50–100 mg/l of Pb²⁺). The analysis of Cu,Zn-SOD activity proved an increase in enzyme activity during Cd²⁺/Pb²⁺ stresses, however in Pb²⁺-treated plants the activity of enzyme was not correlated with respective mRNAs level. Presented data suggest that different metals may act on various level of Cu,Zn-SOD expression in plants exposed to heavy metals stress.     

Simultaneous determination of mevalonate and 7α-hydroxycholesterol in human plasma by gas chromatography—mass spectrometry as indices of cholesterol and bile acid biosynthesis

A very sensitive and specific method for the simultaneous determination of mevalonate and 7α-hydroxycholesterol in human plasma is described. The assay is based on isotope dilution mass spectrometry: the extracts from plasma were treated with benzylamine followed by dimethylethylsilylimidazole, then the resulting dimethylethylsilyl ether derivatives of mevalonylbenzylamide and 7α-hydroxycholesterol were determined by gas chromatography—mass spectrometry using high-resolution selected-ion monitoring. Simple regression analysis revealed significant correlations between the plasma level of mevalonate and the hepatic activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34) (r = 0.83, P < 0.01) and between the plasma level of free 7α-hydroxycholesterol and the hepatic activity of cholesterol 7α-hydroxylase (EC 1.14.13.7) (r = 0.76, P < 0.05).     

Regulation of lysosomal α-mannosidase-1 synthesis during development in Dictyostelium discoideum

The cellular specific activity of lysosomal α-mannosidase-1 increases dramatically during development in Dictyostelium discoideum. α-Mannosidase-1 is composed of two subunits (M_r = 58,000 and 60,000) which are derived from a common precursor polypeptide (M_r = 140,000). Using enzyme-specific monoclonal antibodies we have determined that throughout development (a) the relative rate of precursor biosynthesis closely parallels the rate of accumulation of cellular enzyme activity and (b) the newly synthesized precursor is efficiently processed to mature enzyme (t_1/2 < 10 min). This indicates that the developmental accumulation of α-mannosidase-1 activity is primarily controlled by de novo enzyme synthesis. Furthermore, the change in the relative rate of enzyme precursor synthesis can be accounted for by an increase in the cellular level of functional α-mannosidase-1 mRNA during development.     

Reproducibility of Dual-Energy X-ray Absorptiometry Measurements in Prepubertal Girls

Objective : Regulation of growth and development, clinical assessment, and obesity are among the areas of nutritionrelated research, wherein accurate assessment of body composition is important. We want to test the hypothesis that dual-energy X-ray absorptiometry (DXA) measurements are reproducible in healthy girls. Research Methods and Procedures : We determined total body composition measurements in healthy prepubertal girls using DXA twice, 6 weeks apart. Results : We studied 61 healthy, normal-weight, prepubertal girls, aged 4.8 years to 10.3 years. The girls' DXA-derived mean weight between visits 1 and 2 significantly increased (27.14 kg vs. 27.80 kg, P<0.0001). The increased weight was due to significant increases in total body fat-free mass (FFM) (19.53 kg vs. 19.89 kg,P<O.001), total body bone mass (1.05 kg vs. 1.07 kg, P<0.0001), and total body fat mass (7.61 kg vs. 7.91 kg,P<0.03). The girls' DXA-derived mean total trunk mass between visits 1 and 2 significantly increased (11.23 kg vs. 11.63 kg, p<<0.0001), as did total leg mass (9.33 kg vs. 9.53 kg, p<<0.001), although no significant differences were observed in total arm mass (2.52 kg vs. 2.54 kg, p< = 0.37). The Pearson coefficient of correlation (r) and total coefficient of variation (CV) for intraindividual measurements by DXA were: weight—r = 0.99, CV= 1.97%; total body FFM—r = 0.96, CV = 2.30%; total body bone mass—r = 0.99, CV = 2.08%; total body fat mass—r = 0.96, CV = 6.55%; percentage total body fat— r = 0.91, CV = 5.69%; total trunk mass—r = 0.96, CV = 3.59%; total arm mass—r = 0.95, CV = 4.09%; and total leg mass—r = 0.99, CV = 2.75%. Discussion : Total body FFM, total body bone mass, total body fat mass, percentage of total body fat mass, as well as regional mass determinations by DXA, were highly reproducible in healthy, normal-weight, prepubertal girls. We highly recommend the use of DXA for total body composition studies in girls aged 5 years to 10 years.     

