distinct response of lactating and nonlactating rats exposed to inorganic mercury on hepatic...

Distinct Response of Lactating and Nonlactating Rats Exposedto Inorganic Mercury on Hepatic δ-Aminolevulinic AcidDehydratase Activity

Cláudia S. Oliveira & Alexandre M. Favero &

Carina Franciscato & Sônia C. A. da Luz &

Maria Ester Pereira

Received: 14 January 2014 /Accepted: 3 March 2014 /Published online: 18 March 2014# Springer Science+Business Media New York 2014

Abstract This study investigated if lactating and nonlactatingrats presented differences in relation to hepatic sensitivity toHgCl2 and the potential preventive role of ZnCl2. Lactating(days 3–12 of lactation) and nonlactating rats received27 mg/kg ZnCl2 for five consecutive days and 5 mg/kgHgCl2 for five subsequent days. Lactating and nonlactatingrats exposed to HgCl2 presented a decrease in food intake, adecrease in plasma alanine aminotransferase (ALT), and anincrease in hepatic Hg levels when compared to the controlgroup. Only lactating rats exposed to HgCl2 presented anincrease in hepatic δ-aminolevulinic acid dehydratase activity.On the other hand, only nonlactating rats exposed to HgCl2presented an increase in plasma aspartate aminotransferase(AST). ZnCl2 pre-exposure partially protected the increasein plasma AST activity presented by nonlactating rats andpotentiated the liver Hg accumulation in lactating rats. Pupsfrom the Sal–Hg and Zn–Hg groups showed a decrease inabsolute liver weight and an increase in liver Hg levels.Summarizing, this study demonstrated that lactating rats pre-sented distinct biochemical responses compared to

nonlactating rats exposed to HgCl2 when hepatic parameterswere evaluated.

Keywords Lactation . Liver .Mercuric chloride . Pups . Zincchloride

Introduction

Mercury is a divalent metal that can be released in the envi-ronment through natural phenomena as well as from anthro-pogenic activities [1]. Mercury is found in a number of formsincluding elemental mercury, inorganic salts, and organiccompounds [2, 3]. The inorganic form (HgCl2) is mainlyrecognized to cause nephrotoxicity [2, 4–7], although hepato-toxicity has been suggested since HgCl2 exposure induceshistopathological alterations in the liver and increases aspar-tate and alanine aminotransferase (AST and ALT) activitiesfrom Swiss albino mice [8, 9].

Recently, we verified that young rats exposed to HgCl2presented an increase in hepatic glucose-6-phosphatase andALT activity without alteration on serum ALT activity. Theseeffects were interpreted as an increase of gluconeogenesis[10]. Besides, the animals presented a decrease of hepaticand renal δ-aminolevulinic acid dehydratase (δ-ALA-D) [11].

δ-ALA-D is an enzyme that participates in the second stepof heme biosynthesis [12] and is an important biomarker oftoxicity for many xenobiotics because of potential sites ofinhibition. Particularly, hepatic δ-ALA-D has been assumedto be a target for HgCl2 exposure in both young and adultexperimental animals [6, 7, 13].

During lactation, several physiological conditions arechanged [14, 15] and these certainly influence the distributionand elimination of chemical compounds [16–18]. In fact, theHg biological half-time decreased around 50% in humans and

C. S. Oliveira :A. M. Favero :C. Franciscato : S. C. A. da Luz :M. E. PereiraPrograma de Pós-Graduação em Bioquímica Toxicológica,Universidade Federal de Santa Maria, Santa Maria, RS 97105-900,Brazil

M. E. Pereira (*)Departamento de Bioquímica e Biologia Molecular, Centro deCiências Naturais e Exatas, Universidade Federal de SantaMaria, Av.Roraima, 1000, Prédio 18, sala 2201, Santa Maria, RS 97105-900,Brazile-mail: [email protected]

S. C. A. da LuzDepartamento de Patologia, Centro de Ciências da Saúde,Universidade Federal de Santa Maria, Santa Maria, RS 97105-900,Brazil

Biol Trace Elem Res (2014) 158:230–237DOI 10.1007/s12011-014-9931-9

animals during lactation [19, 20]. Even though organ distri-bution and toxicity of HgCl2 have been studied by numerousinvestigators, differences in susceptibility to the HgCl2-in-duced hepatotoxicity related to the lactation period are limitedin the literature.

Based on the above, the objective of this study was toinvestigate if lactating and nonlactating rats present similarhepatic sensitivity to HgCl2 and the potential preventive roleof ZnCl2. In addition, the effects of metal exposure on pupsthrough maternal milk were assessed.

Materials and Methods

Chemicals

Mercuric chloride (HgCl2), zinc chloride (ZnCl2), sodiumchloride (NaCl), potassium phosphate monobasic (KH2PO4)and dibasic (K2HPO4), absolute ethanol, sodium hydroxide(NaOH), trichloroacetic acid (TCA), nitric acid (HNO3), sul-furic acid (H2SO4), ο-phosphoric acid, perchloric acid, andglacial acetic acid were purchased from Merck (Darmstadt,Germany). δ-Aminolevulinic acid (δ-ALA), bovine serumalbumin, and Coomassie brilliant blue G were obtained fromSigma (St. Louis, MO, USA); p-dimethylaminobenzaldehydewas obtained from Riedel (Seelze, Han, Germany). The kitsfor determination of glucose levels and lactic dehydrogenase(LDH), ALT, and AST activities were acquired from Labtest(Lagoa Santa, MG, Brazil).

