4-Phenylselanyl-7-chloroquinoline attenuates hepatic injury triggered by neonatal exposure to monosodium glutamate in rats

Karline da Costa Rodrigues a, Cristiani Folharini Bortolatto b, Renata Leivas de Oliveira a, Jaini Janke Paltian a, Allya Larroza c, Mauro Pereira Soares d, Diego Alves c, Ethel Antunes Wilhelm a,*,1, Cristiane Luchese a,*,1

Abstract

Aims: Obesity is associated with a spectrum of hepatic abnormalities that can be experimentally induced by injections of monosodium glutamate (MSG) in neonatal rodents. We investigated the protective actions of the repeated therapy with 4-phenylselenyl-7-chloroquinoline (4-PSQ), a quinoline derivative containing selenium, on damage to the liver triggered by early postnatal administration of MSG in male Wistar rats.
Main methods: Neonatal rats received MSG (4 g/kg, subcutaneous route) or saline (1 ml/kg) from 5 to 14 postnatal day (PND) to induce obesity with consequent damages in the liver. 4-PSQ treatment (5 mg/kg) or canola oil (1 ml/kg) was administered from 60 to 76 PND by the intragastric route. On 76 PND, animals were anesthetized for blood and liver collection. Plasma markers of hepatic function, hepatic lipoperoxidation levels and histology analysis of liver tissue were assessed.

Key findings: Our data revealed that treatment with 4-PSQ reverted the increase in plasma transaminases activities observed in MSG rats. Treatment with 4-PSQ reduced plasma lactate levels in obese rats. In the liver, MSG elevated the content of lipoperoxidation which was reverted by 4-PSQ administrations. Lastly, 4-PSQ therapy attenuated the histological alterations induced by MSG.

Significance: Together, the results indicate a hepatoprotective action of repeated treatment with 4-PSQ in obese rats.

Keywords:
Selenium
Quinoline
Hypothalamic obesity
Hepatic damage
Thiobarbituric acid reactive species

1. Introduction

Obesity is characterized by excessive fat accumulation that presents a risk to health. Its prevalence has reached epidemic proportions globally affecting individuals of all sociodemographic strata [1]. Obesity is commonly associated with chronic conditions such as diabetes, metabolic syndrome, non-alcoholic fatty liver (NAFLD) disease, cancer and cardiovascular diseases [1,2]. The majority of the excess energy is stored as fat is stored in enlarged adipocytes, but some lipids may infiltrate other organs such as the liver (ectopic fat) [3].
Monosodium glutamate (MSG) is extensively used as an additive and flavoring agent worldwide for its umami taste and found in a wide range of processed foods. Its use has been questioned due to the incidence of side-effects in human adults, as manifested by the Chinese restaurant syndrome. Although the Food and Drug Administration (FDA) declared MSG safe, clinical trials of human and animal subjects also suggested various potential health hazards [4]. In rodents, MSG can cross the immature brain-blood barrier and lead to hypothalamic lesions triggering disturbances of the central endocrine axis. Neonatal MSG exposure results in adverse effects including obesity, diabetes, gonadal dysfunction and reduced growth [5], as well as hepatotoxic, neurotoxic and genotoxic effects [4]. Toxic doses of MSG affect peripheral tissues including the liver, the largest glandular organ with an important role for detoxification and metabolic homeostasis, causing inflammation, oxidative stress, and hepatocellular damage [6,7]. The inflammation in MSG animals occurs with the increase of gene expression of liver TNF-α and IL-6 as well as elevated plasma transaminases [6,8].
Selenium is an essential trace element acting as a constituent of selenoproteins. Selenoproteins plays critical roles in reproduction, thyroid hormone metabolism, deoxyribonucleic acid synthesis, and protection from oxidative damage and infection [9,10]. In recent years, selenium-based compounds have attracted special attention from the scientific community due to their biological actions including antioxidant, anorexigenic, anti-obesity and hepatoprotective activities [11–15]. In special, 4-phenylselenyl-7-chloroquinoline (4-PSQ), a quinoline derivative containing selenium, has anti-inflammatory, antinociceptive and antioxidant actions in rodents [16] with no apparent systemic toxicity, which is expected, when administered at therapeutic doses [17].
Therapeutic actions of 4-PSQ administrations in MSG rats were recently found by our research group. Improvements in obesity-related parameters, psychiatric symptoms, and cognitive impairments in MSG rats treated with 4-PSQ were observed [18]. However, a possible hepatoprotective action of 4-PSQ in MSG animals has not yet been evaluated. Therefore, the aim of the present study was to investigate the protective effects of repeated treatment with 4-PSQ on hepatic injury induced by neonatal injections of MSG in rats. For this purpose, plasma markers of liver function, as well as liver lipoperoxidation and histology, were assessed.