Increased levels of peroxisomal active oxygen-related enzymes in copper-tolerant pea plants

The effect in vivo of high nutrient levels of copper (240 micromolar) on the activity of different metalloenzymes containing Cu, Mn, Fe, and Zn, distributed in chloroplasts, peroxisomes, and mitochondria, was studied in leaves of two varieties of Pisum sativum L. plants with different sensitivity to copper. The metalloenzymes studied were: cytochrome c oxidase, Mn-superoxide dismutase (Mn-SOD) and Cu,Zn-superoxide dismutase I (Cu,Zn-SOD I), for mitochondria; catalase and Mn-SOD, for peroxisomes; and isozyme Cu,Zn-SOD II for chloroplasts. The activity of mitochondrial SOD isozymes (Mn-SOD and Cu,Zn-SOD I) was very similar in Cu-tolerant and Cu-sensitive plants, whereas cytochrome c oxidase was lower in Cu-sensitive plants. Chloroplastid Cu,Zn-SOD activity was the same in the two plant varieties. In contrast, the peroxisomal Mn-SOD activity was considerably higher in Cu-tolerant than in Cu-sensitive plants, and the activity of catalase was also increased in peroxisomes of Cu-tolerant plants. The higher activities of these peroxisomal active oxygen-related enzymes in Cu-tolerant plants suggest the involvement of reactive oxygen intermediates (O₂⁻, OH) in the mechanism of Cu lethality, and also imply a function for peroxisomal Mn-SOD in the molecular mechanisms of plant tolerance to Cu in Pisum sativum L.     

Effects of dietary copper on growth performance, nutrient digestibility and fiber characteristics in cashmere goats during the cashmere slow-growing period

Thirty-six 2.5-year-old wether Inner Mongolian White Cashmere Goats (IMWG) (BW = 42.7 ± 3.44 kg) were used to determine the effects of dietary copper (Cu) concentration on growth performance, nutrient digestibility and fiber characteristics during the cashmere slow-growing period. Wethers were stratified by weight and randomly assigned to four dietary treatments, which included a control diet containing 5.60 mg Cu/kg DM, the control diet supplied, respectively, with 10, 20 and 30 mg Cu/kg DM (total dietary Cu level of 5.60, 15.6, 25.6 and 35.6 mg/kg DM). The experiment lasted 50 days including a 10-day preliminary trial and 10-day metabolism trial. Average daily feed intake (ADFI) did not differ among treatment groups (P > 0.05), except that the supplement providing 30 mg Cu/kg DM decreased average daily gain and gain efficiency (P < 0.05). Copper supplementation had no influence on digestibility of DM, CP and ADF (P > 0.05), however, NDF digestibility of the treatment group supplemented with 30 mg Cu/kg DM was lower compared with that of other groups (P < 0.05). Length and growth rate of cashmere fiber were higher in the treatment group supplemented with 20 mg Cu/kg DM compared with other groups (P < 0.05), but cashmere diameter was not affected by Cu supplementation (P > 0.05). In conclusion, supplementation of Cu at the levels of 10, 20 and 30 mg/kg DM to the basal diet containing 5.60 mg Cu/kg DM had no influence on ADFI or nutrient digestibility of DM, CP and ADF in cashmere goats, while 30 mg Cu/kg DM supplementation had a negative effect on growth performance and NDF digestibility. However, 20 mg Cu/kg DM supplementation of the basal diet enhanced cashmere growth. Hence, the appropriate supplemental level during the cashmere slow-growing period is deemed to be 20 mg Cu/kg DM (total dietary Cu level of 25.6 mg/kg DM).     

Subunit interaction enhances enzyme activity and stability of sweet potato cytosolic Cu/Zn-superoxide dismutase purified by a His-tagged recombinant protein method