Animals

Adult female Wistar rats (pregnant and nonpregnant), 90 daysold, obtained from the Animal House of the FederalUniversity of Santa Maria were transferred to our breedingcolony and maintained on a 12-h light/dark cycle and at acontrolled temperature (22±2 °C). Pregnant rats were allowedto deliver and nurse their pups until lactational day (LD) 13.One day after birth, litters were culled randomly to eight pupseach. The animals had free access to water and commercialfood (GUABI, RS, Brazil). The animals were used accordingto the guidelines of the Committee on Care and Use ofExperimental Animal Resources, Federal University ofSanta Maria, Brazil (096/2011).

Exposure to Metals

Lactating and nonlactating rats were distributed on a randombasis into four groups of six to seven animals each and housedin individual standard polypropylene plastic cages (41×34×18 cm). Animals were subcutaneously (s.c.) exposed to 0.9 %NaCl (saline solution) or metals dissolved in saline solution ata volume of 1 mL/kg body weight (b.w.) following the

experimental design: Lactating and nonlactating rats weresubmitted to the same protocol of exposure, except that lac-tating rats were exposed on days 3 to 12 of lactation.

Group 1 (Sal–Sal): saline for ten consecutive daysGroup 2 (Sal–Hg): saline for five consecutive days and5 mg/kg HgCl2 for five subsequent daysGroup 3 (Zn–Sal): 27 mg/kg ZnCl2 for five consecutivedays and saline for five subsequent daysGroup 4 (Zn–Hg): 27 mg/kg ZnCl2 for five consecutivedays and 5 mg/kg HgCl2 for five subsequent days

The suckling pups were exposed to metals exclusivelythrough maternal milk. It is important to mention that thetransport of inorganic mercury into milk was previously dem-onstrated in the literature [21, 22].

Animals were weighed daily to adjust the dose. The doses,exposure route, and the time period that lactating (LD 3 to 12)and nonlactating rats were exposed to metals were chosenaccording to previous studies performed by our researchgroup [5–7, 10, 11, 23, 24]. These studies demonstrated that5 mg/kg/day HgCl2 caused toxic effects which were preventedby 27 mg/kg/day ZnCl2 in suckling rats directly exposed tometals and killed 24 h or 21 days after the end of the exposureperiod.

All animals were observed daily throughout the study formortality and signs of toxicity. Water and food consumptionof lactating and nonlactating rats were monitored daily duringthe period of exposure to metals.

Tissue Preparation

Twenty-four hours after the last administration of HgCl2, lac-tating (and their pups) and nonlactating rats were weighed andkilled by decapitation. Blood samples were collected in tubeswith heparin and centrifuged at 2,000×g for 10 min at 4 °C toobtain the plasma. The liver was removed and weighed. For δ-ALA-D activity assay, the liver was quickly placed on ice andhomogenized in seven volumes of NaCl (150 mM, pH 7.4)with 10 up-and-down strokes at ~1,200 rpm in a Teflon glasshomogenizer. The homogenate was centrifuged at 8,000×g for30 min at 4 °C and the supernatant fraction was used in theenzyme assay. For determination of metal Hg and Zn levels,tissue samples were placed into vials and then frozen at −20 °Cuntil analysis.

Biochemical Determinations

Hepatic δ-ALA-D Activity

The enzymatic activity was assayed according to the methodof Sassa [25] with some modifications as described in Peixotoet al. [11]. The incubation was initiated by adding 200 μL of

Response of Rats Exposed to HgCl2 on Hepatic δ-ALA-D Activity 231

8,000×g supernatant of the liver homogenate and was carriedout for 40 min at 39 °C. The specific enzymatic activity wasexpressed as nanomoles of porphobilinogen (PBG) formedper hour per milligram protein. Protein concentrations weredetermined by the Coomassie blue method [26] using bovineserum albumin as a standard.

Plasma Biochemical Determinations

The ALT and AST activity was determined by the method ofReitman and Frankel [27] using the commercial kit fromLabtest. All assays were carried out in triplicate [5]. Theactivity (U/mL) was calculated by comparison with a calibra-tion curve utilizing sodium pyruvate as standard. The LDHactivity (U/L) was determined through the reduction of absor-bance due to the oxidation of NADH and calculated using itsmolar absorption coefficient (6.30×103) as described in detailin Peixoto and Pereira [5]. The quantification of plasma glu-cose (mg/dL) was carried out by measuring of the productformed from the complete oxidation of the glucose [5].

