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Indian Journal of Clinical Biochemistry logoLink to Indian Journal of Clinical Biochemistry
. 2022 Oct 27;39(1):73–82. doi: 10.1007/s12291-022-01093-1

Protein Carbonyl, Lipid Peroxidation, Glutathione and Enzymatic Antioxidant Status in Male Wistar Brain Sub-regions After Dietary Copper Deficiency

Ankita Rajendra Kurup 1,✉, Neena Nair 1
PMCID: PMC10784247  PMID: 38223011

Abstract

Copper a quintessential transitional metal is required for development and function of normal brain and its deficiency has been associated with impairments in brain function. The present study investigates the effects of dietary copper deficiency on brain sub-regions of male Wistar rats for 2-, 4- and 6-week. Pre-pubertal rats were divided into four groups: negative control (NC), copper control (CC), pairfed (PF) and copper deficient (CD). In brain sub regions total protein concentration, glutathione concentration and Cu–Zn SOD activity were down regulated after 2-, 4- and 6 weeks compared to controls and PF groups. Significant increase in brain sub regions was observed in protein carbonyl and lipid peroxidation concentration as well as total SOD, Mn SOD and catalase activities after 2-, 4- and 6 weeks of dietary copper deficiency. Experimental evidences indicate that impaired copper homeostasis has the potential to generate reactive oxygen species enhancing the susceptibility to oxidative stress by inducing up- and down-regulation of non-enzymatic and enzymatic profile studied in brain sub regions causing loss of their normal function which can consequently lead to deterioration of cell structure and death if copper deficiency is prolonged.

Keywords: Copper deficiency, Brain, Protein carbonyl, Lipid peroxidation, Glutathione, Catalase, Superoxide dismutase

Introduction

Trace element copper is involved in cellular mitochondrial respiration, iron metabolism [1], cytochrome C oxidase, lysyl oxidase, ferroxidase I and II, ceruloplasmin, Cu-ZnSOD (SOD1), extracellular SOD (SOD3), tyrosinase, dopamine-β-monoxygenase and peptidylglycine-α-amidating enzyme [2, 3]. Copper deficiency may become more prevalent due to (1) intake of high fructose and fat food [4] (2) consumption of cow milk and highly processed carbohydrate based diets in infants [5] (3) high oral intake of zinc [6] (4) GIT surgery [6] and (5) genetic condition—Menkes disease [7].

Brain is most vulnerable to oxidative stress being composed largely of non-mitotic, highly differentiated cells being irreplaceable once damaged [8]. Copper homeostasis in brain is highly regulated being essential for its normal physiological function as it ensures proper distribution to various proteins involving transporters (CTR1, ATP7A and ATP7B) and chaperones(ATOX1) [9, 10]. Experimental evidence revealed copper stores in various regions of brain: soma of cerebellar granular, cortical pyramidal neurons, neutrophil present within the cerebral cortex, cerebellum, hippocampus, spinal cord [11] and synaptic vesicles [12].

Neurons have the potential to generate free radicals through electron transport chain. Biological system has abundant protein which serves as major target for oxidation from reactive oxygen species (ROS) [13]. Ozcelik and Uzun [14] reported high levels of polyunsaturated (PUFA) lipids in neuronal cell membranes making it highly susceptible to radical –mediated damage in form of lipid peroxidation which can cause loss of membrane fluidity and integrity. Glutathione (GSH), a tripeptide (ϒ–glutamyl-l-cysteinyl-l-glycine), has a protective role against ROS as well as reactive nitrogen species (RNS) and is responsible for maintenance of cellular thiol status [15]. Superoxide dismutase (EC.1.15.1.11) protects cells and tissues from ROS and exists in three forms: extracellular SOD (SOD 3), Cu Zn SOD (SOD 1) and Mn SOD (SOD 2) with Cu–Zn SOD localized in different population of neurons and astrocytes [16] are neuroprotective when overexpressed. Catalase (EC.1.11.1.6) an oxidoreductase and antioxidant is localized mainly in cell peroxisomes and cytoplasm [17]. Since copper has been found localized in various regions of brain hence in the present study attempt has been made to evaluate total protein, protein carbonyl, lipid peroxidation, glutathione and antioxidant enzymes -total superoxide dismutase, Cu–Zn SOD, MnSOD as well as catalase in brain sub regions after dietary copper deficiency.

Materials and Methods

Basal synthetic diet was prepared by using ICN Research Diet Protocol (1999) with slight modifications. The synthetic basal diet was composed of: Egg white/ albumin (180 gm), Corn oil (100 gm), Corn starch (443 gm), Sucrose (200 gm), Cellulose (30gm), Choline chloride (2 gm), DL-methionine (7 gm), AIN-76 salt mixture (35 gm), AIN-76C vitamin-antibody mixture (10 g). The experimental diets differed only in copper content containing 126 nmol Cu/g and 6.3 nmol Cu/g by adding appropriate amount of copper sulphate which was confirmed by GBC 902 atomic absorption spectrophotometer at 324.8 nm in air acetylene flame.

