Animals and diets
The experimental procedures involving rats were performed in accordance with the guidelines of the Brazilian Society of Science in Animals of Laboratory (SBCAL) and were approved by the Ethics Committee at Mato Grosso Federal University (Process Nº 23 108 013713/09-2). Male and virgin female Wistar rats (85–90 d old) were obtained from the university’s breeding colony. Mating was performed by housing one male with four females overnight, and pregnancy was confirmed by examining vaginal smears for the presence of sperm. Pregnant females were randomly separated and from the first day of pregnancy until the end of lactation, were fed isocaloric diets containing 17%
(control [C]) or 6% (low-protein [LP]) casein as the protein source.
Spontaneous delivery occurred on day 22 of pregnancy, and at 3 d of age, large litters were reduced to eight pups to ensure a standard litter size for each mother. After weaning (week 4), the males were divided into five groups: CC (offspring that were fed a C diet following weaning after being born to and suckled by mothers that were fed a C diet), CS (offspring that were fed a soybean diet with 17% protein following weaning after being born to and suckled by mothers that were fed a C diet), LL (offspring that were fed an LP diet following weaning after being born to mothers that were fed an LP diet), LC (offspring that were fed a C diet following weaning after being born of
mothers that were fed an LP diet), and LS (offspring that were fed a soybean diet containing 17% protein following weaning after being born of mothers that were fed an LP diet). The rats were kept four animals per cage, given free access to food and water and kept under standard lighting conditions (12-h light/dark cycle) at 24°C throughout the experimental period.
All diets used in this study are described in Table 1. In the soybean diet, adjustments, which included removing soybean oil and fiber, were made to match the carbohydrate, lipid, and fiber content and the energy value contained in the casein diet. Whole, inactivated soybean flour was obtained by industrial processing (i.e., thermal treatment, peeling, grinding, and micronization), which reduced the content of enzymatic and antitrypsin factors so that the soybean flour contained 80% of the nutritional value of animal casein.
The rats were weighed after weaning and at the end of the experimental period when they were 120 d old. Because it was not possible to evaluate all variables in the same animal, the number of individual experiments varied among the groups, but it was representative of at least four different litters. After decapitation, blood samples were collected and the liver tissue samples were quickly removed, frozen immediately in liquid nitrogen, and stored at – 80 °C until the assays.
Serum metabolic and hormonal profile
The rats were decapitated, the blood samples were collected, the serum was obtained by centrifugation, and aliquots were used to measure serum glucose levels (portable glucose meter, Accu-Chek®, Roche Diagnostics, Germany). Hepatic enzymes and aminotransferase concentrations were also assessed (BT-3000 Plus, Wiener Lab, Rosario, Argentina). Serum norepinephrine, epinephrine and dopamine concentrations
were determined using high performance liquid chromatography (HPLC) (QuantaSep 5000 LX, CA, USA).
RNA preparation and real-time RT-PCR
Total RNA was extracted from the liver samples using Trizol reagent (Invitrogen, USA), according to the manufacturer's instructions, and 3 µg of total RNA were transcribed into cDNA using high capacity reverse transcriptase (Applied Biosystems).
Primers specific for rat catalase (RN00689381_G1), glutathione peroxidase (RN00577994_G1), cytosolic (RN00566938_M1) and mitochondrial (RN00566942_G1) superoxide dismutase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Applied Biosystems. GAPDH was used as an endogenous control. PCR was carried out in duplicate on a Step One using Taqman Gene Expression Master Mix (Applied Biosystems). The cDNA was amplified under the following conditions: denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 15 s, with a final extension at 72 °C for 10 min. Real-time data were analyzed using the Step One System (Applied Biosystems).
Measurement of antioxidant enzyme activity and markers of oxidative stress
The liver CAT activity was measured using the method described by Aebi et al.
1984[11]. The reaction mixture contained 4.5 µg/mL protein and 20 mM hydrogen peroxide (H2O2) in a 50 mM potassium phosphate buffer, pH 7.0. The decomposition of H2O2 was monitored at 240 nm at 25ºC for 3 min and calculated using a molar absorption coefficient of 36 M-1cm-1.
The activity of SOD was measured using the method described by McCord & Fridovich
[12]. The superoxide anion is generated by a xanthine/xanthine oxidase system, and the reduction of cytochrome c was monitored at 550 nm. Enzyme activity was expressed as SOD units, where one unit is defined as the amount of enzyme needed to inhibit 50% of cytochrome c reduction per min and per mg of protein at 25 °C and pH 7.80.