Metal Determinations

Samples weighing around 250 mg each were transferred toquartz vessels. Concentrated HNO3 (6 mL) was added to eachvessel, which was capped and placed into a microwave ovenfor digestion. Then, the samples were diluted with water to25 mL and transferred to graduated polypropylene vials.Metal analyses were carried out using a Model AAS EA 5atomic absorption spectrometer (Analytik Jena, Jena,Germany). For Zn determination, the atomic absorption spec-trometer was equipped with a transversely heated graphitetube atomizer with pyrolytic coated tubes. A batch-operatedchemical vapor generation system, HS 5 (Analytik Jena, Jena,Germany), was adapted to the atomic absorption spectrometerfor Hg determinations. A deuterium background corrector wasused for all determinations. Hollow cathode lamps were op-erated at 4 mA. Wavelength was set at 213.9 nm (Zn) and253.7 nm (Hg) and the spectral band pass at 0.5 nm to bothmetals. Integrated absorbance (peak area) was used for allmeasurements. The heating program for Zn was carried outaccording to the recommendations of the manufacturer. ForHg determination, 3 M HCl and 0.25 % m/v NaBH4 solutionswere used as acid medium and reductant, respectively. Argonwas used as purge gas. Results for all elements determinedwere periodically evaluated by measurements of analyticalstandards (every 10 measurements) as well as by the digestanalysis of the certified reference material SRM NIST 1577(every 3 h of measurements). If the result for standardchecking presented a bias higher than 5 %, a recalibrationprocedure was performed [24].

Statistical Analysis

Results were analyzed by one-way analysis of variance(ANOVA) followed by Duncan’s multiple range.Comparisons among all group treatment means were made.Different letters were used to indicate significant differencesamong groups. Lactating and nonlactating rats were indepen-dently analyzed. A value of p<0.05 was considered to besignificant. Due to discrepancies of values among the samplesof the same group, mercury and zinc data were logarithmicallytransformed and were submitted to ANOVA similar to theother results; the results were shown as real values (nottransformed).

Results

Effects of Exposure to Metals in Lactating and NonlactatingRats

Body and Liver Weight

Body weight gain is presented in Table 1. One-way ANOVArevealed the absence of a significant effect of zinc treatment onbody weight gain (5 days) both in lactating and nonlactatingrats. However, at five subsequent days, one-way ANOVArevealed a significant effect of treatment on both lactating[F(3, 20)=21.35, p<0.001] and nonlactating [F(3, 19)=27.41, p<0.001] rats. This effect was due to loss of bodyweight presented by HgCl2-exposed rats. ZnCl2 pre-exposuredid not prevent this effect. Regarding liver weight, one-wayANOVA revealed that absolute liver weight of lactating ratswas significantly affected by exposure to metals [F(3, 20)=5.65, p<0.01]. Animals from both groups exposed to HgCl2(Sal–Hg and Zn–Hg) presented lower absolute liver weight(around 20 %) when compared to the Zn–Sal group(p<0.05). In contrast, this parameter was not altered in anygroup of nonlactating rats. The relative liver weight was alteredneither for lactating nor for nonlactating rats (Table 1).

Food and Water Intake

Food intake was not altered by zinc pretreatment. However,mercury exposure significantly decreased the food intake(one-way ANOVA) in both lactating [F(3, 16)=21.21,p<0.001] and nonlactating [F(3, 19)=21.61, p<0.001] rats.The reduction was around 45 and 65 %, respectively, whencompared to their respective control groups (p<0.05).Previous exposure to ZnCl2 did not prevent the effects ofHgCl2 on this parameter (Table 1). Water intake was alteredneither in lactating nor in nonlactating rats exposed to metals(data not shown).

232 Oliveira et al.


Hepatic δ-ALA-D Activity Hepatic δ-ALA-D activity was sig-nificantly affected by exposure to metals in lactating rats [F(3,20)=6.70, p<0.001]. Post hoc comparisons showed that lac-tating rats from both groups exposed to HgCl2 (Sal–Hg andZn–Hg) presented an increase (around 50 %) in enzymeactivity when compared to the other groups (Sal–Sal andZn–Sal) (p<0.05) (Table 2). Hepatic δ-ALA-D activity wasnot changed by exposure to metals in nonlactating rats.

Plasma Biochemical Determinations One-way ANOVA re-vealed that ALT activity was significantly changed by expo-sure to metals in both lactating [F(3, 20)=8.83, p<0.001] andnonlactating [F(3, 19)=19.40, p<0.001] rats. Post hoc com-parisons showed that lactating and nonlactating rats exposedto HgCl2 presented ALT activity significantly lower than theirrespective control groups (around 48 and 52 %, respectively,p<0.05). Previous exposure to ZnCl2 did not prevent thealteration induced by HgCl2 on ALT activity (Table 3).

One-way ANOVA revealed that AST activity was significant-ly modified by metals only in nonlactating rats [F(3, 19)=3.32, p<0.05]. Post hoc comparisons showed thatnonlactating rats exposed to HgCl2 presented a slight increasein AST activity (around 20 %) when compared to the control

group. Previous exposure to ZnCl2 partially prevented thisincrease (p<0.05). AST activity was not changed by exposureto metals in lactating rats (Table 3). LDH activity and glucoselevels were not modified by exposure to metals in both lactat-ing and nonlactating rats (Table 3).