Prepubertal male Wistar rats of 30–40 days (weighing 35–50 gm) were divided into four groups: (a) Negative control (NC) fed standard rodent pellet diet (Ashirwad industries; Chandigarh, India) (b) Copper control (CC) group rats were fed with diet containing 126 nmol Cu/gm (c) Pairfed (PF) both were fed 126 nmol Cu/gm of diet was based on diet consumed by copper deficient (CD) group the previous day to eliminate the starvation and stress effect. The above three groups were provided tap water ad libitum (d) Copper deficient (CD) group fed 6.3 nmol Cu/gm of diet and given demineralized water ad-libitum. The experiment was carried out for 2-, 4- and 6-week with 5 animals in each sub groups. This study was carried out following the guidelines for the use and care of laboratory animals set by Committee for the purpose of control and supervision on experiments on animals (CPCSEA) (No. 1678/GO/Re/S/12/CPCSEA dated 16.06.17) and approved by Departmental Animal Ethics Committee, University of Rajasthan, Jaipur, India.

Male Wistar rats were housed individually and maintained in polypropylene cages with stainless steel grills. Cages, grills and water bottles were washed with detergent solution, demineralized water and finally rinsed in 1% EDTA solution prepared in demineralized water so as to avoid contamination and for removal of copper traces. After completion of experiments the animals were anesthetized by intraperitoneal (I.P) injection using sodium thiopentone (20 mg/kg b.wt; Thiosol Sodium: Neon Laboratories Ltd. Mumbai, India). The cranium of each animal was opened; whole brain was carefully removed and separated on a glass plate placed over ice pack, into four sub-regions viz., frontal cortex, parietal, occipital and cerebellum for biochemical estimation.

Biochemical Assays

Total Protein

Total protein of the brain sub-regions was evaluated by Bradford [18] method. Tissue homogenates were centrifuged for 10 min at 1000 rpm. To 5 ml of Bradford solution, 100 μL tissue samples/Bovine serum albumin (standard) were added. The absorbance value was determined at 595 nm against blank. Data were expressed as mg/g protein.

Protein Carbonyl

Protein carbonyl content in brain sub- regions was determined by method given by Levine et al. [19] which involve the formation of stable 2,4-dinitrophenyl (DNP) hydrazone product when 2,4-dinitrophenylhydrazine reacts with protein carbonyls. 1.5 ml of 10 mM 2,4-dinitrophenylhydrazine (DNPH) prepared in 2.5 M HCl was added to the test samples containing brain sub-region tissue samples. The tubes were shaken intermittently every 10 min and incubated for 1 h in the dark at 37 °C. The tubes were then centrifuged at 3000 rpm for 10 min. The supernatant was carefully aspirated and discarded. The pellets were washed three times with ethanol: ethyl acetate (1:1, v/v) solution. Pellet was dissolved in 6 M guanidine HCl, 20 mM potassium phosphate buffer (pH 6.5) and centrifuged. Absorbance of supernatant was read at 395 nm and calculated by using a molar absorption coefficient of 22,000 M−1 cm−1and the data were expressed in nmol/mg protein.

Lipid Peroxidation

Lipid peroxidation was measured according by the method given by Ohkawa et al. [20]. 1, 1, 3, 3-tetramethoxy propane (TMP) (stock-100 μM/ml) was used as standard to the homogenate added 0.2 ml of 8.1% of SDS, 1.5 ml of 20% acetic acid solution (pH 3.5) and 1.5 ml of 0.8% aqueous solution of thiobarbituric acid (TBA). The mixture was finally made up to 4 ml by adding distilled water and heated in a water bath at 95 °C for 60 min. After cooling 1 ml of distilled water and 5 ml of the mixture of n-butanol and Pyridine (15: 1 v/v) were added and centrifuged at 4000 rpm. Absorbance of the upper organic layer was read at 532 nm against blank. Data were expressed in µmole MDA/mg tissue.

Reduced Glutathione (GSH)

Determination of reduced GSH was performed by method given by Brehne and Burch [21] and modified by Palamanda and Kehrer [22]. Rate of reduction of Ellman reagent- 5-5′-dithiobis-(2-nitrobenzoic acid) was measured in an assay involving sodium phosphate buffer adjusted at pH 8.1 containing 0.3 mM 5′,5′-dithiobis (2-nitrobenzoic acid) (DTNB), 15 mM EDTA, 0.04% Bovine Serum Albumin. Homogenates were prepared in 0.01 N HCl. To the homogenate added 1 ml of 0.01 N KOH, mixed thoroughly and centrifuged at 4000 rpm for 5 min. Supernatant was removed and 1 ml of DTNB in buffer was added. GSH (Hi Media, India) was used as standard and graph was plotted (10–70 μM). Absorbance was read spectrophotometrically at 432 nm. The data were expressed in µM GSH/ mg protein.

Total SOD

Brain SOD activity in all sub regions of brain tissue was estimated by the method given by Marklund and Marklund [23] which measures inhibition of auto oxidation of pyragallol (1,2,3-trihydroxybenzene) by Superoxide dismutase (SOD). The reaction mixture contained 2.4 ml of Tris-Cacodylate buffer (50 mM pH 8.2 containing 1 mM Diethylenetriaminepentaacetic acid), 0.05 ml of TrisHCl (50 mM pH 7.4) and 0.1 ml of 2 mM pyrogallol and 0.05 ml of homogenate. The change in absorbance (Ax) of reference cuvette due to auto oxidation of pyragallol and change in absorbance (Ay) of enzyme cuvette was measured at 420 nm. The difference in the rate is compared and expressed as percent inhibition. One unit of SOD activity is defined as the amount required to obtain 50% inhibition of pyrogallol oxidation. The enzyme activity is expressed as Units mg protein/ hr.

