Mitogen-Activated Protein Kinase Signaling and its Association with Oxidative Stress and Apoptosis in Lead-Exposed Hepatocytes Latifah M. Mujaibel, Narayana Kilarkaje Department of Anatomy, Faculty of Medicine, Kuwait University, Kuwait

Received 8 January 2013; revised 13 November 2013; accepted 20 November 2013 ABSTRACT: Lead toxicity has become a serious public health concern all over the world. Previous studies have shown that lead induces biochemical and structural changes in liver. However, although lead is known to alter liver functions, the underlying molecular mechanisms of hepatotoxicity are not yet clear. We hypothesized that a correlation exists between oxidative stress, apoptosis and mitogen-activated protein kinases (MAPKs) in lead-exposed liver. Wistar rats were treated with 0, 0.5%, and 1% lead acetate for 3d, 14d, and 35d and sacrificed the next day. On 4d, oxidative stress and apoptosis were correlated with downregulated expressions of ERK1/2 and p38-MAPKa/b, and upregulated expressions of JNK1/3 in males. In females, the correlation was with downregulated expressions of ERK1/2 and upregulated expressions of p38-MAPKa/b and JNK1/3. On 15d, the correlation was observed with upregulated expressions of p38-MAPKa/b in males and downregulated expressions of p38-MAPKa/b in females. In both sexes, a correlation was observed with upregulated expressions of ERK1/2 and JNK1/3 in 1% groups. On 36d, the correlation was observed with downregulated expressions of p38-MAPKa/b in males and their upregulated expressions in females. Time-dependent increase in lipid peroxidation on 15d and 36d correlated with upregulated expressions of p38-MAPKa/b in females and ERK1/2 in 1% groups in both sexes. The lower dose induced more apoptosis up to 15d in females and the higher dose induced in males on 36d. Generally, the female livers had more p38-MAPKa/b than the male livers. On 36d, the female livers showed more p38-MAPKa/b and JNK1/3 than the male livers. In conclusion, although not clearly defined, a correlation exists among oxidative stress, apoptosis, and the MAPKs in lead-exposed hepatocytes. The sex-dependent effects may be due to differences in hormonal or other physiological mechanisms. In lead-exposed hepatocytes, the apoptosis may be induced via oxidative stress-mediated C 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 513–529, 2015. alterations in the MAPKs. V Keywords: heavy metals; environmental toxicology; oxidative stress; apoptosis; hepatotoxicity; ERKs; JNKs; p38-MAPKa/b

INTRODUCTION

Correspondence to: N. Kilarkaje; e-mail: [email protected] Contract grant sponsor: College of Graduate Studies, Kuwait University. Contract grant number: YS05/09. Contract grant sponsor: General facility. Contract grant number: SRUL02/13. Published online 1 December 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.21928

The mitogen-activated protein kinases (MAPKs), a group of serine-threonine kinases, are involved in regulating several signaling pathways that are crucial for the integration of mitogenic or apoptotic signals in cells (Papa et al., 2009; Yoshizumi et al., 2012). MAPKs regulate many important biological activities by influencing various cellular processes, namely, transcription, cell proliferation and survival, cell differentiation, inflammation, induction of apoptosis, and malignant transformation (Dhalla and M€uller, 2010).

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They are classified into three major groups, the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs) and the p38-MAPK (Dhalla and M€uller, 2010). They operate in cells through a well-organized threetier module system, but with abundant cross talks between the modules (reviewed in Manna and Stocco, 2011). The activated ERK1/2 or MAPK/ERK pathway coordinates a signal from a membrane receptor to the nucleus and induces cell proliferation and differentiation (Thottassery et al., 2004). The ERK1/2 are phosphorylated by a MAPK/ERK Kinase-1/2 (MEK1/2), and the latter, in turn, is activated by an upstream protein called Raf (Cowan and Storey, 2003; Han et al., 2011). The JNKs, also known as SAPKs (stressactivated protein kinases), phosphorylate transcription factors such as c-Jun in response to the extracellular signals (Rao et al., 2003; Bogoyevitch et al., 2009). Varieties of signals such as cytokines, ultraviolet radiation, reactive oxygen species, heat shock, and osmotic shock activate JNK and p38-MAPK pathways leading to cell differentiation and/or apoptosis (Stalheim and Johnson, 2008). The p38-MAPK have four isoforms, namely p38-MAPKa (MAPK14/ SAPK2a), p38-MAPKb (MAPK11), p38-MAPKg (MAPK12/ERK6), and p38-MAPKd (MAPK13/SAPK4) (Haar et al., 2007; Li et al., 2008). The p38-MAPK regulate a variety of cellular processes such as transcriptional regulation, cell growth processes, cell adhesion and spreading, cell differentiation, apoptosis, macrophage and neutrophil functional responses, chemotaxis, granular exocytosis, and cellular responses to inflammation (Son et al., 2011). Lead, one of the most common environmental toxicants, accumulates in bones, kidneys, liver and nervous system organs, especially in the brain (Mehrotra et al., 2008; Kumar et al., 2011). Toxic effects of lead include functional alterations in several proteins, decrease in heme synthesis, gastrointestinal illness, cardiovascular abnormalities such as hypertension, renal malfunctions such as inhibition of glomerular filtration rate, neurological disorders such as encephalopathy, peripheral neuropathy, and neurobehavioral changes (reviewed in ATSDR, 2007). Since lead accumulates in the liver, it imparts extremely deleterious and chronic effects on regulation of cholesterols, phospholipids and free fatty acids (Kojima et al., 2004; Ademuyiwa et al., 2008). Lead-induced liver injuries are characterized by macrovacuolar steatosis, dilatation of the bile canaliculi, bile stasis, and hepatocyte injury (Narayana and Al-Bader, 2011). Lead affects cytochrome P450 (CYP450) in general (reviewed in Mudipalli, 2007; Levesque et al., 2011) and CYP4501A2 in particular (Degawa et al., 1995); it also increases glutathione S transferases indicating its carcinogenic potential (Roomi et al., 1986). The lead effects on mitochondria result in hepatotoxicity due to energy shortage-induced apoptosis of hepatocytes mediated via an upregulation of pro-apoptotic proteins, especially Bax (Xu et al., 2008; Begriche et al., 2011). Lead also interacts with

