Ana Cecilia Anzulovich

Cadmium-induced oxidative stress and histological damage in the myocardium. Effects of a soy-based diet

Cd exposure has been associated to an augmented risk for cardiovascular disease. We investigated the effects of 15 and 100 ppm of Cd on redox status as well as histological changes in the rat heart and the putative protective effect of a soy-based diet. Male Wistar rats were separated into 6 groups and treated during 60 days as follows: groups (1), (2) and (3) were fed a casein-based diet; groups (4), (5) and (6), a soy-based diet; (1) and (4) were given tap water; (2) and (5) tap water containing 15 ppm of Cd²⁺; and (3) and (6) tap water containing 100 ppm of Cd²⁺. Serum lipid peroxides increased and PON-1 activity decreased in group (3). Lipoperoxidation also increased in the heart of all intoxicated groups; however protein oxidation only augmented in (3) and reduced glutathione levels diminished in (2) and (3). Catalase activity increased in groups (3) and (6) while superoxide dismutase activity increased only in (6). Glutathione peroxidase activity decreased in groups (3) and (6). Nrf2 expression was higher in groups (3) and (6), and MTI expression augmented in (3). Histological examination of the heart tissue showed the development of hypertrophic and fusion of cardiomyocytes along with foci of myocardial fiber necrosis. The transmission electron microscopy analysis showed profound ultra-structural damages. No protection against tissue degeneration was observed in animals fed the soy-based diet. Our findings indicate that even though the intake of a soy-based diet is capable of ameliorating Cd induced oxidative stress, it failed in preventing cardiac damage.

Effect of nutritional vitamin A deficiency on lipid metabolism in the rat heart: Its relation to PPAR gene expression.

OBJECTIVE We studied the effect of dietary vitamin A deprivation on lipid composition and mRNA expression of regulatory enzymes involved in rat heart energetic lipid metabolism and its relation to the expression of peroxisome proliferator-activated receptor (PPAR) and retinoid X receptor (RXR) genes. METHODS Male Wistar 21-d-old rats were fed for 3 mo with a vitamin A-free diet (vitamin A-deficient group) and the same diet plus 8 mg of retinol palmitate per kilogram of diet (control group). One group of deficient animals received the control diet 15 d before sacrifice (vitamin A-refed group). Heart ventricular and mitochondrial lipid contents were determined. Lipid synthesis was measured using radioactive precursors and acetyl-coenzyme A carboxylase and mitochondrial carnitine palmitoyltransferase-I (CPT-I) activities using radioactive substrates. Fatty acid composition of mitochondrial phospholipids was analyzed by gas-liquid chromatography. Heart expression of acetyl-coenzyme A carboxylase, CPT-I, PPAR-alpha, PPAR-beta, RXR-alpha, and RXR-beta was assessed by reverse transcriptase polymerase chain reaction, and CPT-I expression was also measured by real-time polymerase chain reaction. RESULTS Vitamin A deficiency induced changes in heart ventricular lipid content and synthesis. Mitochondrial cardiolipin decreased and the proportion of phospholipids/saturated fatty acids increased. Heart activity and mRNA levels of CPT-I and expression of PPAR-alpha and PPAR-beta genes were enhanced, whereas acetyl-coenzyme A carboxylase activity diminished. Furthermore, vitamin A deficiency decreased heart mRNA levels of RXRs. Vitamin A refeeding reverted most of the observed changes. CONCLUSION Lipid metabolism is significantly modified in hearts of vitamin A-deficient rats. Alteration of mitochondrial energetic processes by modifying the activity and gene expressions of the regulatory enzymes is associated with a high PPAR expression induced by vitamin A deprivation.