Cu–Zn SOD

Cu- ZnSOD activity is estimated by the method given by Geller and Winge [24] in which inactivation of MnSOD is carried by addition of sodium dodecyl sulphate (SDS). Assay mixture contained 2.4 ml Tris-cacodylate buffer, 0.05 ml of supernatant (SDS treated) and 0.1 ml of 2 mM pyrogallol. Absorbance measured spectrophotometrically at 420 nm and activity expressed in Units mg protein/ hr.

Mn-SOD

Activity was obtained by subtracting Cu–Zn SOD from total SOD activity. Data expressed in Units mg protein/h.

Catalase

Activity in all sub regions of brain tissue was estimated by the method of Sinha [25] in which the chromic acetate produced by the reduction of dichromate in acetic acid heated in presence of H2O2 is measured. The assay mixture contained 5 ml of phosphate buffer (0.01 M; pH 7.0), 1 ml brain tissue homogenate and 4 ml of H2O2 (400 mM)0.2 ml of color reagent K2Cr2O7 [5% of K2Cr2O7 with glacial acetic acid in 1:3 (v/v)] added to each 10 test tubes. The blank mixture contained 5 ml of phosphate buffer, 1 ml distilled water and 4 ml of H2O2. 1 ml from assay mixture/blank mixture was added to test tubes containing 2 ml color reagent at 1 min interval. Both the mixtures were subjected to hot water bath, cooled and measured spectrophotometrically at 570 nm. H2O2 in the range of 10–600 mM was used as standard and graph was plotted. Data expressed in Kat f.

Copper Estimation

Copper was estimated in all sub-regions of brain by wet assay method. Known quantities of brain sub regions of different groups were digested in diacidic solution (HNO3:HClO4::5:1) and then diluted with distilled water. Copper were estimated at 324 in Varian AA240FS atomic absorption spectrophotometer. Data was expressed in µg Cu/g of wet tissue.

Statistical Analysis

Statistical analysis was performed by Graphpad Prism 7 (Version 7.00). Data were analyzed by using one way Analysis of Variance (ANOVA) and if the difference was found to be significant then post-hoc test (Tukey's multiple comparisons test) was applied. P < 0.05 was considered to be significant. Data expressed as mean ± SEM.

Result

Total protein in all sub-regions of brain decreased significantly (P < 0.05) after 2-, 4- and 6-week of experiment when copper deficient (CD) groups were compared with their respective negative control (NC), copper control (CC) and pairfed (PF) groups. No significant decrease in pairfed groups of 2-week experiment was recorded when compared with negative control (NC) and copper control (CC) groups. However, total protein concentration of 4- and 6-week experiment pairfed groups decreased significantly (P < 0.05) when compared to their respective controls (NC and CC) (Table 1).

Table 1.

Analysis of total protein and protein carbonyl concentration in brain sub-regions of Wistar rats after 2-, 4- and 6-week of dietary copper deficiency (Mean ± SEM)

Groups Total protein (mg/g) Protein carbonyl (nmoles/mg protein)
Frontal Parietal Occipital Cerebellum Frontal Parietal Occipital Cerebellum
2 NC 50.31 ± 1 52.77 ± 0.38 47.37 ± 0.23 52.91 ± 0.57 2.12 ± 0.02 2.22 ± 0.02 2.05 ± 0.02 2.27 ± 0.02
2 CC 49.62 ± 0.27 51.36 ± 0.57 46.76 ± 0.47 51.91 ± 0.54 2.09 ± 0.02 2.16 ± 0.02 2.01 ± 0.02 2.23 ± 0.02
2 PF 49.45 ± 0.3 51.25 ± 0.36 46.44 ± 0.41 51.61 ± 0.58 2.15 ± 0.02 2.25 ± 0.03 2.10 ± 0.02 2.30 ± 0.02
2 CD 40.12 ± 0.38c*e*f* 41.21 ± 0.67c*e*f* 39.43 ± 0.36c*e*f* 41.71 ± 0.31c*e*f* 3.1 ± 0.03c*e*f 3.16 ± 0.02c*e*f* 3.05 ± 0.03c*e*f* 3.2 ± 0.02c*e*f*
4 NC 54.15 ± 0.3 56.14 ± 0.33 53.29 ± 0.21 57.06 ± 0.38 2.48 ± 0.02 2.57 ± 0.02 2.35 ± 0.01 2.66 ± 0.01
4 CC 53.77 ± 0.32 55.93 ± 0.32 52.48 ± 0.19 56.79 ± 0.24 2.41 ± 0.03 2.53 ± 0.03 2.33 ± 0.02 2.6 ± 0.01
4 PF 49.96 ± 0.37b*d* 51.02 ± 0.41b*d* 48.51 ± 0.26b*d* 51.94 ± 0.35b*d* 3.15 ± 0.02b*d* 3.33 ± 0.02b*d* 2.96 ± 0.02b*d* 3.41 ± 0.02b*d*
4 CD 41.96 ± 0.46c*e*f* 43.31 ± 0.42c*e*f* 39.86 ± 0.56c*e*f* 45.41 ± 0.46c*e*f* 3.69 ± 0.02c*e*f* 3.9 ± 0.01c*e*f* 3.6 ± 0.03c*e*f* 3.93 ± 0.02c*e*f*
6 NC 59.05 ± 0.38 61.67 ± 0.28 58.02 ± 0.35 63.22 ± 0.37 2.69 ± 0.01 2.75 ± 0.02 2.62 ± 0.02 2.8 ± 0.03
6 CC 60.39 ± 0.32 60.64 ± 0.44 56.63 ± 0.24 62.39 ± 0.58 2.62 ± 0.01 2.7 ± 0.02 2.53 ± 0.02 2.76 ± 0.01
6 PF 54.42 ± 0.37b*d* 56.43 ± 0.45b*d* 51.49 ± 0.33b*d* 57.61 ± 0.27b*d* 3.8 ± 0.01b*d* 3.97 ± 0.03b*d* 3.69 ± 0.02b*d* 4.05 ± 0.02b*d*
6 CD 43.51 ± 0.31c*e*f* 45.05 ± 0.39c*e*f* 40.99 ± 0.53c*e*f* 45.21 ± 0.35c*e*f* 4.53 ± 0.02c*e*f** 4.65 ± 0.03c*e*f* 4.48 ± 0.04c*e*f* 4.74 ± 0.03c*e*f*
Open in a new tab