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the zinc-binding sites on important DNA-associated proteins and inactivates them, and these changes result in abnormal gene expressions (Yu et al., 2008). Lead upregulates the expression of some protooncogenes and secretion of growth factors that, in turn, stimulate hepatic hyperplasia (Mudipalli, 2007). Lead affects the chromatin structure, induces thermal denaturation of DNA and DNA double-helix destabilization, and inhibits transcription and DNA replication (Rabbani-Chadegani et al., 2009). Moreover, lead also has indirect effects on DNA structure and function; one such effect is mediated through suppression of DNA repair mechanisms, which results in accumulation of damaged DNA (Patra et al., 2011). However, in lead-exposed rats, although hepatocyte nuclear DNA double-strand breaks presenting 30 OH, 30 overhangs and 50 P blunt ends were significantly increased, the activities of 8oxo-dG were not altered indicating the lack of oxidative DNA damage (Narayana and Raghupathy, 2012). Nevertheless, accumulating evidence indicate that lead activates/or inhibits several intracellular signaling pathways involved in both cell survival and cell death (Wozniak and Blasiak, 2003). In astrocytoma cells, lead exposure resulted in protein kinase C-mediated upregulation of MEK1/2 (Lu et al., 2002) and in rat brain, JNKs and caspases were also activated indicating lead-induced cell death (Ramesh et al., 2001). Similarly, lead also induced the phosphorylation of p38MAPKa/b in bovine chromaffin cells (Leal et al., 2002). However, in vivo effects of lead exposure on MAPKs are unknown and there are no reports indicating dose-, duration of exposure (time)-, and sex-dependent effects on these proteins in hepatocytes. In addition, it is also not known whether or not lead-induced hepatotoxicity is also mediated via alterations in expressions of MPAKs. If, in case, the MAPKs activities are altered, are there any relationships between oxidative stress, MAPKs, and induced cell death? The present study was also designed to investigate dosedependent (although only two dose-levels were used), timedependent, and sex-dependent effects of lead on the abovementioned parameters. The results indicate that a correlation exists between lead-induced oxidative stress, apoptosis, and changes in expressions of MAPKs.

MATERIALS AND METHODS Animals Adult male and female (13–15-week-old) Wistar rats were procured from the Animal Resources Center at the Health Sciences Center of our University. They were maintained under controlled temperature (21–24 C) and humidity (50– 55%). The animals were housed in plastic cages (2 rats/ cage) with sterilized saw dust bedding and acclimatized for a week before lead treatment. All animals were given tap water and laboratory chow ad libitum. The experiments

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were conducted in accordance with the Institutional and International guidelines.

Estimation of Total Antioxidant Status (TAS)

Experimental Design and Lead Acetate Exposure

The TAS in the liver was quantified by an assay kit (Rel Assay Diagnostics) as per a previously described procedure (Erel, 2004). A total of 200 mg of liver tissue was homogenized in 93 cold buffer (5 mM potassium phosphate 1 1 mM EDTA; pH 7.0). The homogenate was centrifuged at 10,0003 for 15 min at 4 C and then the supernatant was separated and stored in 280 C, until used for the assay. In a 96 well plate, 125 mL/well of reagent 1 (assay buffer) was added to all wells. The first well was the standard 1 to which 7.5 mL deionized water was added and the second well was the standard 2 to which 7.5 mL standard 2 (1.0 mmol Trolox equivalent/L) was added. To the other wells, 7.5 mL of samples were added and then the initial absorbance was read at 660 nm. Then, 18.75 mL of reagent 2 (2,20 -azido-di-3-ethylbenzothiazoline sulphonate also known as ABTS radical solution) was added to all wells and incubated for 10 min and the second absorbance was read at 660 nm. The D absorbance of standard 1 or standard 2 or sample was the difference between first and second absorbance in each case. The TAS was expressed as a ratio of difference between D absorbance of standard 1 and sample, and difference between D absorbance of two standards, and expressed as mmol/g tissue.

The animals were randomly segregated into groups (G; N 5 8 (4 males 1 4 females in each group) and exposed to lead acetate as follows. Experiment 1: G1 – was given tap water, which served as a control group; G2 – was treated with 0.5%, and G3 – was treated with 1% lead acetate for 3d and sacrificed the next day (4d). Experiment 2: G4–was given tap water, which served as a control group; G5–was treated with 0.5%, and G6–was treated with 1% lead acetate for 14d and sacrificed the next day (15d). Experiment 3: G7–was given tap water, which served as a control group; G8–was treated with 0.5%, and G9–was treated with 1% lead acetate for 35d and sacrificed the next day (36d). The required amount of lead acetate (lead acetate-3hydrate; Riedel-de Haen, 32307) was weighed and dissolved in drinking water and given to rats for specified number of days.