2. Material and methods

2.1. Chemicals and reagents

MSG was obtained from Sigma-Aldrich. 4-PSQ (Fig. 1) was prepared and characterized in our laboratory using the method previously described [19]. The chemical purity of 4-PSQ (99.9%) was determined by gas chromatography–mass spectrometry (GC/MS). 4-PSQ was dissolved in canola oil. All other chemicals were of analytical grade and obtained from standard commercial suppliers.

2.1.1. Chemical compounds in this article

Acetic acid (PubChem CID: 176); Eosin (PubChem CID: 11048); Formalin (PubChem CID: 712); Hematoxylin (PubChem CID: 442514); Isoflurane (PubChem CID: 3763); Monosodium glutamate (PubChem CID: 23672308); Sodium chloride (PubChem CID: 5234); Sodium dodecyl sulfate (PubChem CID: 3423265); 2-Thiobarbituric acid (PubChem CID: 2723628); Tris hydrochloride (PubChem CID:93573).

2.2. Animals

The experiments were carried out using male newborn Wistar rats from a local breeding colony. Animals were kept in a separate animal room, in controlled conditions with a constant temperature (22 ± 1 ◦C), a 12 h light/dark cycle (with the light turned on at 6:00 a.m.) and free access to water and food. At the end of the experimental protocol, the measures of body weight ranged from 250 to 350 g (initial body weight: control – 280 ± 25 g, 4-PSQ – 271 ± 44 g, MSG – 260 ± 43 g, MSG + 4- PSQ – 261 ± 19 g; final body weight: control – 302 ± 30 g, 4-PSQ – 304 ± 47 g, MSG – 268 ± 27 g, MSG + 4-PSQ – 296 ± 21 g) [18]. The experiments were approved by the Ethical Committee on Animal Experimentation of the Federal University of Pelotas, Brazil (CEEA 8358–2017), following the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). All efforts were made to minimize animal suffering and to reduce the number of animals used in the experiments.

2.3. Exposure protocol

The experimental design is illustrated in Fig. 2. Animals were divided into 4 experimental groups (N = 6 rats/group): group I: control, group II: 4-PSQ, group III: MSG and, group IV: 4-PSQ + MSG. Briefly, from the postnatal day (PND) 5 to 14 pups of groups III and IV received administrations of MSG (4 g/kg, by subcutaneous route, s.c., once a day) and pups of groups I and II received 0.9% saline (vehicle), in a fixed volume of 1 ml/kg as described by previous studies [20,21] with a minor modification. On PND 21, the animals were weaned. From PND 66 to 76 rats of groups II and IV were treated with 4-PSQ (5 mg/kg, intragastric route, i.g.) and rats of groups I and III were treated with canola oil (vehicle). The dose of 4-PSQ was based on previous studies of our research group [18,22,23].
At the end of the experimental protocol (PND 76), the animals were anesthetized with inhaled isoflurane. Blood was collected by heart puncture using heparin as an anticoagulant. Liver samples were removed for histology and lipoperoxidation assay. The characterization of obesity in the animals of this study was previously published [18].

2.4. Ex vivo assays

2.4.1. Plasma markers of hepatic function

The blood samples were processed by centrifugation (2500 ×g, 10 min) to obtain heparinized plasma which was used for dosing lactate levels, alanine aminotransferase (ALT) and aspartate aminotransaminase (AST) activities by commercial kits (Bioclin, Brazil), following manufacturers instruction.