Hepatic Hg and Zn Levels

One-way ANOVA revealed significant effects of exposure tometals on hepatic Hg [lactating, F(3, 12)=19.35, p<0.001;nonlactating, F(3, 10)=119.01, p<0.001] and Zn [lactating,F(3, 12)=4.39, p<0.05; nonlactating, F(3, 10)=13.35,p<0.001] levels in rats. The Hg-exposed rats presented asignificant increase in hepatic Hg levels when compared totheir respective control groups. Zn pretreatment intensified thehepatic Hg accumulation in lactating rats. Regarding Znlevels, both lactating and nonlactating rats exposed to bothmetals (Zn–Hg group) presented an increase when comparedto the other groups (p<0.05) (Table 2).

Effects of Exposure to Metals in Suckling Pups

Liver Weight

One-way ANOVA revealed that absolute liver weight ofsuckling pups was significantly altered by exposure to

Table 1 Body weight gain, liver weights (total and relative), and food intake of lactating and nonlactating rats exposed to ZnCl2 (27 mg/kg) or saline forfive consecutive days and exposed to HgCl2 (5 mg/kg) or saline on the five subsequent days

Parameters Sal–Sal Sal–Hg Zn–Sal Zn–Hg

Lactating

Body weight gain (g)

During Zn exposure 15.83±3.40 13.33±2.39 14.17±2.80 21.50±4.67

During Hg exposure 7.17±3.77a −36.83±8.11b 15.00±3.09a −44.00±8.92bLiver weight

Absolute (g) 15.40±0.28a, b 13.53±0.87b 16.68±0.26a 13.91±0.76b

Relative (%) 4.71±0.11 4.84±0.19 4.93±0.14 4.89±0.14

Food intake (g/day/100 g body weight)


During Hg exposure 19.05±1.09a 10.30±0.86b 18.30±1.07a 11.45±0.91b

Nonlactating

Body weight gain (g)


During Hg exposure 8.00±2.28a −27.83±4.55b 5.2±1.2a −29.14±4.54bLiver weight

Absolute (g) 9.64±0.41 8.80±0.51 9.67±0.19 8.84±0.44

Relative (%) 4.13±0.18 4.42±0.27 4.15±0.13 4.36±0.13

Food intake (g/day/100 g body weight)


During Hg exposure 8.38±0.50a 2.55±1.77b 8.33±0.42a 3.29±0.74b

Data are expressed as mean±S.E.M. (n=6–7) and the values followed by different letters in the same line are statistically different (p<0.05)


metals [F(3, 20)=13.08, p<0.001]. Pups from the Sal–Hg andZn–Hg groups showed a decrease in absolute liver weight ofaround 25 % when compared to the control group (p<0.05).Relative liver weights were similar among groups (Table 4).


Hepatic δ-ALA-D, plasma ALT, AST, and LDH activities andglucose levels were not altered by exposure to metals insuckling pups (Table 4).

Metal Determinations

One-way ANOVA revealed significant effects of expo-sure to metals on hepatic Hg [F(3, 8)=29.78, p<0.001]and Zn [F(3, 8)=10.43, p<0.01] levels in pups (Table 4).Suckling pups from the Sal–Hg and Zn–Hg groups presented

a significant increase in hepatic Hg levels when compared tothe other groups (p<0.05). In addition, pups from the Zn–Saland Zn–Hg groups presented a significant increase in hepaticZn levels when compared to the other groups (p<0.05).

Discussion

The objective of this study was to investigate the effects ofHgCl2 exposure on liver metal deposition (Hg and Zn) and onbiochemical parameters indicative of hepatotoxicity in lactat-ing and nonlactating rats as well as the potential preventiverole of ZnCl2. Zn pretreatment did not protect most of thehepatic alterations caused by HgCl2 exposure in both lactatingand nonlactating rats. However, when the same treatmentprotocol (5 days to Zn exposure and subsequently 5 days toHg exposure) was applied in pups, ZnCl2 pre-exposure

Table 2 Hepatic δ-ALA-D activity and mercury and zinc levels of lactating and nonlactating rats exposed to ZnCl2 (27 mg/kg) or saline for fiveconsecutive days and exposed to HgCl2 (5 mg/kg) or saline on the five subsequent days


Lactating

Biochemical determination

δ-ALA-D activity (nmol PBG/mg protein/h) 12.12±0.58a 18.36±2.01b 13.90±0.50a 18.71±1.33b

Metal determinations

Mercury levels (μg Hg/g) 0.03±0.00a 2.38±1.44b 0.05±0.01a 15.28±3.23c

Zinc levels (μg Zn/g) 28.05±1.44a 33.01±3.75a 30.20±1.17a 55.40±7.93b

Nonlactating

Biochemical determination

δ-ALA-D activity (nmol PBG/mg protein/h) 19.23±1.01 20.55±1.73 19.56±1.07 21.93±1.42