Vitamin a deficiency modifies antioxidant defenses and essential element contents in rat heart

Abstract Oxidative stress in the heart of male rats fed on a vitamin A-deficient diet for three months from 21 days of age was evaluated. Vitamin A restriction produced subclinical plasma retinol concentration (0.7 μmol/l), negligible liver retinol stores and a significant decrease of heart retinol concentration, as determined by HPLC, compared with the control group receiving the same diet with 4000 IU of vitamin A (8 mg retinol as retinyl palmitate) per kg of diet. Vitamin A-deficient rats had lower body weight than control rats. No change in the heart weights was observed. In serum of rats fed the vitamin A-deficient diet the concentrations of thiobarbituric acid-reactive substances (TBARS) and reduced glutathione (GSH) increased by ∼65% and 60%, respectively, compared with those fed on the control diet. In hearts of vitamin A-deficient rats the concentration of TBARS and the glutathione peroxidase activity significantly increased. The activity of catalase decreased by ∼30%, while the superoxide dismutase did not change. Additionally, vitamin A-deficient rats showed lower heart concentrations of the trace elements selenium, manganese and chromium. Heart iron, copper and zinc concentrations were not affected. Vitamin A deprivation for 3 months decreased the plasma and heart retinol concentrations associated with changes in non-enzymatic and enzymatic antioxidant defense system components.

Myocardial oxidative stress following sub-chronic and chronic oral cadmium exposure in rats.

We investigated the effect of oral cadmium intoxication on the antioxidant/prooxidant status in serum and heart. Wistar rats, separated into four groups, that received: (1) tap water for 60 days (control); (2), (3) and (4) Cd(2+) (15 ppm)-containing water, during 15, 30 and 60 days, respectively. Lipoperoxidation increased in serum and heart of group 4. Circulating paraoxonase-1 activity was higher in groups 2 and 3. Protein carbonyl-groups increased while total and reduced glutathione levels decreased in the heart after 15 days of cadmium intoxication. Cardiac catalase activity was higher in groups 3 and 4 but glutathione peroxidase activity diminished in the heart of all poisoned groups. Superoxide dismutase transcript levels as well as Nrf2 expression also increased in the heart of groups 2 and 3, while gp91phox and p47phox mRNA levels rose only in group 3. We suggest cadmium intoxication modifies antioxidant/prooxidant ratio in serum and heart in a time-of-exposure-dependent way.

Nutritional vitamin A deficiency alters antioxidant defenses and modifies the liver histoarchitecture in rat.

Oxidative stress and morphologic changes in the liver of female rats fed on a vitamin A-deficient diet for 3 months from 21 days of age were evaluated. Vitamin A restriction produced subclinical plasma retinol concentration (0.6 μmol–L) and negligible liver retinol stores, as determined by high-performance liquid chromatography, compared to the control group, which received the same diet with 4000 IU of vitamin A (8 mg retinol as retinyl palmitate) per kg of diet. Vitamin A-deficient rats had lower body weight than control rats. No change in the liver weights was observed. In the liver of rats fed the vitamin A-deficient diet, the concentrations of thiobarbituric acid-reactive substances increased by ∼50%, and the activities of catalase and glutathione peroxidase increased by 30% and 40%, respectively, compared with those fed on the control diet. Liver superoxide dismutase activity did not change. Additionally, vitamin A-deficient rats showed higher liver concentrations (μg/g tissue) of iron (316.03 ± 12.1 vs 203.12 ± 9.5) and lower concentrations of the antioxidant trace elements zinc (15.50 ± 1.03 vs 26.63 ± 0.25) and copper (2.27 ± 0.22 vs 4.25 ± 0.37) than those of control. Liver selenium concentration was not affected. Morphologically, livers from vitamin A-deficient rats showed a partial hepatic cord disarrangement and an increased number of hepatocytes in process of involution with piknotic nuclei and condensed cytoplasm. A reduced number of fat-hepatocytes distributed at random was observed. Portal spaces showed evidence of edema and a marked infiltration by mononuclear as well as polimorphonuclear cells. Loose portal connective tissue increased, as did the number of fibroblast-like cells in portal spaces. Vitamin A deprivation for 3 months decreased the liver retinol concentration associated with marked histological alterations in the organ, which could be due, at least in part, to the increase in oxidative stress. J. Trace Elem. Exp. Med. 13:343–357, 2000.