2.4.2. Hepatic lipoperoxidation

Levels of thiobarbituric acid reactive species (TBARS) were used as an indicator of lipid peroxidation in liver [24]. The liver of rats was removed and homogenized in 50 mM Tris HCl pH 7.4 (1/10, w/v) and centrifuged at 2500 ×g for 10 min. An aliquot of supernatant (S1) was added to the reaction mixture containing: thiobarbituric acid (0.8%), sodium dodecyl sulfate (SDS), and acetic acid buffer (pH 3.4) and after incubated at 95 ◦C for 2 h. The absorbance was measured at 532 nm. Results were reported as nmol malondialdehyde (MDA)/mg protein.

2.4.3. Protein determination

The protein concentration was measured by the method of Bradford [25], using bovine serum albumin as the standard.

2.4.4. Liver histology analysis

Tissue samples of liver collected and fixed in 10% buffered formalin. For histopathology, fixed tissues (N = 4/group) were processed routinely, cut at 5 μm and stained with hematoxylin and eosin (H&E). Selected hepatic sections were stained with periodic acid Schiff (PAS). These analyses were performed by a veterinary histopathologist.

2.5. Statistical analysis

The normality of data was evaluated by the D’Agostino-Pearson test. Data were analyzed by one-way analysis of variance (ANOVA) followed by Newman-Keuls. All analyses were performed using the GraphPad Prism® software. Data were expressed as the mean ± standard error of the mean (S.E.M). Probability values less than (p < 0.05) were considered statistically significant.

3. Results

3.1. Plasma biochemical markers of hepatic function

AST/ALT activities and lactate levels in the plasma of animals exposed to MSG and treated with 4-PSQ are represented in Fig. 3. One- way ANOVA revealed significant differences among means [AST (F (3,20) Injections of MSG to infant rats resulted in an increase in AST and ALT (p < 0.05) activities measured in adulthood (Fig. 3A and B). Increments in lactate levels were also found in animals exposed to MSG when compared with those of the control group (p < 0.0001) (Fig. 3C). In parallel, repeated oral treatment with 4-PSQ (5 mg/kg) was effective in protecting against the increase of AST and ALT activities (p < 0.05) induced by MSG and partially attenuated the elevated levels of plasma lactate (p < 0.001) (Fig. 3A–C). 4-PSQ per se did not modify AST, ALT and lactate (p > 0.05).

3.2. Hepatic lipoperoxidation levels

Fig. 4 represents the TBARS content measured in the liver of rats injected with MSG and treated with 4-PSQ. One-way ANOVA revealed significant differences among means (F (3, 20) = 9.175; p = 0.0005). Post- hoc comparisons demonstrated that animals of MSG group presented higher levels of hepatic lipoperoxidation (TBARS) compared with those of the control group (p < 0.001). Consecutive administrations of 4-PSQ were able to protect against the lipoperoxidation induced by neonatal exposure to MSG (p < 0.01). TBARS levels remained unmodified by 4- PSQ per se (p > 0.05) when compared to the control.