Mercury levels (μg Hg/g) 0.03±0.00a 30.39±9.57b 0.03±0.00a 25.89±2.04b

Zinc levels (μg Zn/g) 19.73±0.34a 37.42±4.70b 36.88±8.20b 81.81±12.31c

Data are expressed as mean±S.E.M. (n=6–7 for hepatic δ-ALA-D activity and n=3–5 for metal determinations) and the values followed by differentletters in the same line are statistically different (p<0.05, Duncan’s multiple range test)

Table 3 Biochemical determinations in lactating and nonlactating rats exposed to (27 mg/kg) or saline for five consecutive days and exposed to HgCl2(5 mg/kg) or saline on the five subsequent days


Lactating

ALT (U/mL) 125.18±13.27a 65.58±15.87b 133.42±11.28a 62.50±9.67b

AST (U/mL) 56.67±7.68 66.50±6.47 69.08±11.05 72.30±8.78

LDH (U/L) 449.25±30.03 474.59±77.26 439.22±78.83 501.86±71.35

Glucose (mg/dL) 85.59±14.81 114.14±13.67 94.32±10.32 93.86±9.51

Nonlactating

ALT (U/mL) 64.00±4.43a 30.70±2.96b 61.60±3.72a 38.60±3.75b

AST (U/mL) 76.00±3.77a 91.20±5.08b 78.40±3.53a 81.00±1.80a, b

LDH (U/L) 505.50±39.20 537.70±39.04 442.02±27.00 420.25±36.84

Glucose (mg/dL) 120.31±8.89 134.96±9.50 119.45±6.57 131.11±6.53

Data are expressed as mean±S.E.M. (n=6–7) and the values followed by different letters in the same line are statistically different (p<0.05)

234 Oliveira et al.

protected the hepatic alterations induced by HgCl2 [7, 10, 11].On the other hand, other interesting results were observed inthis study.

A pronounced loss of body weight was observed in allgroups of lactating and nonlactating rats exposed to HgCl2.This effect can be directly related to a marked reduction offood intake verified in the groups exposed to HgCl2. Theanorexigenic effects of mercury have been well documentedin the literature [3] reducing food intake without altering waterintake. In fact, lactating and nonlactating rats exposed toHgCl2 had normal water ingestion (data not shown) and didnot present visual symptoms of dehydration.

In this study, both lactating and nonlactating rats presentedan increase in hepatic Hg levels after exposure to HgCl2.Interestingly, it was demonstrated for the first time that previ-ous exposure to ZnCl2 enhanced hepatic Hg accumulation inlactating rats. Recently, Oliveira et al. [28] observed that pre-exposure to one dose of ZnCl2 (27 mg/kg) prevented hepaticHg accumulation 24 h after HgCl2 (one dose, 5 mg/kg) expo-sure. In the present study, both Zn and Hg were administered5 days each. Considering that Zn is an excellent inductor ofscavenger molecules, such as glutathione and metallothionein,treatment for a longer time (5 days) could induce the forma-tion of an inert complex with free Hg increasing the liver Hgcontent, which will be excreted afterward. In fact, Favero et al.[29] observed an increase in kidney Hg content of lactatingrats submitted to the same protocol without any biochemicalalterations. These findings suggest that Zn pre-exposure in-creases Hg excretion.

Despite the high Hg content observed in Zn–Hg, lactatingrats from both groups exposed to HgCl2 (Sal–Hg and Zn–Hg)presented a similar increase on hepatic δ-ALA-D activity

(around 40%), whereas δ-ALA-D activity of nonlactating ratsfrom both groups exposed to HgCl2 was not affected despitethe elevated hepatic Hg levels.

The δ-ALA-D results in the liver of lactating rats wereunexpected. Several lines of evidence have demonstrated thathepatic δ-ALA-D is sensitive to the inhibitory action of inor-ganic mercury poisoning [6, 11, 13], as also shown in thein vitro study [30]. Interestingly, Oliveira et al. [31] found anincrease in hepatic δ-ALA-D activity from fetus exposed toinorganic mercury in utero. Still, methyl mercury, an organicform of Hg, induced an increase of δ-ALA-D (around 48 %)in primary hepatocyte culture from Hoplias malabaricus, awild fish collected from the Amazon Basin [32]. Regardless ofthe mechanism underlying the increased enzyme activity afterHgCl2 exposure, the present data clearly demonstrate that thebehavior of hepatic δ-ALA-D in lactating rats is markedlydistinct from that observed in nonlactating animals.

Aminotransferases and LDH enzymes are sensitive indica-tors of hepatocellular damage [33]. Although the LDH activitywas not affected by Hg exposure, a 20 % increment in plasmaAST activity was observed in nonlactating Hg-exposed rats.This effect was partially prevented by ZnCl2 pre-exposition,indicating Zn pre-exposure as an important preventive treat-ment against mercury-induced hepatotoxicity. An increase inAST activity related to HgCl2 exposure was previously dem-onstrated by several authors [8, 9, 34]. On the other hand, adecrease in plasma ALT activity of lactating (around 48 %)and nonlactating (around 52 %) rats exposed to HgCl2 wasobserved, and this effect was not prevented when the animalswere pre-exposed to ZnCl2. In accordance, Peixoto andPereira [5] showed a reduction of 40 % in ALT activity ofyoung rats exposed to HgCl2. Recently, Franciscato et al. [7]