Multiple comparison procedures were performed for 2-, 4- and 6 weeks experimental groups

*p < 0.05 significant

aNC versus CC

bNC versus PF

cNC versus CD

dCC versus PF

eCC versus CD

fPF versus CD

Protein carbonyl content increased significantly (P < 0.05) in all brain sub-regions after 2-, 4- and 6-week of copper deficiency when compared to their respective controls (NC and CC). Pairfed group of 2-week experiment when compared with control groups no significant change in protein carbonyl content was recorded. However, there were significant rise (P < 0.05) in protein carbonylation in pairfed groups of 4- and 6-week group when compared with their respective controls (NC and CC) (Table 1).

Significant (P < 0.05) increase was observed in lipid peroxidation in copper deficient groups of 2-, 4- and 6-week of experiment when compared with NC, CC and PF groups. Non- significant change was observed when pairfed group of 2-week was compared with control groups. However when 4- and 6-week paired groups when compared with their respective NC and CC groups revealed significant increase (P < 0.05) ( Table 2).

Table 2.

Analysis of lipid peroxidation and glutathione concentration in brain sub regions of wistar rats after 2-, 4- and 6- weeks of dietary copper deficiency (mean ± SEM)

Groups Lipid Peroxidation (µmole MDA/ mg tissue) Glutathione (µmole GSH/ mg protein)
Frontal Parietal Occipital Cerebellum Frontal Parietal Occipital Cerebellum
2 NC 39.24 ± 0.25 40.85 ± 0.45 36.75 ± 0.2 42.55 ± 0.48 24.3 ± 0.08 25.07 ± 0.08 23.71 ± 0.09 25.99 ± 0.1
2 CC 38.74 ± 0.60 40.50 ± 0.20 36.40 ± 0.38 42.20 ± 0.26 24 ± 0.08 24.73 ± 0.11 23.4 ± 0.09 25.63 ± 0.09
2 PF 40.17 ± 0.38 41.61 ± 0.32 37.72 ± 0.40 43.3 ± 0.33 19.62 ± 0.08b*d* 20.13 ± 0.14b*d* 19.21 ± 0.08b*d* 20.65 ± 0.08b*d*
2 CD 80.20 ± 0.25c*e*f* 81.40 ± 0.36c*e*f* 78.25 ± 0.50c*e*f* 82.05 ± 0.23c*e*f* 15.36 ± 0.09c*e*f* 15.83 ± 0.08c*e*f* 14.94 ± 0.06c*e*f* 16.08 ± 0.1c*e*f*
4 NC 41.80 ± 0.28 43.57 ± 0.45 39.95 ± 0.28 45.66 ± 0.20 29.87 ± 0.07 30.28 ± 0.07 29.36 ± 0.07 31.18 ± 0.09
4 CC 41.65 ± 0.35 43.33 ± 0.30 38.33 ± 0.40 44.83 ± 0.66 29.48 ± 0.07 30 ± 0.11 29.18 ± 0.1 30.87 ± 0.11
4 PF 62.45 ± 0.25b*d* 64.16 ± 0.20b*d* 59.66 ± 0.40b*d* 65.41 ± 0.40b*d* 20.64 ± 0.1b*d* 21.44 ± 0.08b*d* 19.88 ± 0.1b*d* 22.32 ± 0.08b*d*
4 CD 140.69 ± 0.33c*e*f* 143.9 ± 0.48c*e*f* 138.6 ± 0.66c*e*f* 145.83 ± 0.3c*e*f* 15.11 ± 0.15c*e*f* 15.79 ± 0.09c*e*f* 14.38 ± 0.11c*e*f* 16.32 ± 0.09c*e*f*
6 NC 45.33 ± 0.63 47.75 ± 0.33 43.62 ± 0.33 49.8 ± 0.83 34.03 ± 0.09 34.45 ± 0.08 32.71 ± 0.14 34.77 ± 0.07
6 CC 44.83 ± 0.50 46.83 ± 0.35 43.53 ± 0.50 49.76 ± 0.20 33.75 ± 0.07 34.19 ± 0.08 32.58 ± 0.12 34.57 ± 0.08
6 PF 69.8 ± 0.83b*d* 71.97 ± 0.27b*d* 67.69 ± 0.75b*d* 73.05 ± 0.45b*d* 21.69 ± 0.07b*d* 20.91 ± 0.1b*d* 20.22 ± 0.09b*d* 22.55 ± 0.09b*d*
6 CD 210.53 ± 0.33c*e*f* 212.65 ± 0.55c*e*f* 207.48 ± 0.33c*e*f* 215.74 ± 0.33c*e*f* 16.94 ± 0.07c*e*f* 16.94 ± 0.0c*e*f* 15.77 ± 0.11c*e*f* 17.3 ± 0.11c*e*f*
Open in a new tab