Retinoic acid receptors move in time with the clock in the hippocampus. Effect of a vitamin-A-deficient diet☆

An endogenous time-keeping mechanism controls circadian biological rhythms in mammals. Previously, we showed that vitamin A deficiency modifies clock BMAL1 and PER1 as well as BDNF and neurogranin daily rhythmicity in the rat hippocampus when animals are maintained under 12-h-light:12-h-dark conditions. Retinoic acid nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), have been detected in the same brain area. Our objectives were (a) to analyze whether RARα, RARβ and RXRβ exhibit a circadian variation in the rat hippocampus and (b) to investigate the effect of a vitamin-A-deficient diet on the circadian expression of BMAL1, PER1 and retinoic acid receptors (RARs and RXRβ) genes. Holtzman male rats from control and vitamin-A-deficient groups were maintained under 12-h-light:12-h-dark or 12-h-dark:12-h-dark conditions during the last week of treatment. RARα, RARβ, RXRβ, BMAL1 and PER1 transcript and protein levels were determined in hippocampus samples isolated every 4 h in a 24-h period. Regulatory regions of RARs and RXRβ genes were scanned for clock-responsive sites, while BMAL1 and PER1 promoters were analyzed for retinoic acid responsive elements and retinoid X responsive elements. E-box and retinoid-related orphan receptor responsive element sites were found on regulatory regions of retinoid receptors genes, which display an endogenously controlled circadian expression in the rat hippocampus. Those temporal profiles were modified when animals were fed with a vitamin-A-deficient diet. Similarly, the nutritional vitamin A deficiency phase shifted BMAL1 and abolished PER1 circadian expression at both mRNA and protein levels. Our data suggest that vitamin A deficiency may affect the circadian expression in the hippocampus by modifying the rhythmic profiles of retinoic acid receptors.

Ovaric physiology in the first oestral cycle: influence of noradrenergic and cholinergic neural stimuli from coeliac ganglion.

Both peripheral innervation and nitric oxide (NO) participate in ovarian steroidogenesis. The aims of the work were (1) to investigate whether ganglionic noradrenergic (NE) and cholinergic (Ach) stimulus modify the ovarian steroids and NO release and (2) to examine the effect of those stimuli on the mRNA expression of 3beta-HSD and P450 aromatase in the ovary. The experiments were carried out using the ex vivo coeliac ganglion-superior ovarian nerve-ovary (CG-SON-O) system of rats in the first oestral cycle. The system was incubated in a buffer solution for 120min, with the ganglion and ovary located in different compartments and linked by the SON. NE and Ach were added into the ganglion compartment. Both NE and Ach predominantly induced ovarian release of androstenedione and oestradiol while inhibited progesterone release. Ovarian NO release increased after ganglionic stimulation during proestrous while its secretion decreased during the diestrous. Noteworthily, 3beta-HSD and P450 aromatase expression were modulated by neural stimulation. In the follicular phase, Ach in CG increased 3beta-HSD and NE increased P450 aromatase. In the luteal phase both neurotransmitters increased 3beta-HSD and Ach increased P450 aromatase transcript levels. All above observations suggest that the preponderancy of an either noradrenergic or cholinergic effect would depend on the stage of the first oestral cycle in the rat. The ovarian response to noradrenergic and cholinergic stimuli on GC, via SON, is strongly linked to oestral-stage-specific ovarian structures and their secretion products.