Animal Sacrifice and Sample Collection All animals were weighed before the onset of lead exposure, once a week during the treatment period and, at the end of the experiment just before the sacrifice. The animals were anesthetized with ether and sacrificed by CO2 asphyxiation. In each animal, thoracotomy was conducted and blood was collected from the right ventricle. Whole body perfusion by infusing through the left ventricle of heparinized phosphate buffered saline (PBS, pH 7.4) was carried out to flush out blood from the liver. The livers were removed and diced into two parts; one part was stored in 280 C and the other part was fixed in 10% neutral buffered formalin.

Lipid Peroxidation This assay was conducted as per the procedure described previously (Ohkawa et al., 1979). A homogenate of 100 mg liver tissue was prepared in 1.15% KCl. Tetra-methoxyl propane (TMP) was used as a standard. A total of 100 lL of the homogenate or equal volume of water (blank) was mixed with 200 lL of a mixture of 8.1% sodium dodecyl sulfate and 1.5 mL of 20% acetic acid (pH 3.0), and 1.5 mL of 0.8% of thiobarbituric acid. Required amount of dH2O was added to make up 4 mL mixture and heated at 95 C for 1 h. A total of 1 mL of dH2O and 5 mL of a mixture of n-butanol and pyridine (15:1 v/v) were added to the mixture and shaken vigorously and centrifuged at 40003 for 10 min. The superficial organic layer was extracted and its absorbance was read at 530 nm. Lipid peroxidation was directly proportional to an increase in malondialdehyde (MDA) concentration in nmol units/g tissue (Ohkawa et al., 1979).

Terminal Deoxynucleotidyl TransferaseMediated dUTP Nick End Labeling (TUNEL Assay) The TUNEL assay was conducted according to the procedures described in the assay manual and in our previous paper (Narayana et al., 2012). Three mm thick paraffin sections were de-waxed in xylene, rehydrated in descending grades of alcohol, and washed with dH2O and PBS. The antigen retrieval was done in citrate buffer for 5 min in a microwave oven. The tissue sections were kept in PBS for 5 min, in 0.1% Triton X for 8 min and washed with PBS. The section was covered by 50 mL of TUNEL reaction mixture (50 mL of enzyme solution: terminal deoxynucleotidyl transferase in storage buffer 1 450 mL of labeling solution; In situ Cell Death Detection Fluorescein Kit, Roche). The negative control section was covered with only 50 mL of labeling solution. The slides were incubated in a humidified chamber for 1 h at 37 C and then washed with PBS. Freshly prepared DAPI (40 ,6-diamidino-2-phenylindole, 1:4000; Sigma Chemicals) was added onto the tissue section for 1 min and the sections were mounted in Vectashield (Vector Laboratories). The edges of the cover slips were sealed by nail polish. The slides were observed under a confocal microscope (ZEISS LSM 510 META) and selected areas were photographed. Ten fields for each liver were observed and the TUNEL positive hepatocyte nuclei were counted and the mean value was calculated.

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Western Blotting for MAPKs The Western blotting for the proteins was conducted as per the procedure described in our previous paper (Narayana et al., 2012). The total protein in the tissue lysates was estimated by the Bradford assay with bovine serum albumin as a standard and the proteins were separated by SDS–PAGE at 120 V. A 10% gel (1 mm; Invitrogen) was immersed in 13 running buffer [50 mL of 203 MOPS–SDS running buffer (Invitrogen) 1 dH2O up to 1 L] and 45 mg protein was loaded into each well; the electric circuit (140 V) was completed and electrophoresed. The gel was transferred to PVDF membrane (Polyvinylidene fluoride, Biorad) immersed in the transfer unit filled with the 13 transfer buffer (50 mL of 103 transfer buffer 1 200 mL methanol 1 required amount of dH2O) at 30 V for 75 min. The membrane was blocked with 5% non-fat milk in 13 Tris buffered saline for 1 h and probed with rabbit polyclonal primary antibodies for ERK1/2, p38-MAPKa/b and JNK1/3 (1:200; Santa Cruz Biotechnology) overnight at 4 C. The membrane was washed thrice with Tris buffered saline-0.1% Tween 20 (Sigma Chemicals) for 10 min each and incubated with appropriate secondary antibodies diluted with blocking solution (1:500) for 1–2 h. The protein bands were labeled with luminol reagent (ECL reagent; Santa Cruz Biotechnology) for 1 min and exposed to a photographic film (Hyperfilm) for 3–4 min. The density of the protein bands was quantified using GS-800 Calibrated Densitometer (Bio-Rad Quantity One 4.6.3) at sensitivity of 11 and band width of 3.302 mm. b-actin was used as an endogenous control. To normalize the band densities of the proteins, their density values were divided by respective b-actin values.

Confocal Microscopy for MAPKs The confocal microscopy was performed as per the procedures described previously (Renshaw, 2007). Three mm thick paraffin sections were mounted on poly-L-lysine-coated slides. The sections were de-waxed in xylene, hydrated in descending grades of alcohol and washed with dH2O and PBS (pH 7.35). The slides were submerged in antigen retrieval solution (0.01 M sodium citrate buffer) for 5 min in a microwave oven with medium power, removed and brought to room temperature. The slides were immersed in PBS for 5 min, treated with 0.1% Triton X (Sigma–Aldrich) for 15–30 min and then treated with 3% H2O2 for endogenous peroxidase quenching. The tissue was treated with 50 mM glycine and 1% sodium borohydride for 30 min each and washed with PBS. The slides were incubated in blocking solution (5% bovine serum albumin 1 10% normal goat serum 1 0.1% Triton X in PBS) for an hour and covered with diluted rabbit primary antibodies for ERK1/2, p38MAPKa/b and JNK1/3 (1:200; Santa Cruz Biotechnology) and incubated overnight in humidified chamber at 4 C. Negative control sections were covered with antibody diluents

Environmental Toxicology DOI 10.1002/tox

and appropriate positive controls were used. Next day, the slides were incubated in dark with appropriate diluted secondary antibodies (1:250) in a humidified chamber for an hour and treated with freshly prepared DAPI (40 ,6-diamidino-2-phenylindole; 1:4000 in PBS; Sigma Chemicals) for 1 min. The slides were mounted with Vectashield (Vector Laboratories) and the coverslip edges were sealed with nail polish and the slides were viewed under a confocal microscope and representative photomicrographs were taken with 603 objective (ZEISS LSM 510 META Confocal microscope).