Table 4 Absolute and relative liver weights, biochemical determinations, and liver metal determinations in pups from dams exposed to ZnCl2(27 mg/kg) or saline for five consecutive days and exposed to HgCl2 (5 mg/kg) or saline on the five subsequent days during lactation


Liver weight

Absolute (g) 0.68±0.03a 0.50±0.04b 0.69±0.03a 0.50±0.03b

Relative (%) 2.70±0.10 2.58±0.09 2.74±0.08 2.63±0.06

Biochemical determinations

δ-ALA-D activity (nmol PBG/mg protein/h) 27.23±0.74 26.61±1.98 29.04±1.76 29.56±1.92

ALT (U/mL) 28.25±2.68 31.66±3.98 26.50±1.42 29.42±2.89

AST (U/mL) 40.33±3.00 47.42±7.15 43.58±8.40 40.50±3.04

LDH (U/L) 318.38±22.76 348.23±39.74 335.80±31.97 430.15±50.49

Glucose (mg/dL) 86.92±5.73 86.04±6.08 98.14±11.04 83.38±11.24


Mercury levels (μg Hg/g) 0.07±0.01a 0.48±0.09b 0.10±0.02a 0.49±0.11b

Zinc levels (μg Zn/g) 30.33±1.32a 38.50±2.17a 62.97±11.72b 68.66±8.05b

Data are expressed as mean±S.E.M. (n=6–7 for weight and biochemical determinations and n=3 for metal determinations) and the values followed bydifferent letters in the same line are statistically different (p<0.05)


demonstrated that the effect of Hg on reducing ALT activitycontinues for a long time after cessation of metal intoxication.The effect of Hg on ALT activity cannot be defined as being ahepatotoxicity event since liver lesion is a feature by theincrease, and not a decrease, of such activity [33]. A recentstudy from our research group demonstrated that HgCl2 in-duced an inhibition on serum ALT activity in vitro [10]. It hasbeen suggested that the chemical modification of the sulfhy-dryl group of cysteine is involved in the inactivation of ALTactivity [35]. In fact, Hg is a typical reagent of sulfhydrylgroups and its large affinity with these groups contributes toits toxicity [2].

The distinctive responses between lactating andnonlactating rats observed in this study (hepatic δ-ALA-Dand AST activities) could be associated with some of theseveral physiological changes that occur during lactation[14, 15]. Indeed, the physiological changes that occurredduring this period, such as an increase in blood and plasmavolume, a decrease in total plasma protein, and increases incardiac output and blood flow, could affect the pharmacoki-netics of any chemicals, including inorganic mercury [16–18].It was previously demonstrated that during lactation, the bio-logical half-time of inorganic mercury decreases by 50 % dueto a higher rate of excretion (i.e., 3.5 days in lactating ratscompared to approximately 7 days in nonlactating adult fe-male rats) [20].

The sensitivity of young animals to various compounds(including metals) may differ from that observed in adults [36,37]. Mammals present a high sensitivity to external insultswhen these are applied in the early phases of life. In rodents,this phase happens from birth to weaning [38] and is associ-ated with intense development. Breast milk is nutritionallybeneficial to the neonate; however, it may also be a repositoryfor contaminants from the environment. Based on the datadescribed above, we evaluated the effects of Hg and Znexposure on neonates via breast milk. In fact, the transportof inorganic Hg into milk was previously demonstrated in rats[21] and in guinea pigs [22]. In the present study, an increasein Hg levels was observed in the liver of suckling pups fromboth groups exposed to HgCl2. In accordance, Sundberg et al.[21] demonstrated that Hg concentration in milk was line-arly correlated to the levels in the liver of suckling pupsafter exposure to Hg via milk. Interestingly, our resultsshowed that the increase in Hg levels observed in the liverof pups seems to be insufficient to cause alterations onbiochemical parameters related to hepatic damage.Indeed, Hg hepatic levels in suckling pups exposed toHg via milk were lower than encountered in maternallivers. In contrast, when young rats were directly exposedto HgCl2 (at the same dose and developmental period), anincrease in liver Hg levels [6, 11, 23] followed by alter-ations in hepatic functions and glycemia [5] and inhibitionof hepatic δ-ALA-D activity [6, 11] were observed.

The levels of zinc, an essential trace element which iscritical to normal growth and development [39], also weremeasured in the liver of suckling pups from lactating damsexposed to metals during lactation. In accordance with previ-ous studies, it was demonstrated that zinc could be transferredto the pups through maternal milk [40, 41]. In fact, Zn levelsincreased about twofold in the liver of pups from both groupsof lactating rats exposed to ZnCl2 (Zn–Sal and Zn–Hg). It isknown that Zn is an essential metal to δ-ALA-D activity [42],and there are a number of studies showing that Zn can activatethis enzyme [43, 44]. In this study, however, the increase of Znlevels in the liver of pups was unable to induce any changes inδ-ALA-D activity.