Daily oscillation of glutathione redox cycle is dampened in the nutritional vitamin A deficiency

Examples of hormonal phase-shifting of circadian gene expression began to emerge a few years ago. Vitamin A fulfills a hormonal function by binding of retinoic acid to its nuclear receptors, RARs and RXRs. We found retinoid- as well as clock-responsive sites on regulatory regions of Glutathione reductase (GR) and Glutathione peroxidase (GPx) genes. Interestingly, we observed retinoid receptors, as well as GSH, GR, and GPx, display daily oscillating patterns in the rat liver. We also found that feeding animals with a vitamin A-free diet, dampened daily rhythms of RARα and RXRβ mRNA, GR expression and activity, GSH, BMAL1 protein levels, and locomotor activity. Differently, day–night oscillations of RXRα, GPx mRNA levels and activity and PER1 protein levels, were phase-shifted in the liver of vitamin A-deficient rats. These observations would emphasize the importance of micronutrient vitamin A in the modulation of biological rhythms of GSH and cellular redox state in liver.

Circadian rhythms of locomotor activity and hippocampal clock genes expression are dampened in vitamin A-deficient rats

The main external time giver is the day-night cycle; however, signals from feeding and the activity/rest cycles can entrain peripheral clocks, such as the hippocampus, in the absence of light. Knowing that vitamin A and its derivatives, the retinoids, may act as regulators of the endogenous clock activity, we hypothesized that the nutritional deficiency of vitamin A may influence the locomotor activity rhythm as well as the endogenous circadian patterns of clock genes in the rat hippocampus. Locomotor activity was recorded during the last week of the treatment period. Circadian rhythms of clock genes expression were analyzed by reverse transcription-polymerase chain reaction in hippocampus samples that were isolated every 4 hours during a 24-hour period. Reduced glutathione (GSH) levels were also determined by a kinetic assay. Regulatory regions of clock PER2, CRY1, and CRY2 genes were scanned for RXRE, RARE, and RORE sites. As expected, the locomotor activity pattern of rats shifted rightward under constant dark conditions. Clock genes expression and GSH levels displayed robust circadian oscillations in the rat hippocampus. We found RXRE and RORE sites on regulatory regions of clock genes. Vitamin A deficiency dampened rhythms of locomotor activity as well as modified endogenous rhythms of clock genes expression and GSH levels. Thus, vitamin A may have a role in endogenous clock functioning and participate in the circadian regulation of the cellular redox state in the hippocampus, a peripheral clock with relevant function in memory and learning.

Daily rhythms of catalase and glutathione peroxidase expression and activity are endogenously driven in the hippocampus and are modified by a vitamin A-free diet

Abstract Objectives Alterations in enzymatic antioxidant defense systems lead to a deficit of cognitive functions and altered hippocampal synaptic plasticity. The objectives of this study were to investigate endogenous rhythms of catalase (CAT) and glutathione peroxidase (GPx) expression and activity, as well as CREB1 mRNA, in the rat hippocampus, and to evaluate to which extent the vitamin A deficiency could affect those temporal patterns. Methods Rats from control and vitamin A-deficient (VAD) groups received a diet containing 4000 IU of vitamin A/kg diet, or the same diet devoid of vitamin A, respectively, during 3 months. Rats were maintained under 12-hour-dark conditions, during 10 days before the sacrifice. Circadian rhythms of CAT, GPx, RXRγ, and CREB1 mRNA levels were determined by reverse transcriptrase polymerase chain reaction in hippocampus samples isolated every 4 hours during a 24-hour period. CAT and GPx enzymatic activities were also determined by kinetic assays. Regulatory regions of clock and antioxidant enzymes genes were scanned for E-box, RXRE, and CRE sites. Results E-box, RXRE, and CRE sites were found on regulatory regions of GPx and CAT genes, which display a circadian expression in the rat hippocampus. VAD phase shifted CAT, GPx, and RXRγ endogenous rhythms without affecting circadian expression of CREB1. Discussion CAT and GPx expression and enzymatic activity are circadian in the rat hippocampus. The VAD affected the temporal patterns antioxidant genes expression, probably by altering circadian rhythms of its RXR receptors and clock factors; thus, it would impair the temporal orchestration of hippocampal daily cognitive performance.