Statistical Analysis The data were expressed as mean 6 S.D. for each group and subjected to statistical analysis by SPSS software version 17. The groups were compared by one way ANOVA followed by Fisher’s least square difference test. In each experiment, control and experimental groups were compared (the male and female rats were combined to form a single group; merged group). The comparisons were between control and all experimental groups, as well as among the experimental groups to observe time- and dose-dependent responses. For this purpose, we combined both male and female rats together as one group. To analyze the sex-dependent response, in each group, male and female rats were compared. In addition, dose- and time-dependent effects were also investigated in each sex. In all cases, P < 0.05 was considered significant.

RESULTS The present study investigated the effects of two dose-levels of lead on oxidative stress, cell death and MAPKs signaling in the liver. We included both male and female rats as one group in each treatment category (merged group) and compared with respective time-matched control groups. In addition, the male and female rats were separately compared to time- and sex-matched controls in each experiment. The summary of results from merged groups and sex-response study are indicated in Tables I and II, respectively.

Lead Exposure Reduces Body Weight Gain The body weights of the rats were measured in 36d groups on three time points on 4d, 15d, and 36d. In merged groups, the body weights of lead-exposed rats did not show significant differences with respective control groups possibly due to high variations [Fig. 1(A); Table I]. Thus, we investigated sex-dependent effects on percentage body weight changes, which revealed significant decreases in lead-treated groups as compared to time- and sex-matched control groups (P < 0.05), except in female rats on 36d [Fig. 1(B)]. A dosedependent decrease in weight gain was observed in male rats on 15d (P < 0.05), but not in other experiments [Fig. 1(B)].

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TABLE I. Summary of the results in merged groups

4d

15d

36d

Dose

Body Weight

Liver Weight

LPX

TAS

Apoptosis

ERK1/2

p38-MAPKa/b

JNK1/3

0 0.5% 1% 0 0.5% 1% 0 0.5% 1%

N NC NC N NC NC N NC NC

N NC NC N NC NC N NC NC

N " " N " " Di Ti N " " Di Ti

N # # N # # N C #

N " " N " " N " "

N # # N NC Ti " Di Ti N # Td " Di Ti

N NC NC N NC NC N NC NC

N " " N NC " N NC "

The data in control groups are considered normal (N) and the data in other groups are compared to the respective control data. If no significant changes are observed, then the effects are considered ‘no change’ (NC). The significant increase (") and decrease (#) against respective control values are indicated. Di, dose-dependent increase; Td, time-dependent decrease; and Ti, time-dependent increase.

Time-dependent effects on body weight changes were discordant. The male rats in 0.5% dose group on 36d showed a recovery in weight gain compared with the corresponding group on 4d (P < 0.05); however, the weight gain was still less than that in the control group (P < 0.05). In 1% dose groups, the male rats showed a decrease in weight gain on 15d and an increase on 36d compared with those on 4d (P < 0.05). In 0.5% dose groups, the female rats showed a decrease on 15d and an increase on 36d in the weight gain as compared to that on 4d (P < 0.05). But in 1% dose groups, the female rats showed a significant improvement in weight gain on 36d over the previous two experiments (P < 0.05). In fact, the female rats in the control groups consistently showed less weight gain than the male rats did (P < 0.05), but such a difference was not observed in experimental groups, except in 0.5% group on 15d [P < 0.05; Fig. 1(B); Table II].

pared to that on 4d (P < 0.05), but such a difference was not seen between 15d and 36d [Fig. 3(A); Table I]. The sex– response study indicated significant increases in the lipid

Lead Does Not Affect Liver Weight in Rats The liver weights did not show any significant changes in lead-exposed merged groups as compared to their respective time-matched control groups [Fig. 2(A); Table I]. The sexresponse study indicated that, generally, the male rats had heavier livers than the female rats had in both control and experimental groups [P < 0.05; Fig. 2(B)]. Only the male rats in 1% dose group on 4d showed significantly more liver weight than the time-matched control male rats [P < 0.05; Fig. 2(B); Table II].

Lead Induces Lipid Peroxidation in a Dose- and Time-Dependent Pattern but Without Any Sex-Dependent Effects In merged groups, the MDA levels significantly increased following lead exposure (P < 0.05) as compared to timematched control groups indicating lipid peroxidation [Fig. 3(A)]. The latter occurred in a dose-dependent manner on 15d and 36d (P < 0.05). In 1% dose groups, a timedependent increase was observed on 15d and 36d as com-

Fig. 1. Effects of lead acetate on body weights of rats. (A) The initial (IW) and final body weights (FW) of rats (male and female rats are together; merged groups) are indicated (N 5 8; mean 6 SD) without any significant differences. (B) Percentage body weight changes in control and leadexposed rats (N 5 4/sex/dose/time point). The weight changes in male and female rats were plotted separately. *P < 0.05, control male rats versus lead-exposed male rats; # P < 0.05, control female rats versus lead-exposed female rats. uP < 0.05, time-dependent effects in male rats in 0.5% dose groups; rP < 0.05, time-dependent effects in male rats in 1% dose groups; $P < 0.05, time-dependent effects in female rats in 0.5% dose groups; dP < 0.05, time-dependent effects in female rats in 1% dose groups; uP < 0.05, male rats versus female rats.