Reduced glutathione (SH) was estimated based on the determination of non-protein thiol contained in the sample. Tissue samples were homogenized in 500 µL of 0.02 M EDTA and maintained on ice. Samples were protected from direct light for the remainder of the procedure. The homogenate was centrifuged at 4,000 g for 15 minutes at 4 ºC, and the supernatant was incubated with 50 µL of 10% TCA at 4 ºC for 60 minutes. Following the incubation, the samples were centrifuged at 12,000 g for 5 minutes at 4ºC, and the supernatant was collected to determine the non-protein thiol content in the sample. The reaction was started by adding 200 µL of Tris/HCl (200 mM, pH 8.9) and 20 µL of DTNB (2.5 mM) to 100 µL of sample in a microplate and incubating at room temperature for 5 minutes[13]. The resulting color change was measured at 405 nm with a FisherBiotech Microkinetics Reader BT 2000 (Pittsburgh, PA). Cysteine was used as a standard, and the thiol (SH) content in the samples was corrected based on the total protein content of the sample[14].
Lipid peroxidation was quantified using the Calbiochem® Lipid Hydroperoxuide (LPO) assay kit, according to the manufacturer's instructions.
The thiobarbituric acid reactive species (TBARS) reaction was performed as described elsewhere[15, 16]. The solution color resulting from the reaction was measured at 535 nm using a Hitachi U-2001 Spectrophotometer (Sataiama, Japan). Malondialdehyde (MDA)
was used as a standard to determine the MDA level in the sample. The TBARS content of the sample was corrected based on the protein content of the samples.
Protein carbonyl levels were assayed using a previously described method[17], with minor modifications. Initially, each liver sample was homogenized in 500 µL of phosphate buffer (50 mM, pH 7.4), and the total amount of protein was measured and adjusted to 1 mg/mL[14]. Then, 0.5 mL of 10% trichloroacetic acid was added, and the sample was centrifuged at 5000×g for 5 min. The supernatant was discarded.
Afterwards, 0.5 mL of 10 mM 2,4-dinitrophenylhydrazine (DNPH) was added to the test group tube, and 0.5 mL of HCl (2 M) was added to the white tube. Samples were then incubated at room temperature for 1 h. After the incubation, 0.5 mL of 10% TCA was added, and the mixture was centrifuged at 5000×g for 5 min. After centrifugation, the pellets were washed three times with 1 mL of ethanol-ethyl acetate (1:1, v/v). The precipitate was dissolved in 1.5 mL of protein-dissolving solution (10% SDS) and incubated for 15 min. The color intensity of the supernatant was measured using a spectrophotometer at 370 nm. The carbonyl content was calculated using a molar extinction coefficient (22×10−3 M cm−1), and the results were expressed as nmol/mg protein.
To determine the nitric oxide content in each liver sample, we used the method described by Miranda et al. 2001[18] for nitrite/nitrate (NO) measurement, which is a stable metabolite of nitric oxide. Briefly, the liver samples were homogenized in phosphate buffer (50 mM, pH 7.4), and 200 µL of sample was incubated with 100 µL of acetonitrile and 100 µL of ZnSO4 (200 mM). Then, the samples were incubated on ice for 1 hour and centrifuged at 16,000 x g for 10 min at 4°C. The reaction was started by mixing 75 µL of the sample with 25 µL of Gries Reagent (1:1 NEED 0.16 and sulfanilamide 3.3%) and 25 µL of VCl3 (3.3%); the mixture was then incubated at
37°C, protected from light, for 1 hour. NaNO3 and NaNO2 were used as standards, and the color that formed was read at 570 nm.
Statistical analysis
The results are expressed as the mean±standard deviation for the number of rats indicated. Leven’s test for the homogeneity of variances was initially used to determine whether the data complied with the assumptions necessary for a parametric analysis of variance. When necessary, the data were log transformed to correct for variance in heterogeneity or non-normality[19]. A two-way analysis of variance (effect of previous nutritional status and diet) was used to compare the data from the CC, CS, LC and LS groups. A one-way analysis of variance was used to assess whether the diets were effective in improving the nutritional status of the LC, LS and LL groups. When necessary, these analyses were complemented by the least significant difference (LSD) test to determine the significance of the individual differences. A P<0.05 indicated statistical significance. All statistical comparisons were conducted using the Statistic Software package (Stat-soft, Tulsa, OK, USA).
