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Mercury chloride increases hepatic alanine aminotransferase and glucose 6-phosphatase activities in newborn rats in vivo
Lucélia Moraes‑Silva*, Tania Maria Bueno*, Carina Franciscato*, Cláudia Sirlene de Oliveira†, Nilce Coelho Peixoto* and Maria Ester Pereira*†1
*Programa de PsGraduao em Cincias Biolgicas, Bioqumica Toxicolgica, Universidade Federal de Santa Maria, 97105900, Santa Maria, RS, Brasil, and †Departamento de Qumica, Centro de Cincias Naturais e Exatas, Universidade Federal de Santa Maria, 97105900, Santa Maria, RS, Brasil
This work investigated the in vivo and in vitro effects of HgCl2 and ZnCl2 on metabolic enzymes from tissues of young rats to verify whether the physiological and biochemical alterations induced by mercury and prevented by zinc are related to hepatic and renal glucose metabolism. Wistar rats received (subcutaneous) saline or ZnCl2 (27 mg/kg/day) from 3 to 7 days old and saline or HgCl2 (5.0 mg/kg/day) from 8 to 12 days old. Mercury exposure increased the hepatic alanine aminotransferase (∼6-fold) and glucose 6-phosphatase (75%) activity; zinc pre-exposure prevented totally and partially these mercury alterations respectively. In vitro, HgCl2 inhibited the serum (22%, 10 μM) and liver (54%, 100 μM) alanine aminotransferase, serum (53%) and liver (64%) lactate dehydrogenase (10 μM), and liver (53%) and kidney (41%) glucose 6-phosphatase (100 μM) from 10- to 13-day-old rats. The results show that mercury induces distinct alterations in these enzymes when tested in vivo or in vitro as well as when different sources were used. The increase of both hepatic alanine aminotransferase and glucose 6-phosphatase activity suggests that the mercury-exposed rats have increased gluconeogenic activity in the liver. Zinc prevents the in vivo effects on metabolic changes induced by mercury.
Key words: alanine aminotransferase, gluconeogenesis, glucose-6-phosphatase, lactate dehydrogenase, mercuric chloride, young rats, zinc chloride
Abbreviations: ALT, alanine aminotransferase, G6Pase, glucose 6-phosphatase, LDH, lactate dehydrogenase, PBG-synthase, porphobilinogen synthase, Pi, inorganic phosphorus
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Inorganic mercury is a toxic metal, and the exposure to this form of mercury is mainly occupational, such as occurring in industrial activities and mining (Nevado et al., 2003). This form is primarily nephrotoxic (Emanuelli et al., 1996; Clarkson, 1997); however, it causes several other biochemical alterations (Peixoto et al., 2003; Agarwal et al., 2010), such as renal insufficiency, changes in the body and organ weights, and a decrease in PBG-synthase (porphobilinogen synthase) activity of young rats exposed to metal during the precocious phase of development (8–12 days of age) (Peixoto et al., 2003, 2007a). Several physiological and biochemical changes presented by young rats exposed to HgCl2 are prevented by pre-treatment with ZnCl2 (Peixoto and Pereira, 2007; Franciscato et al., 2009, 2011). Liver and kidney of animals exposed to zinc have high content of metallothioneins (Peixoto et al., 2003, 2007b).
We showed recently that young rats exposed to mercury have increased serum urea and creatinine levels, as well as inhibited serum ALT (alanine aminotransferase) activity, but no alteration in serum LDH (lactate dehydrogenase) activity. Pre-exposure to zinc was effective in preventing renal toxicity and ALT inhibition (Peixoto and Pereira, 2007; Franciscato et al., 2011). The lower serum ALT activity does not characterize hepatic lesion (Devlin, 1997), whereas increased serum ALT and LDH activity is considered an indicator of liver cell damage (Kopperschlager and Kirchberger, 1996). In addition, increased liver ALT activity is related to an increase in the gluconeogenic pathway (Rosen et al., 1959; Hagopian et al., 2003). In many mammalian cells, glucose is the major energy source; its supply is therefore strictly regulated (Nordlie et al., 1999). Liver plays a central role in this process since one of its important functions, and to lesser extent the renal cortex, is to provide glucose during starvation. Glucose is formed from gluconeogenic precursors in both tissues, and also from glycogen in liver. The major precursors of glucose in animals are lactate, pyruvate, glycerol and most of the amino acids (Lehninger et al., 2008). However, amino acids, mainly alanine, and lactate are the main substrates for gluconeogenesis (Stryer, 1995), even in rats (Rémésy et al. 1978), and may be considered as a major source of cytosolic pyruvate (Fafournoux et al., 1983). The initial reactions of these processes are catalysed by ALT and LDH respectively (Lehninger et al., 2008).