Multiple comparison procedures were performed for 2-, 4- and 6 weeks experimental groups

*p < 0.05 significant

aNC versus CC

bNC versus PF

cNC versus CD

dCC versus PF

eCC versus CD

fPF versus CD

Reduced Glutathione concentration declined significantly (P < 0.05) in all brain sub-regions after 2-, 4- and 6-week of copper deficiency when compared with their respective NC, CC and PF groups. Decrease was also significant when pairfed group of 2-, 4- and 6-week experiment were compared with respective NC and CC groups (Table 2).

After 2-, 4- and 6-week of dietary copper deficiency total SOD activity increased significantly (P < 0.05) in CD groups compared with their respective NC, CC and PF groups. Comparison of three experimental PF groups with respective NC and CC groups also revealed significant (P < 0.05) increase (Table 3).

Table 3.

Analysis of total SOD and Cu- Zn SOD activity in brain sub regions of wistar rats after 2-, 4- and 6- weeks of dietary copper deficiency (Mean ± SEM)

Groups Total SOD (units mg protein/h) Cu–Zn SOD(units mg protein/h)
Frontal Parietal Occipital Cerebellum Frontal Parietal Occipital Cerebellum
2 NC 7.88 ± 0.2820 9.22 ± 0.039 6.49 ± 0.1808 10.20 ± 0.0069 4.89 ± 0.0866 5.21 ± 0.0738 4.53 ± 0.1219 6.94 ± 0.0565
2 CC 7.90 ± 0.0317 9.22 ± 0.034 6.48 ± 0.23 10.29 ± 0.1118 4.97 ± 0.0986 5.20 ± 0.0639 4.52 ± 0.1363 6.99 ± 0.038
2 PF 8.91 ± 0.0036b*d* 10.04 ± 0.019b*d* 7.82 ± 0.390b*d* 11.40 ± 0.0212b*d* 4.70 ± 0.1516 4.74 ± 0.0721b*d* 3.54 ± 0.1224b*d* 5.86 ± 0.1282b*d*
2 CD 9.69 ± 0.0178c*e*f* 11.81 ± 0.040c*e*f* 9.65 ± 0.3809c*e*f* 13.90 ± 0.010c*e*f* 3.68 ± 0.01232c*e*f* 3.93 ± 0.0547c*e*f* 2.93 ± 0.040c*e*f* 5.26 ± 0.1884c*e*f*
4 NC 8.96 ± 0.0202 10.34 ± 0.023 8.66 ± 0.1811 14.71 ± 0.0115 5.98 ± 0.0244 6.88 ± 0.0131 5.5 ± 0.113 7.21 ± 0.1415
4 CC 8.99 ± 0.0168 10.36 ± 0.0229 8.63 ± 0.3301 14.72 ± 0.012 5.99 ± 0.0564 6.90 ± 0.0212 5.51 ± 0.0149 7.25 ± 0.1668
4 PF 9.98 ± 0.0227b*d* 11.68 ± 0.1974b*d* 9.92 ± 0.1834b*d* 17.21 ± 0.014b*d* 3.83 ± 0.0159b*d* 3.54 ± 0.0159b*d* 2.41 ± 0.0157b*d* 4.90 ± 0.0241b*d*
4 CD 13.44 ± 0.0197c*e*f* 13.73 ± 0.1805c*e*f* 11.97 ± 0.019c*e*f* 25.71 ± 0.028c*e*f* 2.05 ± 0.0182c*e*f* 2.94 ± 0.0386c*e*f* 1.07 ± 0.0176c*e*f* 3.88 ± 0.0173c*e*f*
6 NC 9.84 ± 0.0193 14.38 ± 0.2401 9.17 ± 0.010 29.82 ± 0.0123 7.87 ± 0.0193 10.72 ± 0.0113 7.53 ± 0.114 14.09 ± 0.0121
6 CC 9.87 ± 0.0260 14.40 ± 0.2569 9.24 ± 0.036 29.82 ± 0.0125 7.89 ± 0.0260 10.74 ± 0.0154 7.62 ± 0.0125 14.11 ± 0.0134
6 PF 10.57 ± 0.0100b*d* 17.08 ± 0.1056b*d* 12.51 ± 0.215b*d* 36.75 ± 0.0244b*d* 2.54 ± 0.0100b*d* 2.57 ± 0.0134b*d* 1.89 ± 0.0171b*d* 3.61 ± 0.0116b*d*
6 CD 19.44 ± 0.0270c*e*f* 22.02 ± 0.3754c*e*f* 17.51 ± 0.1887c*e*f* 39.33 ± 0.0133c*e*f* 1.39 ± 0.0192c*e*f* 1.64 ± 0.0154c*e*f* 0.36 ± 0.003c*e*f* 1.99 ± 0.0103c*e*f*
Open in a new tab

Multiple comparison procedures were performed for 2-, 4- and 6 weeks experimental groups

*p < 0.05 significant

aNC versus CC

bNC versus PF

cNC versus CD

dCC versus PF

eCC versus CD

fPF versus CD

Significant (P < 0.05) decline in Cu-SOD was observed in copper deficient groups when compared to their respective NC, CC and PF groups. However, when PF groups of 4- and 6-week of experiment were compared with their respective NC and CC groups significant (P < 0.05) decrease in enzymatic activity was observed (Table 3).