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TABLE II. Summary of the results of the sex-response study Body Weight Dose

M

F 2

Liver Weight M

N 0 N N 0.5% # # NC 1% # # " 15d 0 N N2 N 0.5% # #2 Td NC 1% # Dd # NC 36d 0 N N2 N 0.5% # Ti NC Ti NC 1% # Dd NC Ti NC 4d

F 2

LPX M

F

N N N NC2 " " NC2 " " N2 N N NC2 " " NC2 " Di Ti " Ti N2 N N NC2 " " NC2 " Di Ti " Di Ti

TAS

Apoptosis

M F

M

N # # N # # N # #

F

ERK1/2 M

p38-MAPKa/b F

M

F 2

JNK1/3 M

N N N N N N N N # " "1 # # #2 "1 " # " " Dd # NC # " Dd " N N N N N2 N N2 N 1 2 # " " Ti NC Ti NC Ti # Td " Td NC # " " Dd " Di Ti " Di Ti #2 Di Ti " Di Ti " Di N N N N N2 N N2 N # " Ti " # Dd Td # Dd Td # Ti " Td # # " Di Ti "2 Dd " Di Ti " Di Ti # Di " Di Ti NC Di

F N2 " " N2 " " Di N2 # " Di

The data in control groups are considered normal (N). The significant increase (") and decrease (#) in lead-exposed groups are indicated. The lack significant changes in results in lead-exposed groups as compared to the respective control groups are indicated as ‘no changes’ (NC). The sex-dependent increase (1) and decrease (2) are indicated wherever the differences are significant. Dd, dose-dependent decrease; Di, dose-dependent increase; Td, timedependent decrease; and Ti, time-dependent increase.

peroxidation in both sexes in all lead-exposed groups [P < 0.05; Fig. 3(B)]. The male rats showed dose-dependent increases in lipid peroxidation on 15d and 36d, whereas the female rats showed dose-dependent increases only on 36d (P < 0.05), although a trend for dose-dependent increase was observed on 15d. Both male and female rats showed timedependent increases in the lipid peroxidation in 1% groups on 15d and 36d as compared to that on 4d [P < 0.05; Fig.

Fig. 2. Effects of lead-exposure on liver weight (mean 6 SD). (A) The liver weight in merged groups (N 5 8) without any significant effects. (B) Sex-dependent effects on the liver weight of lead exposure (N 5 4/sex/dose/time point). *P < 0.05, control male rats versus lead-exposed male rats; u P < 0.05, male rats versus female rats.

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3(B)]. On the other hand, lead exposure did not show any sex differences in induction of the lipid peroxidation [Fig. 3(B); Table II].

Fig. 3. Effects of lead-exposure on malondialdehyde (MDA) concentration in the liver (mean 6 SD). (A) The MDA activities in merged groups (N 5 8). *P < 0.05, control versus experimental groups; *P < 0.05, dose- and time-dependent effects are diagrammatically indicated. (B) Sex-dependent effects of lead exposure on MDA activities (N 5 4/sex/dose/ time point). *P < 0.05, control male rats versus leadexposed male rats; #P < 0.05, control female rats versus lead-exposed female rats. eP < 0.05, dose-dependent effects in male rats; /P < 0.05, dose-dependent effects in female rats; rP < 0.05, time-dependent effects in male rats in 1% dose groups; dP < 0.05, time-dependent effects in female rats in 1% dose groups.

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[P < 0.05; Fig. 6(A); Table I]. The sex-response study indicated higher incidences of TUNEL positive hepatocytes in both male and female rats in all three experiments [P < 0.05; Fig. 6(B)]. A dose-dependent increase in cell death was observed in male rats only on 36d (P < 0.05). In female rats, the 1% dose groups showed less cell death than the 0.5% dose groups did; thus, there was a dose-dependent decrease in cell death [P < 0.05; Fig. 6(B)]. A time-dependent increase in cell death was observed in male rats in both dose groups on 36d as compared to the other two experiments (P < 0.05). The female rats showed a time-dependent increase in cell death in 0.5% dose group on 15d when compared to the other two experiments (P < 0.05), but 1% dose groups did not show any time-dependent effects. On 4d and 15d, the female rats in 0.5% dose groups showed more cell death than the male rats did (P < 0.05); but conversely, the male rats in 1% dose group on 36d showed more cell death than the female rats did [Fig. 6(B); Table II].

Fig. 4. Effects of lead-exposure on total antioxidant status (TAS) in livers (mean 6 SD). (A) The effects on merged groups (N 5 8). *P < 0.05, control versus lead-exposed groups. (B) Sex-dependent effects on TAS (N 5 4/sex/dose/ time point). *P < 0.05, control male rats versus leadexposed male rats; #P < 0.05, control female rats versus lead-exposed female rats.

Lead Reduces TAS Without Any Dose-, Time-, or Sex-Dependent Effects The hepatic TAS was significantly decreased in leadexposed merged groups when compared to time-matched control groups (P < 0.05). However, there were no dose- or time-dependent effects on hepatic TAS [Fig. 4(A); Table I]. The sex-response study also did not show any significant sex differences in TAS levels [Fig. 4(B)]. However, both male and female rats in lead-exposed groups showed less TAS than did the time- and sex-matched controls, similar to the effects observed in merged groups [P < 0.05; Fig. 4(B); Table II].