ALT (EC 188.8.131.52) has an important role in gluconeogenesis and amino acid metabolism, particularly during fasting and intense exercise (Felig, 1973). On the other hand, LDH (EC 184.108.40.206) plays an important metabolic role in glycolytic metabolism, since the reoxidation of NADH provides NAD+ to the glyceraldehyde phosphate dehydrogenase reaction, according to the metabolic needs of the tissue (Lehninger et al., 2008). Another important enzyme for glucose metabolism is the G6Pase (glucose-6-phosphatase; EC 220.127.116.11), which is found mainly in the liver and kidney, and plays a key role in glucose homoeostasis, catalysing the terminal step of both gluconeogenesis and glycogenolysis (Schaftingen and Gerin, 2002).
Thus, considering the effects of mercury on young rats cited above that lead to a high content in the rat liver and kidney (Peixoto et al., 2003, 2008; Franciscato et al., 2011), this study investigates the effect of mercury exposure on the hepatic and renal ALT, LDH and G6Pase activity, blood glucose and tissue glycogen levels. In addition, its relationship with physiological and biochemical alterations induced by mercury and prevented by zinc (Peixoto et al., 2003; Peixoto and Pereira, 2007; Franciscato et al., 2009, 2011) were studied. The in vitro effects of these metals on these enzymes have also been investigated. There is little information on hepatic and renal glucose metabolism alterations induced by mercury in young rats, which is the reason why they were chosen as the subjects of this study.
2. Materials and methods
Mercury (HgCl2) and zinc (ZnCl2) were obtained from Labsynth. BSA, Coomassie Brilliant Blue G and d-glucose-6-phosphate barium hydrate (C6H11O9PBa.H2O) were purchased from Sigma Chemical Company. Glycogen was purchased from Merck. The kits for determination of ALT and LDH were acquired from Labtest and for glucose from Bioclin. All other chemicals were of analytical grade and purchased from local supplier.
2.2. Experimental animals
Wistar rats obtained from the Animal House of the Federal University of Santa Maria were transferred to our breeding colony and maintained on a 12 h light/12 h dark cycle and controlled temperature (22±2°C). The animals had free access to water and commercial food. The breeding regimen consisted of grouping three females (90–120 days old) and one adult male for 20 days. After this period, pregnant rats were selected and housed individually in opaque plastic cages (50 cm×25 cm×18 cm). Pregnant rats were checked once a day between 3:00 and 6:00 p.m. to check for the presence of pups.
2.3. Newborn animals
The day of birth was defined as day 0. At day 1, the litters were reduced to 8 pups to avoid undernutrition due to the number of teats. Males and females were used randomly.
2.4. Pre-treatment and intoxication
Three-day Wistar rats were treated with NaCl 90 mg/kg/day (saline, control) or ZnCl2 27 mg/kg/day for 5 consecutive days (from 3 to 7 days old). From 8 to 12 days of age they received one daily dose of saline or HgCl2 5.0 mg/kg. The doses of HgCl2 (5 mg/kg/day) and ZnCl2 (27 mg/kg/day) were selected according to previous studies performed with suckling rats (Peixoto et al., 2003, 2007b, 2008; Peixoto and Pereira 2007; Franciscato et al., 2009, 2011). Treatments were administered by subcutaneous injections in a constant volume of 10 ml/kg body weight. Animals were weighed daily to adjust the dose.