Mn-SOD activity significantly increased (P < 0.05) in all brain sub-regions of 2-, 4- and 6-week of copper deficiency when compared to their respective NC, CC and PF groups. Similar pattern of increase was observed when pair fed groups of the three experiments was compared with their respective NC and CC groups (Table 4).

Table 4.

Mn SOD and catalase activity in brain sub regions of Wistar rats after 2-, 4- and—weeks of dietary copper deficiency (Mean ± SEM)

Groups Mn SOD (Units mg protein/h) Catalase (Kat.f)
Frontal Parietal Occipital Cerebellum Frontal Parietal Occipital Cerebellum
2 NC 2.99 ± 0.0125 4.03 ± 0.0198 1.99 ± 0.0186 3.27 ± 0.0155 0.1126 ± 0.018 0.1318 ± 0.034 0.1019 ± 0.018 0.1789 ± 0.030
2 CC 2.94 ± 0.0175 4.00 ± 0.0250 1.96 ± 0.02 3.29 ± 0.0134 0.1120 ± 0.0259 0.1317 ± 0.037 0.1017 ± 0.037 0.1779 ± 0.0118
2 PF 4.22 ± 0.0188b*d* 5.31 ± 0.0216b*d* 4.28 ± 0.035b*d* 5.54 ± 0.0145b*d* 0.1127 ± 0.067 0.1328 ± 0.020 0.1028 ± 0.051 0.1781 ± 0.0135
2 CD 6.05 ± 0.0164c*e*f* 7.89 ± 0.0192c*e*f* 6.75 ± 0.03c*e*f* 8.64 ± 0.0161c*e*f* 0.1218 ± 0.024c*e*f* 0.1429 ± 0.016c*e*f* 0.1132 ± 0.024c*e*f* 0.1991 ± 0.043c*e*f*
4 NC 2.99 ± 0.0427 3.49 ± 0.0318 3.12 ± 0.021 7.51 ± 0.0324 0.1165 ± 0.014 0.1331 ± 0.065 0.102 ± 0.01 0.1814 ± 0.010
4 CC 3.02 ± 0.0454 3.49 ± 0.0309 3.47 ± 0.03 7.46 ± 0.0516 0.1162 ± 0.025 0.1332 ± 0.014 0.1021 ± 0.03 0.1811 ± 0.023
4 PF 6.16 ± 0.0403b*d* 8.16 ± 0.0451b*d* 7.54 ± 0.03b*d* 12.34 ± 0.0525b*d* 0.1192 ± 0.034b*d* 0.1405 ± 0.05b*d* 0.1049 ± 0.02b*d* 0.1984 ± 0.096b*d*
4 CD 11.37 ± 0.0279c*e*f* 10.78 ± 0.0393c*e*f* 10.97 ± 0.052c*e*f* 21.82 ± 0.0458c*e*f* 0.1316 ± 0.061c*e*f* 0.1622 ± 0.057c*e*f* 0.1280 ± 0.096c*e*f* 0.2684 ± 0.014c*e*f*
6 NC 2.01 ± 0.0461 3.68 ± 0.0447 1.7 ± 0.0369 15.85 ± 0.0572 0.1171 ± 0.037 0.1356 ± 0.064 0.107 ± 0.0369 0.1827 ± 0.034
6 CC 1.98 ± 0.0407 3.65 ± 0.0432 1.64 ± 0.0387 15.78 ± 0.0525 0.1176 ± 0.051 0.1349 ± 0.038 0.1077 ± 0.029 0.1831 ± 0.071
6 PF 8.04 ± 0.0381b*d* 14.54 ± 0.0378b*d* 10.67 ± 0.0604b*d* 33.32 ± 0.0318b*d* 0.1228 ± 0.046b*d* 0.1456 ± 0.063b*d* 0.1279 ± 0.081b*d* 0.1978 ± 0.081b*d*
6 CD 18.06 ± 0.0465c*e*f* 20.38 ± 0.0526c*e*f* 17.34 ± 0.147c*e*f* 37.75 ± 0.0401c*e*f* 0.2732 ± 0.035c*e*f* 0.2998 ± 0.018c*e*f* 0.1873 ± 0.047c*e*f* 0.4629 ± 0.058c*e*f*
Open in a new tab

Multiple comparison procedures were performed for 2-, 4- and 6 weeks experimental groups

*p < 0.05 significant

aNC versus CC

bNC versus PF

cNC versus CD

dCC versus PF

eCC versus CD

fPF versus CD

Catalase activity in all brain sub- regions increased significantly (P < 0.05) when copper deficient groups of 2-, 4- and 6-week experiment were compared with their respective NC,CC and PF groups. No significant change observed was observed when pairfed group of 2-week experiment was compared with NC and CC groups. However, when PF groups of 4- and 6-week of experiment were compared with their respective NC and CC groups a significant (P < 0.05) increase was observed (Table 4).