RESULTS
At the beginning of the recovery phase, the offspring born to and suckled by mothers fed a low protein diet (LC, LS and LL groups) had similar body weights that were significantly lower than those of offspring born to and suckled by mothers fed a control diet (CC and CS groups). At the end of the experimental period, the body weights were significantly lower in the LS and the LC groups than in the CS and the CC groups (F1,27
= 106.5, P < 0.0001). In addition, the rats in the LS and CS groups had lower final body weights than those in the LC and CC groups (F1,27 = 4.53, P < 0.05). The assessment of nutritional recovery showed that the LS and LC rats had similar final body weights, and
both were significantly higher than the LL rats (P < 0.0001). In the basal state, the serum glucose concentration in the LS rats was lower than in the LC rats, and both exhibited higher glycemia than the CC rats. The serum glucose concentration was similar in the CS and CC rats. The serum ALT concentration was similar in the LS, LC and CC groups; the CS group showed a higher serum ALT concentration in relation to the CC and LS rats, but not different to the LC rats. The serum aspartate aminotransferase (AST), γglutamyl transpeptidase (γGT), epinephrine, norepinephrine and dopamine levels did not differ among groups. Serum alkaline phosphatase (ALP) concentrations were similar in the LS and LC rats and both were lower than the LL rats (P<0.02) (Table 2).
The expression of CAT mRNA was higher in liver samples from the LS and CS groups than in the LC and CC groups (F1,15 = 6.95, P < 0.02). The LS group had higher levels of liver CAT mRNA transcripts than the LC group and the latter was higher than the LL group (Figure 1A). The liver GPx mRNA expression did not differ among the LS, LC, CS and CC groups. Both the LS and LC rats had higher liver GPx mRNA levels than the LL rats (Figure 1B). The expression of SOD1 and SOD2 mRNA did not differ among groups (data not shown).
SOD activity in the liver was lower in the LS and LC groups than in the CS and CC groups (F1,12 = 6.24, P < 0.03), as well as in the LS and CS groups compared to the LC and CC groups (F1,12 = 10.44, P < 0.01). There was no difference in the SOD activity among the LC, LS and LL groups (Figure 2A). In the LS and CS rats, liver CAT activity was lower compared to the LC and CC rats (F1,16 = 5.68, P < 0.03). Liver CAT activity was similar in the LS and LC groups, and both were higher compared to the LL group (P<0.01) (Figure 2B). The liver SH content was lower in the LS and CS rats than
in the LC and CC rats (F1,16 = 7.10, P < 0.02). The LS groups had lower liver SH levels than the LC and LL groups (Figure 2C).
The lipid hydroperoxide content in the liver was similar in all groups (Figure 3A). The liver MDA (Figure 3B), protein carbonyl (Figure 3C) and NO (Figure 3D) concentrations did not differ among the LS, LC, CS and CC groups. However, these parameters were lower in the LS and LC groups compared to the LL group (P<0.0001) (Figure 1B-1D).
DISCUSSION
In the present study, we investigated whether a soybean flour diet used for nutritional recovery in male Wistar rats subjected to a low protein diet during intrauterine life and lactation interferes with liver antioxidant defenses and prevents hepatocellular damage.