2.5. Sample preparation
Twenty-four hours after the last dose of saline or mercury the pups were killed. Blood and muscle (hind paws) samples as well as the liver and kidneys (total organs) were quickly collected and placed on an inverted Petri dish on ice. For biochemical analysis, blood serum obtained by total blood centrifugation at 3000 g was frozen (−20°C) until analyses (∼5 days). Liver and kidney were dissected, weighed and portions of each organ were used for determination of enzyme activity. Samples of liver and kidney were homogenized in 10 volumes of cold Tris/HCl buffer (4°C) 10 mM plus MgSO4 1 mM, pH 7.4 to determine ALT; liver samples were homogenized in 20 volumes of the same buffer to determine LDH activity. For G6Pase, liver and kidney samples were homogenized in 39 and 20 volumes of cold citrate buffer (4°C) 0.1 mM, pH 6.5 respectively. Glycogen was assayed in liver, kidney and muscle tissues. An aliquot of each tissue was removed, weighed and stored at −20°C until glycogen extraction and quantification.
2.6. Biochemical determinations
2.6.1. ALT and LDH
ALT and LDH activity were determined using the commercial kit Labtest and expressed as units of enzyme activity per mg of protein. All assays were carried out in triplicate (Peixoto and Pereira, 2007).
G6Pase was assayed as described by Bergmeyer (1965). The determination of the Pi (inorganic phosphorus) released was made according to the Fiske and Subbarow method (1925). The absorbance were measured at 660 nm and compared with phosphate standard curve. The enzyme activity was expressed as nmol Pi formed per hour per mg of protein.
Glucose was quantified using the commercial Bioclin kit, and was given as mg of glucose per dl of serum (Peixoto and Pereira, 2007).
Glycogen was extracted as described by Peixoto and Pereira (2007). Samples were frozen until quantification analyses (∼5 days). Glycogen content was determined by the Krisman method (1962). The resultant absorbance was determined at 460 nm and compared with glycogen standard curve. Tissue glycogen contents were expressed as g glycogen per 100 g tissue.
2.6.5. Protein dosage
Protein content was determined according to Bradford (1976), with BSA as the standard.
2.6.6. In vitro zinc and mercury assay
Blood samples as well as liver and kidney tissues were collected from 10- to 13-day-old rats and prepared as described in tissue preparations (section 2.5). The in vitro effects of mercury chloride on serum, liver and kidney ALT activity were tested at the following concentrations: 0, 0.1, 1, 10 and 100 μM; and for zinc chloride at: 0, 1, 10 and 100 μM. For thermoequilibration, the metals and substrate were pre-incubated for 2 min and the enzymatic reaction was conducted as described above (section 2.6.1). For LDH activity in serum and liver, the effect of mercury was tested at: 0, 1, 5, 10 and 100 μM; and for zinc at: 0, 1, 10 and 100 μM. Metals, enzymes and substrates were incubated, and enzyme reaction was carried out as described above (section 2.6.1). The effects of mercury (0, 10, 50, 100 and 1000 μM) and zinc (0, 10, 100 and 1000 μM) on G6Pase activity of the liver and kidney were also tested. The metals and enzyme sources were pre-incubated for 5 min, and the enzymatic reaction was carried out as described above (section 2.6.2).
2.7. Statistical analysis
Results were analysed by 1-way or 2-way ANOVA, followed by Duncan's multiple range tests as necessary. Effects were considered significant when P≤0.05. Data are presented as means±S.E.M. For in vivo exposure, each litter contributed only with one animal (n) for each one of the determinations. For in vitro assays, pools of 2 or more pups were necessary to provide enough serum enzymatic material.
The study conformed with the University Ethics Committee Guidelines for experiments on animals (Process number 23081.014805/2007-68).
3. Results and discussion
Animals treated only with mercury grew more slowly (Figure 1) and had higher kidney weights (Table 1) than the other groups. The 2-way ANOVA gave significant effects of treatments on body weight [F(5,180) = 1239.91, P<0.001] and treatment×day interaction [F(15,180) = 22.42; P<0.001]. The mercury treated rats did not gain body weight after the 2nd dose, but a significant difference between groups was verified only 24 h after the end of the treatment [one-way ANOVA: F(3,36) = 9.78; P<0.001]. Kidney weight was significantly increased by mercury exposure [F(4,15) = 6.60; P<0.004]. Previous exposure to zinc partially prevented the effects of mercury on body and kidney weights. Others have reported alterations in body and kidney weights of animals exposed to this metal (Rocha et al., 1995; Peixoto et al., 2003; Roza et al., 2005). Increase in kidney size and weight in Hg-exposed rats may be related to structural changes due primarily to an increase in proximal tubule volume (Madsen and Maunsbach, 1981).