3.3. Histological analysis of liver tissue

The histopathology of liver tissues extracted from rats of the present study is depicted in Fig. 5. Fig. 5A shows hepatocyte cytoplasm with a homogeneous appearance and without vacuolation in the liver of control animals (group I) by the H&E method. PAS staining of control tissues (Fig. 5B) showed hepatocytes with a discrete distribution of glycogen dispersed in small clusters in the cytoplasm and localized in the three distinct regions of the hepatic lobules, i.e., centrilobular, medizonal and periportal regions.
The liver tissue of group II (4-PSQ) presented hepatocytes with homogenous cytoplasm and no pathological alterations by H&E histology analysis (Fig. 5C). PAS staining revealed that the glycogen distribution in the liver of 4-PSQ animals was similar to that of the control group (Fig. 5D).
H&E staining of animals exposed to MSG (group III) demonstrated enlarged hepatocytes with unclear cytoplasmic borders and presenting intracytoplasmic vacuoles characterizing a steatosis state (Fig. 5E) In the periportal region, it was observed the proliferation of cells of the bile ducts (Fig. 5I). See the swelling of hepatocytes by the PAS method where enlarged cytoplasm can be observed due to glycogen accumulation with all regions of the hepatic lobules being affected (Fig. 5F).
Unlike group III, H&E staining demonstrated that animals exposed to MSG and treated repeatedly with 4-PSQ (Group IV) had CR-marginal hepatocytes preserved limiting the lesion to the hepatocytes of the medizonal and periportal region (Fig. 5G). In these regions there was more discreet steatosis when compared to the MSG group (Fig. 5H). In the PAS staining, there was cytoplasmic vacuolization and glycogen accumulation, but in a smaller amount than in the animals of the MSG group (Fig. 5H). The periportal region also presented proliferation of bile duct cells (Fig. 5J).