The coding region of copper/zinc-superoxide dismutase (Cu/Zn-SOD) cDNA from sweet potato, Ipomoea batatas (L.) Lam. cv. Tainong 57, was introduced into an expression vector, pET-20b(+). The Cu/Zn-SOD purified by His-tagged technique showed two active forms (dimer and monomer). The amount of proteins of dimer and monomer appeared to be equal, but the activity of dimeric form was seven times higher than that of monomeric form. The enzyme was dissociated into monomer by imidazole buffer above 1.0 M, acidic pH (below 3.0), or SDS (above 1%). The enzyme is quite stable. The enzyme activity is not affected at 85 °C for 20 min, in alkali pH 11.2, or in 0.1 M EDTA and also quite resistant to proteolytic attack. Dimer is more stable than monomer. The thermal inactivation rate constant k _dcalculated for the monomer at 85 °C was 0.029 min^-1 and the half-life for inactivation was about 28 min. In contrast, there is no significant change of dimer activity after 40 min at 85 °C. The enzyme dimer and monomer retained 83% and 58% of original activity, respectively, after 3 h incubation with trypsin at 37 °C, while those retained 100% and 31% of original activity with chymotrypsin under the same condition. These results suggest subunit interaction might change the enzyme conformation and greatly improve the catalytic activity and stability of the enzyme. It is also possible that the intersubunit contacts stabilize a particular optimal conformation of the protein or the dimeric structure enhances catalytic activity by increasing the electrostatic steering of substrate into the active site.     

Diastolic Dysfunction Is an Independent Predictor of Cardiovascular Events in Incident Dialysis Patients with Preserved Systolic Function

Background

Diastolic heart failure (HF), the prevalence of which is gradually increasing, is associated with cardiovascular (CV) morbidity and mortality in the general population and, more specifically, in patients with end-stage renal disease (ESRD). However, the impact of diastolic dysfunction on CV outcomes has not been studied in incident dialysis patients with preserved systolic function.

Methods

This prospective observational cohort study investigates the clinical consequence of diastolic dysfunction and the predictive power of diastolic echocardiographic parameters for CV events in 194 incident ESRD patients with normal or near normal systolic function, who started dialysis between July 2008 and August 2012.

Results

During a mean follow-up duration of 27.2 months, 57 patients (29.4%) experienced CV events. Compared to the CV event-free group, patients with CV events had a significantly higher left ventricular (LV) mass index, ratio of early mitral flow velocity (E) to early mitral annulus velocity (E’) (E/E’), LA volume index (LAVI), deceleration time, and right ventricular systolic pressure, and a significantly lower LV ejection fraction and E’. In multivariate Cox proportional hazard analysis, E/E’>15 and LAVI>32 mL/m² significantly predicted CV events (E/E’>15: hazard ratio [HR] = 5.40, 95% confidence interval [CI] = 2.73–10.70, P< .001; LAVI>32 mL/m²: HR = 5.56, 95% CI = 2.28–13.59, P< .001]. Kaplan-Meier analysis revealed that patients with both E/E’>15 and LAVI>32mL/m² had the worst CV outcomes.

Conclusion

An increase in E/E’ or LAVI is a significant risk factor for CV events in incident dialysis patients with preserved LV systolic function.     

Hypertension: does impaired endothelium-dependent relaxation affect superoxide scavenging?

The purpose of this study was to investigate the effects of acute nitric oxide synthase inhibition on mean arterial blood pressure, oxidative stress markers such as plasma malondialdehyde (MDA) concentration, intracellular antioxidant enzyme activities such as copper-zinc superoxide dismutase (Cu/Zn SOD) and catalase and on trace elements important for activity and stability of Cu/Zn-SOD. Wistar-Kyoto rats (approx 150 g) (n=11) were treated with N ^ω-nitro-l-arginine methyl esther (l-NAME) (0.5 mg/mL) for 2 d. Age- and bodyweight-matched rats (n=10) were used for control group. Their systolic blood pressures and heart rates were recorded daily during the experimental period and also before their blood samples were drawn. Plasma MDA, plasma and red cell zinc and copper concentrations, and red cell Cu/Zn-SOD and catalase activities were determined. A progressive rise in systolic arterial blood pressure was observed compared to the control group (p<0.001). The heart rate of the experimental group was reduced on the third day (p<0.05). Plasma MDA concentration and red cell catalase activity were increased in the experimental group (p<0.001 and p<0.001, respectively). Plasma copper and red cell zinc concentrations were also increased significantly in the experimental group (p<0.001 and p<0.01, respectively). In conclusion, impairment in endothelium-derived relaxation altered mean arterial blood pressure, oxidant status, and trace element concentrations. Presented at the Advanced Course (sponsored by NATO-ASI, SFRR, FEBS, UNESCO-MCBN, IUBMB) “Free Radicals, Nitric Oxide, and Inflamation: Molecular, Biochemical, and Clinical Aspects,” Lara, Antalya, Turkey, September 23–October 3, 2001.     