Table 1 Liver and kidney weight, liver LDH activity and liver and kidney ALT activities from young rats treated with ZnCl2 (27 mg/kg/day; subcutaneous) for 5 consecutive days (3–7 days old) and with HgCl2 (5 mg/kg/day; subcutaneous) for 5 subsequent days (8–12 days old)
Results are presented as means±S.E.M. Duncan's multiple range test: different letters confer significant statistical difference between groups in the same line (P<0.01).
Hg-intoxicated rats undergo changes in their hepatic metabolism, since these animals showed a 6-fold increase in liver ALT activity [F(3,12) = 21.45, P<0.01], whereas liver LDH and kidney ALT activity were unaltered (Table 1). This effect of mercury seems to be strongly related to some type of compensatory metabolic mechanism, such as the increase in gluconeogenic activity (Rosen et al., 1959; Hagopian et al., 2003). This hypothesis is important when we consider that serum ALT activity in vivo (Peixoto and Pereira, 2007) as well as serum and liver ALT activities in vitro (as discussed below) were inhibited by mercury. Rosen et al. (1959) showed an increase of ALT activity related to 4 conditions associated with gluconeogenesis: protein intake, diabetes, fasting and hydrocortisone treatment. However, Hg-exposed rats were not included in any of these metabolic situations. They were lactating rats and had not been separated from their dams to induce starvation. In an attempt to clarify this, we investigated the effect of Hg-exposure on G6Pase, the last enzyme of the gluconeogenic pathway. Hg-exposed rats had increased liver G6Pase activity of ∼75% [F(3,12) = 5.10, P<0.02] without renal enzyme alteration (Table 2).
Table 2 Liver and kidney G6Pase activity, serum glucose, liver, kidney and muscle glycogen of young rats treated as described in Table 1
Results are presented as mean±S.E.M. Duncan's multiple range test (n = 4): different letters confer significant statistical difference between groups in the same line (P<0.01).
Thus, considering the increase of both liver ALT and G6Pase activity, we suggest that Hg-exposed rats show increased hepatic, but not renal, gluconeogenesis. This increase in the liver may be related to a nutritional factor, since animals poisoned with mercury had decreased body weight gain, similarly to another study (Peixoto et al., 2003). Hg induces anorexigenic effects (Counter and Buchanan, 2004) and can cause alteration in absorption of nutrients (Sastry et al., 1982; Farmanfarmaiam et al., 1989). Moreover, it was possible to see that the Hg-exposed rats had lower fat deposit and size of skeletal muscle. Muscle proteolysis could be providing amino acids for gluconeogenesis. In fact, the Hg-exposed animals have increased creatinine and urea levels (Peixoto and Pereira, 2007; Franciscato et al., 2011).
The glucose level and the liver, kidney and muscle glycogen contents were unaltered by exposure to mercury (Table 2). These parameters were analysed to rule out any effect on glycogen store. The absence of effect also corroborates evidence of an increased hepatic gluconeogenesis, maintaining the normal blood glucose levels in rats exposed to mercury. These results also suggest that the increase in liver G6Pase activity is unrelated to the increase of glycogenolysis, since the levels of the liver glycogen were unaltered.
In relation to zinc exposure, this did not induce metabolic alterations in the parameters studied. Zinc was chosen because it does not have harmful effects on the enzymes studied and on physiological parameters, and because it still prevents several of the toxic effects of mercury (Peixoto et al., 2003; Peixoto and Pereira, 2007; Franciscato et al., 2009, 2011).
Pre-treatment with zinc partially impeded the loss in body weight gain and the increase of kidney weight of Hg-exposed rats. Nevertheless, this metal prevented increased liver ALT activity and partially the increase in liver G6Pase activity. Others have shown that several preventive effects occur in parallel with increased metallothionein content in the liver and kidney (Peixoto et al., 2003, 2007b).