Lead Induces Cell Death of Hepatocytes: Dose-, Time- and Sex-Dependent Effects In the livers of control rats, the number normal nuclei (DAPI-labeled; blue) was more than that of TUNEL positive (green) nuclei indicating cell death of a few hepatocytes (Fig. 5). However, in lead-exposed rat livers, the number of green fluorescent hepatocyte nuclei was more than the blue nuclei indicating an increase in cell death (Fig. 5). In merged groups, quantification of TUNEL positive nuclei indicated an increased cell death as compared to their time-matched controls without any dose- or time-dependent effects

MAPK Expression Alters in Lead-Exposed Hepatocytes Lead Affects the ERK1/2 Expression Confocal microscopy showed both cytoplasmic and nuclear localization of ERK1/2 in hepatocytes (Fig. 7). In livers of control rats, a very few hepatocytes showed high intensity protein expression as compared to other hepatocytes. In livers from 1% lead-treated rats, the protein expression and its nuclear translocation increased in hepatocytes (Fig. 7). In merged groups, the protein activities decreased in both dose groups on 4d (P < 0.05) as compared to the time-matched control group [Figs. 8(A) & (B)]. On 36d, a significant decrease in 0.5% dose group and on 15d and 36d, an increase in 1% dose groups were observed when compared to their respective control groups [P < 0.05; Fig. 8(B)]. In 0.5% groups, the expression of the protein decreased from 4d to 36d, but with an interim recovery on 15d. On 15d and 36d, a time-dependent increase in the protein expression was observed in 1% groups as compared to that on 4d (P < 0.05; Table I). The results of the sex-response study were similar to that observed in merged groups [Fig. 8(C)]. Sex difference was observed only in control groups on 15d and 36d; the female rats showed less protein expression than the male rats did (P < 0.05). Dose- and time-dependent effects in both sexes were similar to the results observed in merged groups (Table II).

Lead Induces Sex-Dependent Effects on P38MAPKa/b This protein was not only localized in cytoplasm, but also in nuclei of hepatocytes (Fig. 9). Similar to the ERK1/2 expression, some hepatocytes in livers of control rats showed high intensity staining, whereas the others showed low intensity staining. In merged groups, although some changes in

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Fig. 5. Effects of lead exposure on apoptosis in the liver. The apoptosis was evaluated by the TUNEL assay. Representative merged confocal images of the livers showing TUNEL positive hepatocyte nuclei in control and experimental livers. Blue nuclei (DAPI-stained) are normal and green (fluorescin-stained) are the ones undergoing apoptosis. Note that a few nuclei stained with both TUNEL and DAPI indicating that not whole nuclei experienced DNA damage. Original magnification, 6003. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

protein activities were observed in lead-treated groups, the effects were not statistically significant as compared to respective control groups [Fig. 10 (A) & (B); Table I]. The sex-response study indicated that the effects were significantly different in both sexes as compared to their sex- and time-matched controls [P < 0.05; Fig. 10(C)]. The male rats showed less protein levels in all experiments at both doselevels than their respective controls [P < 0.05; Fig 10C). The male rats on 15d and 36d showed less protein expression in 0.5% groups than in 1% groups (P < 0.05). The female rats showed higher protein activities in 0.5% group than in 1% group on 4d, but on 15d and 36d, a dose-dependent increase was observed (P < 0.05). In male rats, an intermittent decrease in protein activities was observed in 0.5% group on 15d when compared to the other two experiments. In 1%

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dose groups, a decrease on 15d and an increase on 36d were observed as compared to that on 4d. The female rats showed a time-dependent decrease in 0.5% groups and a timedependent increase in 1% groups. The female rats in control groups showed less protein expression than did the male rats [P < 0.05; Fig. 10(C)]. Interestingly, in the experimental groups, whenever sex-dependent effects were observed, the female rats showed more protein expression than did the male rats [P < 0.05; Fig. 10(C); Table II].

Lead Interferes with the JNK1/3 Expression in Hepatocytes The protein was expressed not only in cytoplasm, but also in nuclei of hepatocytes (Fig. 11). Hepatocytes in control rat

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decrease in 0.5% groups, but no such effects were observed in 1% groups. The female rats showed a time-dependent decrease in 0.5% groups and a time-dependent increase in 1% groups [P < 0.05; Fig. 12(C)]. In control groups, the female rats showed less JNKs expression than the male rats did (P < 0.05). In experimental groups, generally, no sex difference was observed in the protein expression, except in 1% dose group on 36d, on which day, the male rats showed less protein expression than did the female rats (P < 0.05; Table II).

DISCUSSION

Fig. 6. Effects of lead-exposure on incidence of TUNEL positive hepatocytes (mean 6 SD). (A) The incidence of TUNEL positive cells in merged groups (N 5 8). *P < 0.05, control groups versus lead-exposed groups. (B) Sex-dependent effects on cell death of hepatocytes (N 5 4/sex/dose/time point). *P < 0.05, control male rats versus lead-exposed male rats; #P < 0.05, control female rats versus leadexposed female rats; eP < 0.05, dose-dependent effects in male rats; /P < 0.05, dose-dependent effects in female rats; u P < 0.05, time-dependent effects in male rats in 0.5% groups; rP < 0.05, time-dependent effects in male rats in 1% groups; $P < 0.05, time-dependent effects in female rats in 0.5% dose groups; uP < 0.05, male rats versus female rats.