As previously reported[20,9], using the same animal model, nutritional rehabilitation after weaning did not correct the body weight deficit, regardless of the protein type, and a soybean diet reduced the final body weight of both control and recovered rats. The lean profile of the rats maintained on a soybean diet was accompanied by a reduced liver lipid content that was attributed to decreased lipogenesis and increased β-oxidation[9]. It has been reported that the oxidation of fatty acids is an important source of ROS[8,21]
and affects the expression of scavenger enzymes, especially CAT[22]. Based on these observations, our animals that were fed a soybean diet exhibited increased CAT mRNA levels; however, CAT enzyme activity decreased. SOD and GPx mRNA transcription was not altered, but SOD activity was decreased in the soybean diet-fed animals. Our results are consistent with the observation that soy protein increases CAT mRNA expression and that soy isoflavones decrease SOD activity[23]. Long-term protein restriction decreased CAT and GPx mRNA expression, reduced CAT activity and did
not affect SOD mRNA expression and activity. These animals also exhibited elevated oxide nitric levels. It is known that nitric oxide interacts with the heme group of CAT, inhibiting its activity[24]. Interestingly, nutritional recovery increased CAT and GPx mRNA expression as well CAT activity, independently of protein type. Alterations in the mRNA levels of these enzymes could arise from changes in transcription, mRNA processing or mRNA turnover. SOD, CAT and GPx play an important role in free radical detoxification and their absence or a decrease in their activity may induce noxious metabolic outcomes. The superoxide anion formed during the detoxification process is removed by CAT and GPx[25]. Thus, it is reasonable to propose that nutritional recovery implies more efficient detoxification of H2O2 compared to the protein restriction status. This hypothesis was confirmed by reduced levels of malondialdehyde and protein carbonyls, known markers of lipid peroxidation and protein oxidation, respectively[26], as well as decreased nitric oxide levels (reactive nitrogen species) in recovered rats compared to low-protein rats. The apparent protection of oxidative damage observed in recovered animals and in those maintained on a soybean diet may be related to the presence of non-enzymatic antioxidants provided by the mineral and vitamin mix from the diet (such as vitamin E and selenium, both present in the diets) that compensated for the reduced antioxidant enzyme activities. Vitamin E is a major antioxidant responsible for terminating free radical chain reactions that result from the oxidation of polyunsaturated fatty acids[27]. Selenium has been identified as an essential cofactor for selenoproteins[28]. Dietary deficiencies of selenium decrease tissue GPx activity by 90% and result in peroxidative damage and mitochondrial dysfunction[29]. Another plausible explanation is that the reduced amount of peroxidizable lipids in cellular membranes and/or decreased liver lipid contents may have contributed to lower free radical damage in our recovered
animals. Although the low-protein rats also benefited from the dietary antioxidants, the presence of fatty liver as previously observed[9] may have contributed to increased oxidative damage.
The apparent protective effect of a soybean diet against oxidative damage was ruled out by the reduction in the non-protein thiol levels in the animals maintained on a soybean diet as well as the increase in the serum ALT concentration in the control soybean group. The major non-protein thiol compound, both intracellular and extracellular, is reduced glutathione (GSH). Thiol compounds, especially glutathione, are responsible for neutralizing several types of reactive species, such as nitric oxide and superoxide[30]. Thus, a reduction in non-protein thiol levels in soybean-treated animals may indicate the consumption of non-protein thiol by enhanced free radical generation. An increased activity of ALT is an indicator of liver damage. When the liver is damaged, these enzymes leak out of liver cells in large quantities and the concentration in the blood is increased[31].
CONCLUSION
Based on these results it is reasonable to conclude that a soybean diet (with sulphuretted amino acid addition) suppressed the hepatic antioxidant defense capacity and compromised the hepatic function in offspring that were fed a soybean diet following weaning after they were born to and suckled by mothers fed a control diet. However, it was as efficient as casein for recovering weight gain and the antioxidant status of the liver, without alteration of stress parameters (epinephrine, norepinephrine, and dopamine) or signs of toxicity (i.e., alterations in serum γGT, AST, and ALP). Although some studies suggest increased β-oxidation in soy-maintained animals, we found no
support for this as well as no indication that the soy diet is superior to casein in stimulating the antioxidant/scavenger system.
Acknowledgments
The authors are grateful to Celso Roberto Afonso for his excellent technical assistance.
This work was supported by the Brazilian foundations CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Grant number: 620106/2008-5), FAPEMAT (Fundação de Amparo à Pesquisa do Estado de Mato Grosso, grant number:40175/2009) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) . Celso Roberto Afonso provided technical assistance for the study.
This work is part of a dissertation presented by Ilkilene Taques Camargo Oliveira as a partial requirement for the Master’s degree in Biosciences at the College of Nutrition, UFMT.
Contributors
ITCO, SRLR and KCS carried out the conduced experiments, performed Real time PCR. MFR and JF conduced the analysis of stress oxidative markers. ITCO, MQL and LFS contributed equally to this paper in various aspects of this study. LFS and MQL conceived of the study and designed this study. ITCO drafted the manuscript along with the other authors. All authors read and approved the final manuscript. The authors have no conflict of interest.
Conflict of interest
The authors declare no conflict of interest.
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