In summary, this study demonstrated that lactating ratspresented distinct biochemical responses compared tononlactating rats exposed to HgCl2 when hepatic parameterswere evaluated. Of particular importance, HgCl2 exposureinduced an increase in hepatic δ-ALA-D activity of lactatingbut not of nonlactating rats. Furthermore, Hg intoxication ofdams resulted in an increase in liver Hg levels without affect-ing any hepatic parameter evaluated in pups.

Acknowledgments Financial support was provided by FINEP researchgrant “Rede Instituto Brasileiro de Neurociência (IBN-Net)” No.01.06.0842-00. M.E.P. (503867/2011-0) and A.M.F. (142297/2006-2)are recipients of CNPq fellowships; C.F. is a recipient of CAPESfellowship.

Conflict of Interest The authors declare that there are no conflicts ofinterest.

References

1. Holmes P, James KA, Levy LS (2009) Is low-level environmentalmercury exposure of concern to human health? Sci Total Environ408:171–182

2. Clarkson TW (1997) The toxicology of mercury. Crit Ver Clin LabSci 34:369–403

3. Counter SA, Buchanan LH (2004) Mercury exposure in children: areview. Toxicol Appl Pharmacol 198:209–230

4. Goyer RA (1996) Toxic effects of metals. In: Klaassen CD, AmdurMO, Doull J (eds) Casarett & Doull’s toxicology—the basic scienceof poisons. McGraw-Hill, pp 691-736

5. Peixoto NC, Pereira ME (2007) Effectiveness of ZnCl2 in protectingagainst nephrotoxicity induced by HgCl2 in newborn rats. EcotoxicolEnviron Saf 66:441–446

6. Peixoto NC, Kratz CP, Roza T, Morsch VM, Pereira ME (2007)Effects of HgCl2 on porphobilinogen-synthase (E.C. 4.2.1.24) activ-ity and on mercury levels in rats exposed during different precociousperiods of postnatal life. Cell Biol Int 31:1057–1062

7. Franciscato C, Moraes-Silva L, Duarte FA, Oliveira CS, Ineu RP,Flores EMM, Dressler VL, Peixoto NC, Pereira ME (2011) Delayedbiochemical changes induced by mercury intoxication are preventedby zinc pre-exposure. Ecotoxicol Environ Saf 74:480–486

8. KumarM, SharmaMK, Kumar A (2005) Spirulina fusiformis: a foodsupplement against mercury induced hepatic toxicity. J Health Sci 51:424–430

236 Oliveira et al.

9. Sharma MK, Sharma A, Kumar A, Kumar M (2007) Spirulinafusiformis provides protection against mercuric chloride induced oxi-dative stress in Swiss albino mice. Food Chem Toxicol 45:2412–2419

10. Moraes-Silva L, Bueno TM, Franciscato C, Oliveira CS, Peixoto NC,Pereira ME (2012) Mercury chloride increases hepatic alanine ami-notransferase and glucose 6-phosphatase activities in newborn ratsin vivo. Cell Biol Int 36:561–566

11. Peixoto NC, Roza T, Flores EMM, Pereira ME (2003) Effects of zincand cadmium on HgCl2 delta-ALA-D inhibition and Hg levels intissues of suckling rats. Toxicol Lett 146:17–25

12. Jaffe EK (1995) Porphobilinogen synthase, the first source of heme’sasymmetry. J Bioenerg Biomembr 27:169–179

13. Emanuelli T, Rocha JBT, Pereira ME, Porciuncula LO, Morsch VM,Martins AF, Souza DO (1996) Effect of mercuric chloride intoxicationand dimercaprol treatment on delta-aminolevulinate dehydratase frombrain, liver and kidney of adult mice. Pharmacol Toxicol 79:136–143

14. Hanwell A, Linzell JL (1973) The time course of cardiovascularchanges in lactation in the rat. J Physiol 233:93–109

15. Suzuki K, Hirose H, Hokao R, Takemura N, Motoyoshi S (1993)Changes of plasma osmotic pressure during lactation in rats. J VetMed Sci 55:561–564

16. Hallén IP, Jönsson S, Karlsson MO, Oskarsson A (1996)Toxicokinetics of lead in lactating and nonlactating mice. ToxicolAppl Pharmacol 136:342–347

17. Houpert P, Federspiel B, Milhaud G (1997) Toxicokinetics of cad-mium in lactating and nonlactating ewes after oral and intravenousadministration. Environ Res 72:140–150

18. Sundberg J, Jonsson S, KarlssonMO, Hallén IP, Oskarsson A (1998)Kinetics of methylmercury and inorganic mercury in lactating andnonlactating mice. Toxicol Appl Pharm 151:319–329

19. Greenwood MR, Clarkson TW, Doherty RA, Gates AH, Amin-ZakiL, Elhassani S, Majeed MA (1978) Blood clearance half-times inlactating and nonlactating members of a population exposed to meth-ylmercury. Environ Res 16:48–54

20. Prester L, Blanusa M, Kostial K (1994) Half-time and distribution ofinorganic mercury in lactating dams and their pups. Toxicol Lett 74:66–67

21. Sundberg J, Oskarsson A, Bergman K (1991) Milk transfer of inor-ganic mercury to suckling rats—interaction with selenite. Biol TraceElem Res 28:27–38