livers showed low intensity staining as compared to those in lead-treated rat livers. Some hepatocytes in livers of experimental rats showed higher intensity staining than the other hepatocytes did (Fig. 11). In merged groups, a significant increase in protein expression was observed at both doselevels on 4d and in 1% dose groups on 15d and 36d when compared to time-matched control groups [P < 0.05; Fig. 12(A), (B)]. The effects were dose-dependent on 15d and 36d (P < 0.05). There were no time-dependent effects in any experiment [Fig. 12(B); Table I]. The sex-response study indicated discordant but significant effects in both male and female rats [Fig. 12(C)]. A significant increase in both dose groups was observed in male rats on 4d, but on 15d, only 1% dose group showed an increase and on 36d, a decrease in 0.5% group was also observed [Fig. 12(C); P < 0.05]. The female rats showed significant increases in protein levels in both dose groups on 4d, but on 15d, a dose-dependent increase, and on 36d, an increase only in 1% dose group was observed compared with that in sex- and time-matched controls (P < 0.05). The male rats showed a time-dependent

The present study investigated dose-, time-, and sexdependent effects of lead toxicity on livers. We observed that lead exposure resulted in induction of oxidative stress, apoptosis, and alterations in MAPKs expression in hepatocytes. Lead exposure, up to 5-week-long period, did not affect the body weight or the liver weight in merged groups possibly due to high variations in the weights between the animals. These findings are in agreement with the results of previous studies, which indicated a lack of lead effects on body weight (Smith et al., 2008; Narayana and Al-Bader, 2011; Narayana and Raghupathy, 2012). Therefore, we analyzed percentage weight changes and observed a significant decrease in weight gain in lead-treated rats, except in female rats on 36d. These findings indicate lead-induced general toxicity in rats. The normal female control rats showed less weight gain than normal male rats did, possibly due to sex differences in physiological mechanisms. The inherently less weight gain observed in control female rats was not observed in lead-treated ones; in fact, on 36d, the weight gain recovered to sex-matched control levels indicating that lead actually increased the weight gain in female rats. In other words, as the number of treatments increased, the weight gain also increased in a time-dependent manner. However, there was no animal mortality in any experiment indicating that the dose-levels used in this study were not terminally toxic to the animals. An increase in MDA activities and a decrease in TAS indicate that lead exposure resulted in generation of free radicals, which led to membrane damage in hepatocytes and depletion in endogenous antioxidant status (Martindale and Holbrook, 2002). In general, an increase in lipid peroxidation and a decrease in endogenous levels of antioxidants such as superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase result in an oxidative stress status (Pagliara et al., 2003; Abdel-Moneim et al., 2011; Kilikdar et al., 2011). Lead binds sulfhydryl groups in enzymatic antioxidants such as superoxide dismutase leading to inhibition of their activities (Liu et al., 2012). The decrease in TAS observed in our study may be due to reduction by lead of antioxidant levels in livers. For our knowledge, this is the first study to report the effects of lead on TAS. Further,

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Fig. 7. Effects of lead exposure on expression of ERK1/2 in the liver. Merged images of hepatocytes showing ERK1/2 immunolocalization in the cytoplasm (green, FITC). The nuclei were counterstained with DAPI. The nuclear translocation was observed in 1% dose groups on 15d and to a lesser extent on 36d. Original magnification, 6003. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

there are no studies, which investigated dose-, time-, and sex-dependent effects on oxidative stress of lead. The absence of sex-dependent effects on lipid peroxidation and TAS indicates the lack of influence of physiological differences, hormonal or otherwise, on lead-induced mechanisms responsible for upregulation of oxidative stress. The results obtained on 15d and 36d indicate that hepatocyte membrane damage occurs in a dose- and time-dependent manner in lead-exposed rats. But a lack of dose- and time-dependent effects on TAS indicate that the antioxidant levels, although found decreased, do not get affected by increasing strength of the doses or the number of doses, may be because of continuous exogenous supply of antioxidants through the food. The induced apoptosis of hepatocytes showed a positive correlation with upregulated oxidative stress. On 4d and

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15d, that is, within 2 weeks of continuous treatment, 0.5% dose-level had induced more hepatocyte apoptosis than 1% dose-level did. This discrepancy was due to more apoptosis in female livers than that in male livers exposed to 0.5% dose-level. This increase in apoptosis in female livers exposed to the lower dose indicates the existence of an inverse relationship between cell death and the strength of the dose. The reason for this discrepancy is not clear, but it may be related to sex hormonal actions. Moreover, less apoptosis in 1% groups than in 0.5% groups in females may be due to higher dose-induced hepatocyte proliferation and survival (Narayana and Al-Bader, 2011). In fact, lead simultaneously induces hepatocyte proliferation and cell death in rats (Narayana and Raghupathy, 2012), but repeated lead exposures result in progressive decrease in cell proliferation

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Fig. 8. Effects of lead-exposure on ERK1/2 in livers. (A) Representative Western blotting bands for ERK1/2 (44 kDa). B) The ERK1/2 expression in merged groups (N 5 8; mean 6 SD), *P < 0.05, control versus lead-exposed groups; *P < 0.05, dose- and time-dependent effects are diagrammatically indicated. (C) Sex-dependent effects on ERK1/2 expression in livers (N 5 4/sex/dose/time point), *P < 0.05, control male rats versus lead-exposed male rats; #P < 0.05, control female rats versus lead-exposed female rats; e P < 0.05, dose-dependent effects in male rats; /P < 0.05, dose-dependent effects in female rats. uP < 0.05, timedependent effects in male rats in 0.5% dose groups; r P < 0.05, time-dependent effects in male rats in 1% dose groups; $P < 0.05 and dP < 0.05, time-dependent effects in female rats in 0.5% and 1% dose groups, respectively; u P < 0.05, male rats versus female rats.