Liver Lobe Volumes and the Ratios of Liver Lobe Volumes to Spleen Volume on Magnetic Resonance Imaging for Staging Liver Fibrosis in a Minipig Model

Objective

To investigate liver lobe volumes and the ratios of liver lobe volumes to spleen volume measured with magnetic resonance imaging (MRI) for quantitatively monitoring and staging liver fibrosis.

Methods

Animal study was approved by Institutional Animal Care and Use Committee. Sixteen minipigs were prospectively used to model liver fibrosis, and underwent abdominal gadolinium-enhanced MRI on 0, 5^th, 9^th, 16^th and 21^st weekend after modeling this disease staged by biopsy according to METAVIR classification system. On MRI, volume parameters including left lateral liver lobe volume (LLV), left medial liver lobe volume (LMV), right liver lobe volume (RV), caudate lobe volume (CV), and spleen volume (SV) were measured; and LLV/SV, LMV/SV, RV/SV and CV/SV were calculated. Statistical analyses were performed for staging this fibrosis.

Results

LLV and CV increased with increasing stage of fibrosis (r = 0.711, 0.526, respectively; all P < 0.05). RV and LMV increased from stage 0 to 2 and decreased from 2 to 4; and RV/SV decreased from 0 to 1, increased from 1 to 2, and decreased from 3 to 4 (all P > 0.05). LLV/SV, LMV/SV and CV/SV decreased from stage 0 to 4 (r = -0.566, -0.748 and -0.620, respectively; all P < 0.05). LLV, CV, LLV/SV, LMV/SV, RV/SV, and CV/SV could distinguish stage 0–1 from 2–4 and 0–2 from 3–4 (all P < 0.05). Among these parameters, LLV and LMV/SV could best classify stage ≥2 and ≥3, respectively (area under receiver operating characteristic curve = 0.893 and 0.946, respectively).

Conclusion

LLV and LMV/SV complement each other in staging liver fibrosis, and both parameters should be used to stage this disease.     

Regio-distribution of stearic acid is not conserved in chylomicrons after ingestion of randomised, stearic acid-rich fat in a single meal

Stearic acid from conventional food is well absorbed, but the fate of synthetic randomized stearic acid in fat absorption and subsequent metabolism is unclear. In this study, we examined the postprandial triglyceridemia following an ingestion of randomized stearic acid-rich fat. Following a 12-h fast, nine healthy young males ate a hamburger meal with 16.7 g of stearic acid (30% in triacylglycerol (TAG) sn-2 position, fully randomized). Postprandial blood samples were collected for 450 min, and the stearic acid content in chylomicron (CM, Svedberg flotation rate >400) TAG and the proportion of stearic acid in the sn-2 position were measured by tandem mass spectrometry at peak (180 min) and late (360 min) triglyceridemia. Of all stearic acid in CM TAG, 23% and 22% were in the sn-2 position at peak and late triglyceridemia (P<.004 and P<.001, respectively). This suggests a 68% and 62% conservation of sn-2 stearic acid, respectively. Peak postprandial TAG concentration and incremental area under the TAG curve showed a higher correlation with the fasting CM TAG (r=0.88, P<.01 and r=0.72, P<.05, respectively) than with total fasting plasma TAG (r=0.73, P<.05 and r=0.24, nonsignificant, respectively). In an earlier study, we showed that the absorption efficiency of the stearic acid of the meal was normal, with only marginal amounts of mainly sn-1,3 stearic acid found in the feces. In conclusion, we showed that sn-2 stearic acid is underrepresented in the postprandial CM TAG following an ingestion of fully randomized fat.     