In vitro data showed that almost all the enzymes studied were inhibited by mercury (except kidney ALT). Serum and hepatic ALT activity were inhibited by 22 and 54% respectively, in the presence of 10 and 100 μM [serum: F(4,15) = 8.31, P<0.001; liver: F(4,10) = 6.18, P<0.009] (Figure 2). LDH was the most sensitive enzyme; its hepatic activity was inhibited by 64% and serum activity by 53% at 10 μM [serum: F(4,27) = 30.77 P<0.001; liver: F(4,27) = 11.66 P<0.001] (Figure 3). Hepatic and renal G6Pase activities were also inhibited by mercury in 53 and 41% respectively in the presence of 100 μM [liver: F(4,18) = 17.13, P<0.001; kidney: F(4,14) = 14.31, P<0.001] (Figure 4). Data from elsewhere have suggested that the chemical modification of sulfhydryl group of cysteine is involved in the inactivation of ALT (Vedavathi et al., 2004), LDH (Zheng et al., 2002) and G6Pase (Clottes et al., 2002) activities. Mercury is a typical reagent of sulfhydryl groups and its large affinity with these groups contributes to its toxicity (Clarkson, 1997). However, the in vitro effect corroborates the hypothesis that increased in vivo activity may be related to a metabolic change caused by mercury, and not to a direct effect of mercury on these enzymes as verified by in vitro inhibition.
Since LDH activity in vitro was substantially inhibited and in vivo it was unaltered by mercury exposure, our results suggest that these different effects may be due to Hg levels reaching lower levels in vivo than those incubated in vitro. However, as hepatic LDH activity was not increased by Hg exposure (differently from ALT and G6Pase activity), these results suggest that the pyruvate proceeding from lactate by the Cori Cycle is not the principal substrate for the increase of gluconeogenesis in these animals (Stryer, 1995).
In relation to the effect of zinc chloride in vitro on the activity of enzymes ALT, LDH and G6Pase, zinc does not affect the activity of most of these enzymes. Furthermore, we studied whether the pre-incubation of the enzymes with ZnCl2 could modify the effect of HgCl2 on LDH activity in vitro (data not shown). However, zinc did not alter the inhibitory mercury effect on LDH activities. This result shows that the preventive effect of zinc is not related to a direct effect on this metal.
In conclusion, mercury induces alteration in almost all enzymes analysed from different sources in vivo and in vitro (except liver LDH in vivo and kidney ALT in vivo and in vitro). Increased hepatic ALT and G6Pase activities suggest that animals exposed to mercury have an increased gluconeogenic activity in this tissue. The effects of toxic metal on these enzymes (marker of liver function) were not expressed in the form of increased levels of serum activity (Peixoto and Pereira, 2007), but as in vitro and in vivo inhibition of serum ALT activity, and increase in in vivo hepatic activities. Zinc prevents, even in some cases partially, the in vivo effects of mercury on the metabolic changes confirming its preventive role. These results agree with our previous studies that verified that zinc exposure prevents several biochemical, behavioural and essential metal level alterations induced by inorganic mercury (Peixoto et al., 2003, 2008; Peixoto and Pereira, 2007; Franciscato et al., 2009, 2011).
Maria Ester Pereira is the advising Professor; Nilce Coelho Peixoto and Carina Franciscato are collaborating Professors. Lucélia Moraes-Siva is a doctoral Student and Tania Maria Bueno and Claudia Sirlene de Oliveira are graduate students. All collaborators shared in some degree of responsibility with the paper. Maria Ester Pereira, Nilce Coelho Peixoto and Lucélia Moraes-Siva contributed to the design of research, performed the research and data analysis, and wrote the paper. Carina Franciscato, Tania Maria Bueno and Claudia Sirlene de Oliveira carried out the laboratory experiments.
This work was supported by a
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Received 30 June 2010/18 January 2012; accepted 14 March 2012
Published as Cell Biology International Immediate Publication 14 March 2012, doi:10.1042/CBI20100475
© The Author(s) Journal compilation © 2012 International Federation for Cell Biology
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 1 Body weight of young rats treated with ZnCl2 (27 mg/kg/day; subcutaneous) for 5 consecutive days (3–7 days old) and with HgCl2 (5 mg/kg/day; subcutaneous) for 5 subsequent days (8–12 days old)
Figure 2 In vitro effects of HgCl2 on the serum (a), liver (b) and kidney (c) and ZnCl2 on the serum (d), liver (e) and kidney (f) ALT activities of young rats
Figure 3 In vitro effects of HgCl2 on the serum (a) and liver (b) and ZnCl2 on the serum (c) and liver (d) LDH activities of young rats