22. YoshidaM,Watanabe C, Satoh H, Kishimoto T, Yamamura Y (1994)Milk transfer and tissue uptake of mercury in suckling offspring afterexposure of lactating maternal guinea pigs to inorganic or methyl-mercury. Arch Toxicol 68:174–178

23. Peixoto NC, Serafim MA, Flores EMM, Bebianno MJ, Pereira ME(2007)Metallothionein, zinc, andmercury levels in tissues of young ratsexposed to zinc and subsequently to mercury. Life Sci 81:1264–1271

24. Peixoto NC, Rocha LC, Moraes DP, Bebianno MJ, Dressler VL,Flores EMM, Pereira ME (2008) Changes in levels of essentialelements in suckling rats exposed to zinc and mercury.Chemosphere 72:1327–1332

25. Sassa S (1982) Delta-aminolevulinic acid dehydratase assay. Enzyme28:133–145

26. Bradford MM (1976) A rapid and sensitive method for the quantita-tion of microgram quantities of protein utilizing the principle ofprotein-dye binding. Anal Biochem 72:248–254

27. Reitman S, Frankel S (1957) A colorimetric method for the determi-nation of serum glutamic oxalacetic and glutamic pyruvic transami-nases. Am J Clin Pathol 28:56–63

28. Oliveira VA, Oliveira CS, Ineu RP, Moraes-Silva L, Siqueira LF,Pereira ME (2014) Lactating and non-lactating rats differ in sensitiv-ity to HgCl2: protective effect of ZnCl2. J Trace Elem Med Biol. doi:10.1016/j.jtemb.2014.01.005

29. Favero AM, Oliveira CS, Franciscato C, Oliveira VA, Pereira JSF,Bertoncheli CM, Luz SCA, Dressler VL, Flores EMM, Pereira ME(2014) Lactating and non-lactating rats differ to renal toxicity inducedby mercuric chloride: the preventive effect of zinc chloride. doi: 10.1002/cbf.3032

30. Peixoto NC, Roza T, Pereira ME (2004) Sensitivity of δ-ALA-D (E.C. 4.2.1.24) of rats to metals in vitro depends on the stage of postnatalgrowth and tissue. Toxicol in Vitro 18:805–809

31. Oliveira CS, Oliveira VA, Ineu RP, Moraes-Silva L, Pereira ME(2012) Biochemical parameters of pregnant rats and their offspringexposed to different doses of inorganic mercury in drinking water.Food Chem Toxicol 50:2382–2387

32. FilipakNeto F, Zanata SM, Silva de Assis HC, Nakao LS, RandiMA,Oliveira Ribeiro CA (2008) Toxic effects of DDT and methyl mer-cury on the hepatocytes from Hoplias malabaricus. Toxicol in Vitro22:1705–1713

33. Devlin TM (1997) Textbook of biochemistry with clinical correla-tions. Wiley, New York

34. El-Demerdash FM (2001) Effects of selenium and mercury on theenzymatic activities and lipid peroxidation in brain, liver, and bloodof rats. J Environ Sci Health B 36:489–499

35. Vedavathi M, Girish KS, Kumar MK (2004) Isolation and character-ization of cytosolic alanine aminotransferase isoforms from starvedrat liver. Mol Cell Biochem 267:13–23

36. Kostial K, Kello D, Jugo S, Rabar I, Maljković T (1978) Influence ofage on metal metabolism and toxicity. Environ Health Perspect 25:81–86

37. Webb M, Holt D (1982) Endogenous metal-binding proteins inrelation to the differences in absorption and distribution of mercuryin newborn and adult rats. Arch Toxicol 49:237–245

38. Gottlieb A, Keydar I, Epstein HT (1977) Rodent brain growthstages—analytical review. Biol Neonate 32:166–176

39. Krebs NF (1999) Zinc transfer to the breastfed infant. J MammaryGland Biol 4:259–268

40. Krebs NF, Hambidge KM, Jacobs MA, Rasbach JO (1985) Theeffects of a dietary zinc supplement during lactation on longitudinalchanges in maternal zinc status and milk zinc concentrations. Am JClin Nutr 41:560–570

41. Moser-Veillon PB (1995) Zinc needs and homeostasis during lacta-tion. Analyst 120:895–897

42. Bevan DR, Bodlaender P, Shemin D (1980) Mechanism ofporphobilinogen synthase—requirement of Zn2+ for enzyme-activity. J Biol Chem 255:2030–2035

43. Thompson J, Jones DD, BeasleyWH (1977) The effect of metal ionson the activity of delta-aminolevulinic acid dehydratase. Br J IndMed 34:32–36

44. Nelson HM, Hughes MA, Meredith PA (1981) Zinc, copper anddelta-aminolaevulinic acid dehydratase in vitro and in vivo.Toxicology 21:261–266


http://dx.doi.org/10.1016/j.jtemb.2014.01.005

http://dx.doi.org/10.1002/cbf.3032

http://dx.doi.org/10.1002/cbf.3032

distinct response of lactating and nonlactating rats exposed to inorganic mercury on hepatic...

Documents