4. Discussion

Obesity is a crucial factor in the development of metabolic abnormalities including metabolic syndrome, low-grade inflammation and oxidative stress [26]. MSG, a controversial flavor enhancer, is experimentally employed as an inductor of hypothalamic obesity, besides causing acute liver injury and hepatocyte ultrastructural alterations [27]. Data of the present study demonstrated for the first time the therapeutic effects of a repeated treatment with 4-PSQ on hepatic injury induced by exposure of neonatal rats to MSG. Oral administrations of 4- PSQ were effective in normalizing plasma transaminases (AST and ALT) and hepatic TBARS levels of MSG obese rats. Moreover, 4-PSQ therapy attenuated the increase of plasma lactate and alterations of liver morphology triggered by MSG.
The liver is involved in detoxification and metabolism and may be directly affected by toxic chemicals or their metabolites as MSG at high doses [4]. Increased transaminases are considered as a diagnostic index for assessing hepatotoxicity [28]. Injuries to hepatocytes result in increased membrane permeability modifying the plasma enzyme profile, i.e., transaminases are released from the liver into the bloodstream [28]. Here, neonatal MSG exposure caused an increase of AST and ALT activities in plasma of adult rats, results that are in accordance with other MSG studies [7,25,28–30]. Indeed, rats exposed to MSG during early life develop obesity with metabolic abnormalities including disruption of liver homeostasis [31,32].
On the other hand, 4-PSQ treatment was effective in reversing the elevation of transaminase activities (AST and ALT) observed in the plasma of MSG rats, suggesting a hepatoprotective action by this selenium-functionalized quinoline compound. This fact is also demonstrated when comparing the blood biochemistry with the histological pattern of the livers, where the animals of the group treated with MSG are vacuolated and engulfed by the accumulation of glycogen and there is an increase in the activities of the aminotransferases while the animals treated with 4-PSQ present the centrilobular region of the organ preserved and the activity of AST and ALT do not change. The hepatoprotection afforded by other organoselenium compounds [14,33,34] or even by selenium [35–37] and quinoline derivatives [38] have also been documented. Moreover, research aimed at investigating the 4-PSQ pharmacology have expanding in recent years [15,16,39–45], but this is the first study dedicated to evaluating the protective role of 4-PSQ on a hepatic injury model.
In addition, higher levels of plasma lactate were found in MSG rats. Under normal conditions, lactate is rapidly cleared by the liver with a small amount of additional clearance by the kidneys [46,47]. Lactate levels are commonly evaluated in acutely ill patients. Although most commonly used in the context of evaluating shock, lactate can be elevated for many reasons, including liver failure [48]. Blood lactate levels are often elevated as a result of both impaired tissue perfusion, which increases production, and decreased clearance by the liver [49]. Thus, hepatic damage by MSG could reduce the ability of the liver to clear circulating lactate. Elevated lactate could also represent secondary mitochondrial dysfunction occurring as a result of severe liver disease [50]. Reinforcing our data, Quines et al. [51] also demonstrated increased plasma lactate levels in MSG rats. In the current study, we demonstrated that 4-PSQ therapy partially reducing the lactate levels in hypothalamic obese rats, corroborating the hepatoprotection hypothesis.
Liver dysmetabolism accompanied by inflammation and oxidative stress have been described in the MSG model [7,31]. The metabolism is displaced to increased lipid production due to higher glucose flux throughout increased glucokinase activity and enhanced lipogenic gene expression in MSG animals [52]. As previously reported, it is evidenced a marked increase of hepatic lipid peroxidation and elevated plasma transaminases in hypothalamic obese rats [53–56]. Choudhary and collaborators [57] found that early postnatal administration of MSG induced oxidative stress in hepatic microsomes of adult rats. Here, the content of TBARS measured in the liver of MSG rats was increased in relation to the control group, indicating oxidative damage to lipids (lipoperoxidation) of this tissue. In addition, moderate and patchy hepatocellular damage has been histologically observed in the MSG model [5]. MSG in excess could to act as toxins to the hepatocytes, thereby affecting their cellular integrity and causing defect in membrane permeability and cell volume homeostasis [58]. It was found atrophic and degenerative changes in the liver of adult rats that received MSG during early life in an extension that may affect the liver functions [59]. Corroborating these studies, we demonstrated that MSG caused a distortion in the cytoarchitecture of the liver, vacuolated and engulfed by the accumulation of glycogen, as evidenced by the histological analysis. It should be noted that the histological changes found in the liver of animals treated with MSG are in line with the changes found in plasma biochemical markers.
In the present study, 4-PSQ treatment reverted the hepatic lipid peroxidation in animals exposed to MSG by reducing the TBARS levels. Further, 4-PSQ per se not altered the liver histology; and when administered to obese rats, it partially reduced the morphological changes in liver architecture caused by MSG, with centrilobular region of the organ preserved and unchanged plasma transaminases activities. Together, these findings give support to the hepatoprotective action of 4-PSQ. We did not rule out the possibility that a larger dose of the compound 4-PSQ or a higher frequency of administration could be necessary to obtain more effective histological protection against MSG.
Regarding mechanistic hypotheses, antioxidant effects could contribute to 4-PSQ hepatoprotective action since it reduced the hepatic lipoperoxidation in MSG rats. Indeed, 4-PSQ has been described as an antioxidant molecule. A study previously reported that subacute treatment with 4-PSQ (5 mg/kg) played antioxidant action against the oxidative stress associated with aging in different rat tissues, including the liver [45]. In addition, 4-PSQ elicited antioxidant actions in a model of Parkinson-like disease induced by rotenone in Drosophila melanogaster [40]. In addition to 4-PSQ, other antioxidants have elicited antioxidant effects associated to hepatoprotection such as vitamins E and C, glutathione, quercetin, N-Acetyl-l-Cysteine and, p-chloro-diphenyl diselenide [7,26,51,53,54].
Chronic inflammation plays a central role in the pathogenesis of a number of diseases and is closely related to the oxidative stress. Evidently, oxidative stress and inflammation are important features of MSG-induced liver damage [7,8]. In this way, antioxidant and anti- inflammatory actions of 4-PSQ could be acting together to reduce liver injury by MSG. Previous studies about anti-inflammatory and antioxidant actions of 4-PSQ bring some light to this supposition. Firstly, 4-PSQ caused a reduction of edema formation in the ear tissue in mice challenged with croton oil and also reduced myeloperoxidase activity and reactive species levels [15]. Also, 4-PSQ ameliorated inflammatory and oxidative parameters in an atopic dermatitis model induced by 2,4-dinitrochlorobenzene [44].
It is important to note that this work has certain limitations, since more dosages are required to really provide information about oxidative disbalance. However, our results provide a good indication of the potential effect of the 4-PSQ compound in a MSG-induced obesity model, being considered relevant for the scientific research.

5. Conclusion

In summary, 4-PSQ treatment attenuated the increase of plasma transaminases, lactate, lipoperoxidation as well as alterations in liver morphology of MSG adult rats. These results suggest a hepatoprotective action of 4-PSQ against the hepatic injury triggered by neonatal exposure to MSG.

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