Copper concentration in all brain sub-regions decreased significantly (P < 0.05) in copper deficient groups of 2-, 4- and 6-week in comparison to their respective controls (NC and CC) and PF groups. Similar pattern of significant decrease was evident when PF groups (4- and 6-week) were compared with their respective control (NC and CC) groups but in 2PF the change was not significant (Table 5).

Table 5.

Copper concentration (µg Cu/g of wet tissue) in brain sub regions of WISTAR rats after 2-, 4- and 6-weeks of dietary copper deficiency (mean ± SEM)

Groups Frontal Parietal Occipital Cerebellum
Copper (µg Cu/g of wet tissue)
2 NC 3.27 ± 0.019 3.47 ± 0.016 3.17 ± 0.012 2.39 ± 0.013
2 CC 3.25 ± 0.026 3.47 ± 0.014 3.16 ± 0.015 2.37 ± 0.010
2 PF 3.23 ± 0.014 3.42 ± 0.015 3.14 ± 0.011 2.34 ± 0.012
2 CD 2.45 ± 0.015c*e*f* 2.77 ± 0.011c*e*f* 2.52 ± 0.017c*e*f* 2.09 ± 0.012c*e*f*
4 NC 3.49 ± 0.027 3.70 ± 0.017 3.26 ± 0.026 2.65 ± 0.017
4 CC 3.47 ± 0.016 3.69 ± 0.021 3.27 ± 0.019 2.65 ± 0.021
4 PF 2.88 ± 0.011b*d* 3.37 ± 0.011b*d* 2.62 ± 0.020b*d* 2.20 ± 0.013b*d*
4 CD 1.98 ± 0.021c*e*f* 2.29 ± 0.012c*e*f* 1.83 ± 0.014c*e*f* 1.55 ± 0.017c*e*f*
6 NC 3.74 ± 0.02 3.95 ± 0.015 3.72 ± 0.013 2.90 ± 0.010
6 CC 3.76 ± 0.04 3.91 ± 0.018 3.68 ± 0.015 2.89 ± 0.012
6 PF 2.59 ± 0.010b*d* 3.19 ± 0.011b*d* 2.31 ± 0.015b*d* 2.17 ± 0.013b*d*
6 CD 1.22 ± 0.014c*e*f* 1.83 ± 0.017c*e*f* 1.39 ± 0.014c*e*f* 0.96 ± 0.016c*e*f*
Open in a new tab

Multiple comparison procedures were performed for 2-, 4- and 6 weeks experimental groups

*p < 0.05 significant

aNC versus CC

bNC versus PF

cNC versus CD

dCC versus PF

eCC versus CD

fPF versus CD

Discussion

Copper distribution varies in different regions in brain with variation being evident not only in species to species but also in age groups [26, 27]. Experimental evidence demonstrates copper dyshomeostasis (excess/deficiency) can enhance the generation of free radicals and oxidative insult which can result in neurodegenerative processes [28] and functional disorders [29, 30]. Dietary copper deficiency for 2-, 4- and 6-week in Wistar rats resulted in significant decrease in total protein levels of all brain sub-regions compared to their respective control groups (NC and CC) and pair fed groups (PF).These results are indicative of decreased protein synthesis of some proteins, nitrogen loss with abnormal utilization of amino acids and consequently modification of proteins leading to abnormal and damaged proteins due to misfolding and increased hydrophobicity of proteins leading to their aggregation [31, 32]. Increased hydrophobicity and oxidation of these modified proteins makes them susceptible to proteolysis [32] enhances its sensitivity to degradation by 20S proteasomes which in general would consequently decrease levels of the proteins [33].There was no marked change observed in pairfed groups of 2-week when compared with negative control and copper control groups this may happen as short term starvation/stress do not have any significant effect on protein content of brain since brain obtains a significant share of its oxidative energy from the ketone bodies, beta-hydroxybutyrate and acetoacetate, thus decreasing the consumption of glucose during starvation [34].

Proteolysis resistant aggregates of protein carbonyls in tissues have been implicated in the pathogenesis of various neurodegenerative diseases [35]. Further our study revealed significant increase in protein carbonyl in all the sub regions of brain tissue of copper deficient groups when compared to respective control groups (NC) and (CC) as well as pairfed (PF) groups which increased progressively with increase in duration of copper deficiency. This increase would lead to increased chemical modification of proteins and consequently to oxidative stress [36] disrupting the normal cellular function and physiological decline of body functions. Authors [37] observed that cytoskeleton proteins β-actin and β-tubulin are vulnerable to carbonylation resulting in proteolysis resistant aggregates of these in cells which further leads to their abnormal polymerization and depolymerization, leading to altered axonal growth, arborization and synaptic function of neurons.

The enhanced metabolism of free fatty acids in the myelin sheath makes the rodent brain susceptible to lipid peroxidation [38]. Copper deficiency reduced antioxidant potential of brain generating free radicals and it was reported that brain is the organ with highest lipid peroxidation among other organs tested [39]. Similar to their observation in whole brain significant increase in lipid peroxidation (MDA levels) in sub-regions of brain of copper deficient groups was observed which indicates oxidative damage due to increased free radical generation.