DNA damage induced by copper deficiency in cattle assessed by the Comet assay

Cattle hypocuprosis is a well-known endemic disease in several parts of the world. In a previous paper, the clastogenic effect of copper deficiency in cattle has been described although the occurrence of DNA damage was not directly tested. For this reason, the relation between DNA damage assessed by the Comet assay and Cu plasma concentration was studied in Aberdeen Angus cattle.Blood samples were obtained in heparinized Vacutainer^® tubes from 28 female Aberdeen Angus cows during pregnancy or immediately after to give birth. Each sample was divided into two aliquots for Comet assay and Cu plasma determination, respectively. From the 28 cattle sampled, 17 were normocupremic and 11 were hypocupremic.Results obtained showed that whereas the average plasma Cu level in normocupremic cattle was 67.6 μg/dl, in hypocupremic cattle it was 32.1 μg/dl. The increase of DNA damage was mostly evidenced by the decrease of comet degree 1 cells and an increase of comet degree 2 cells. Correlation analysis comparing plasma Cu levels and degree 1 cells showed a correlation coefficient 0.72 (P<0.01). The comparison between plasma Cu levels and comet degree 2 cells was −0.65 (P<0.01). The comparison between plasma Cu levels and the comet length-head diameter medians determined in 23 out of 28 animals showed a correlation coefficient of −0.54 (P<0.01).The induction of DNA damage was clearly supported by the fact that the decrease of plasma Cu levels was correlated with the increase of comet length-head diameter. These findings could be considered as a contribution to the hypothesis that DNA and chromosome damage are a consequence of the higher oxidative stress suffered by hypocupremic animals.     

Physiological and biochemical adaptations to training inRana pipiens

Summary FifteenRana pipiens were trained on a treadmill thrice weekly for 6.5 weeks to assess the effects of training on an animal that supports activity primarily through anaerobiosis. Endurance for activity increased 35% in these frogs as a result of training (P=0.006, Fig. 1). This increased performance was not due to enhanced anaerobiosis. Total lactate produced during exercise did not differ significantly for the trained or untrained animals in either gastrocnemius muscle (2.77±0.21 and 2.82±0.13 mg/g, respectively) or whole body (1.32±0.10 and 1.47±0.06 mg/g, respectively). Glycogen depletion also did not differ between the two groups (Fig. 2c). The primary response to training appeared to involve augmentation of aerobic metabolism, a response similar to that reported for mammals. Gastrocnemius muscles of trained frogs underwent a 38% increase over those of untrained individuals in the maximum activity of citrate synthase (14.5±1.0 and 10.5±0.9 moles/(g wet wt·min);P=0.008). This enzyme was also positively correlated with the level of maximum performance for all animals tested (r=0.61,P<0.01) and with the degree of improvement in the trained animals (r=0.72,P<0.05). In addition to an increased aerobic capacity, the trained animals demonstrated a greater removal of lactate from the muscle 15 min after fatigue (Fig. 2b).     

The chlorophenoxy herbicide dicamba and its commercial formulation banvel induce genotoxicity and cytotoxicity in Chinese hamster ovary (CHO) cells

The sister chromatid exchange (SCE) frequency, the cell-cycle progression analysis, and the single cell gel electrophoresis technique (SCGE, comet assay) were employed as genetic end-points to investigate the geno- and citotoxicity exerted by dicamba and one of its commercial formulation banvel^® (dicamba 57.71%) on Chinese hamster ovary (CHO) cells. Log-phase cells were treated with 1.0–500.0 μg/ml of the herbicides and harvested 24 h later for SCE and cell-cycle progression analyses. All concentrations assessed of both test compounds induced higher SCE frequencies over control values. SCEs increased in a non-dose-dependent manner neither for the pure compound (r = 0.48; P > 0.05) nor for the commercial formulation (r = 0.58, P > 0.05). For the 200.0 μg/ml and 500.0 μg/ml dicamba doses and the 500.0 μg/ml banvel^® dose, a significant delay in the cell-cycle progression was found. A regression test showed that the proliferation rate index decreased as a function of either the concentration of dicamba (r = −0.98, P < 0.05) or banvel^® (r = −0.88, P < 0.01) titrated into cultures in the 1.0–500.0 μg/ml dose-range. SCGE performed on CHO cells after a 90 min pulse-treatment of dicamba and banvel^® within a 50.0–500.0 μg/ml dose-range revealed a clear increase in dicamba-induced DNA damage as an enhancement of the proportion of slightly damaged and damaged cells for all concentrations used (P < 0.01); concomitantly, a decrease of undamaged cells was found over control values (P < 0.01). In banvel^®-treated cells, a similar overall result was registered. Dicamba induced a significant increase both in comet length and width over control values (P < 0.01) regardless of its concentration whereas banvel^® induced the same effect only within 100.0–500.0 μg/ml dose range (P < 0.01). As detected by three highly sensitive bioassays, the present results clearly showed the capability of dicamba and banvel^® to induce DNA and cellular damage on CHO cells.