Wistar rats after copper deficiency resulted in significant decrease in glutathione concentration in all sub- regions of brain of copper deficient groups. The consumption of glutathione (GSH) in the present study accounts for overwhelming generation of free radical which led to its depletion and enhances the vulnerability towards oxidative stress. Similar observation was recorded by Ognik et al. [39], although they studied the whole brain and sub regions. GSH acts as the main potent antioxidant in the brain and plays a crucial role in cellular protection during oxidative stress, especially in neurons and glia [40]. In granule cells of cerebellum and PC12 cells depletion of GSH increased the oxidative stress and affected the normal mitochondrial functioning [41, 42]. Various studies have reported that a continuous decrease in reduced glutathione (GSH) makes the brain susceptible to oxidative stress mediated apoptosis of neurons [43].

Current study has shown a significant increase in activities of total SOD and Mn-SOD in brain sub-regions of copper deficient groups whereas Cu/Zn SOD significantly decreased in copper deficient groups as compared to their respective control groups (NC and CC) and PF groups suggesting a counteracting mechanism towards elevated oxidative stress accompanied by copper deficiency. Copper plays a crucial part in superoxide dismutase mediated antioxidant defense [44]. It has been reported that both zinc and copper deficiencies have been linked to reduced activities of the enzyme Cu–Zn SOD [45]. Oxidative stress increased after copper deficiency and when the O2 − is released further increase in the generation of ROS is evident, as a result there is not sufficient copper ions present for the dismutation reaction with the lysine residues in the enzymatic active site of Cu–Zn SOD [46]. Embryos grown in serum low in copper revealed a significant decline in the activity of superoxide dismutase and these embryos revealed high levels of superoxide anion concentrated in some parts of brain with malformations [47]. During the development period, i.e., gestation and lactation of offspring, the female rats were given Cu-inadequate diets, which continued even after weaning period, a moderate decrease in cerebellar Cu/Zn-SOD activity was observed [26]. Similar results were observed in other animal models. In native sheep a marked decline in the activities of Cu/Zn superoxide dismutase was observed in copper deficient group as compared to control groups [48]. Prohaska et al. [49], reported decreased levels of SOD1 protein in brain of Holtzman rats and ND4 Swiss Webster mice from the copper deficient groups as compared to their normal counterparts. Immunohistochemical staining indicated decreased expression of Cu–Zn SOD in the Purkinje cells of MD patients as a consequence of oxidative insult caused by free radicals [50]. Decline in Cu–Zn SOD activity in brain sub regions after dietary copper deficiency could probably be due to (1) low availability of copper to maintain its expression (2) being highly sensitive to oxidative stress and (3) neuronal nitration on account of endogenous formation of O2.− / peroxynitrite [51]. MnSOD is found in mitochondria that plays a crucial role in antioxidant defense in the central nervous system against oxidative stress [52], protecting neurons against apoptosis and neuronal degeneration [53]. The mitochondrial protein influx (MPI) can increase the expression of the MnSOD gene and enhance MnSOD activity by post-translational modifications induced by increased ROS [54]. In Obex tissue of copper deficient calves the antioxidant status was not compromised but a significant decrease in Cu–Zn SOD activity and increased MnSOD activity was observed [55]. Copper deficiency has been found to increase transcriptional rate of MnSOD [56]. Enhanced MnSOD in the present study would probably protect the brain cells from degeneration by decreasing the superoxide anion levels concentration as well as peroxynitrite formation.

Studies have reported that hydrogen peroxide formation due to mitochondrial superoxide leakage is involved in oxidative stress induced neuronal injury with catalase being more effective than SOD as well as glutathione peroxidase in neurons against oxidative stress [8]. Catalase is involved in supressing oxidative stress activated TRP channel activities thus preventing unwanted cellular remodelling that leads to neurological dysfunction [57]. Catalase activity was enhanced in all brain sub regions after dietary copper deficiency which might be attributable to (1) enhanced formation of hydrogen peroxide, which in turn has the potential to form hydroxyl radicals by Fenton reaction, or (2) counteracting against increased ROS by disposing of hydrogen peroxide.

In present study, a marked decline in copper concentration in brain sub-regions of copper deficient groups after 2-, 4- and 6-week was observed. These results are consistent with previous reports by Zucconi et al. [58] and Gybina et al. [59]. Dietary copper intake directly affects tissue copper levels [29, 60] The observed decrease in copper levels of PF groups can be due to starvation effect as food restriction can affect copper management [61].

Various data suggest that the level of antioxidant enzymes varied in different regions of the brain [62] as it shows regional variations in function and metabolic activities. In the present study, cerebellum appeared to be more sensitive to oxidative insult compared to other brain sub-regions evaluated. Especially granule cells of cerebellum are more vulnerable to oxidative stress in study carried out by Wang et al. [63] and as a result these are proposed to be the first to undergo degeneration. The present study suggests that dietary copper deficiency increases the susceptibility to oxidative stress of the brain in a region-specific and duration-dependent manner.

Conclusion

Dietary copper deficiency during the prepubertal period can have detrimental effects and modify the biochemical state of the Wistar rat brain sub-regions. The increase in oxidative stress accompanying copper deficiency may probably be responsible for altered antioxidant status of brain which can subsequently lead to neurodegeneration. These findings indicate that the copper has a critical role in brain.

Acknowledgements

The authors gratefully acknowledge Department of Zoology, Centre for Advanced Studies, University of Rajasthan, Jaipur, India for providing the necessary facilities.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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