as compared to acute exposures (Ledda-Columbano et al., 1983). Because of this tilted balance between cell proliferation and cell death, the latter increases in livers after longterm lead treatment. Further, during involution of leadinduced hyperplasia, the frequency of hepatocytes undergoing cell death increases (Columbano et al., 1985). Thus, in male rats, during early exposure, apoptosis was induced without any correlation to the strength of doses, but longterm treatment led to a dose-dependent increase in cell death. Hepatocytes in the livers of female rats exposed to 0.5% dose seem to be more sensitive and respond better to cell death inducing properties of lead than those exposed to 1% dose; we believe that this discrepancy is probably due to more cell proliferation in 1% groups than in 0.5% groups. Although the female rats are more sensitive to apoptosis inducing properties of lead up to 15d, following long-term exposure, the male rats become more sensitive as seen on

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36d. Lead-induced increase in TUNEL positive cells was due to induced DNA damage in hepatocyte nuclei (Pagliara et al., 2003; Xu et al., 2008; Narayana and Raghupathy, 2012). The TUNEL assay identifies DNA strand breaks presenting with 30 OH ends, therefore, the assay is widely used to label apoptotic cells (Didenko et al., 1998). Because the assay may label DNA damage in some necrotic cells (Didenko et al., 1998), a previous study compared an apoptosis-specific labeling of 30 overhangs, which is a confirmatory marker for apoptosis (Hornsby and Didenko, 2012), in lead-exposed hepatocytes and found similar results in both assays (Narayana and Raghupathy, 2012). This indicates that the cell death of hepatocytes observed in this study was indeed apoptosis. Activation of ERKs stimulates cell proliferation and survival (Hetman and Gozdz, 2004; Roskoski, 2012), but some evidence also indicate a role for them in cell death induction (Chu et al., 2004). A decrease in ERK1/2 expression and an increase in cell death on 4d in merged groups indicate an inverse relationship between the two endpoints. A decreased protein expression in 0.5% group on 36d may also have a similar relationship with apoptosis. Increased protein expression in 1% dose groups on 15d and 36d may well be correlated with higher dose-induced cell proliferation (Hommes et al., 2003; Narayana and Al-Bader, 2011), which may lead to the onset of carcinogenesis, although the ability of lead to induce cancers has not been unequivocally confirmed (ATSDR, 2007). These effects indicate that both down- and upregulation of ERK1/2 expression may be possible in the liver as lead concomitantly induces cell death of some hepatocytes and proliferation of some others (Narayana and Raghupathy, 2012). These interesting findings indicate that chronic exposure to a higher dose results in upregulation and to a relatively lower dose results in inhibition of the ERK pathway. The ERK1/2 expression is directly related to the strength of the doses and the duration of exposure as seen in 1% groups on 15d and 36d. Moreover, during early phase of lead exposure, the ERK1/2 expression is downregulated, but as the number of treatment increases, the protein expression is increased in a dose-dependent manner. Interestingly, the effects on ERK1/2 of lead are not exerted in any sexdependent pattern, but in untreated control female rats the hepatic expression of the protein is less than that in the male rats, which might be related to female sex hormone effects, but this conjecture has to be further elucidated. Confocal microscopy showed both nuclear and cytoplasmic localization of the protein indicating the occurrence of nuclear translocation in 1% groups, especially on 15d. This may result in induction of transcription of several genes with some consequences (Chang and Karin, 2001). Upregulated ERK1/2 pathway might be a consequence of induced oxidative stress (Martindale and Holbrook, 2002; Leonard et al., 2004), but we did not observe any particular relation between the oxidative stress and the ERK1/2 expression. Although there are no studies on lead-induced effects on the

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Fig. 9. Effects of lead exposure on expression of p38-MAPKa/b in the liver. The merged images of hepatocytes showing immunolocalization of the proteins in the cytoplasm (green, FITC). The nuclei were counterstained with DAPI. Perinuclear translocation of the protein was also seen. Original magnification, 6003. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ERK1/2 pathway in hepatocytes, the heavy metal activates ERK1/2 and p38-MAPK in fish cerebellum, but without affecting total MAPKs levels (Leal et al., 2006). Effects of lead on MAPKs are studied in vitro, especially in glia, in which an upregulation of the ERK1/2 expression was observed and, fibroblast growth factor alleviated this effect (Zhang et al., 2007). In rat C6 glioma cells, lower dose of lead upregulated the ERK pathway by 24 h and p38-MAPK and JNK pathways by 48 h (Posser et al., 2007). In vivo, in immature rat hippocampal neurons, lead exposure (2–12 mg/ kg) upregulated the ERK and p38-MAPK pathways without any effects on cell viability (Cordova et al., 2004). However, these studies evaluated the effects of short-term exposure on cells of the nervous system and our results cannot be compared to them as the experimental protocol and target tissues

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are different, but it is clear from these afore-mentioned studies that the lead exposure has an effect on ERK pathway. The p38-MAPK and JNKs may act as anti- or proapoptotic proteins depending upon the stimuli, but they are definitely involved in induction of cell death (Chen and Tan, 2000). These two kinases, although largely work as independent modules, are simultaneously upregulated by many exogenous stimuli including ischemia, oxidative stress, and DNA damage (Wada and Penninger, 2004). The lack of significant changes in p38-MAPKa/b activities in merged groups was due to high inter-sample differences. The sexresponse study showed an increase in female rats and a decrease in male rats of p38-MAPKa/b activities with significant sex differences. In females, the increase in p38MAPKa/b activities happened in a dose-dependent manner

MAPK SIGNALING IN LEAD-EXPOSED HEPATOCYTES

Fig. 10. Effects of lead-exposure on p38-MAPKa/b expression in livers. (A) Western blotting showed a band at 38 kDa level. (B) Densitometric data (mean 6 SD) for p38MAPKa/b in merged groups (N 5 8) did not show any significant differences with time-matched control groups in any experiment. (C) Sex-dependent effects of lead-exposure on the protein expression in livers (N 5 4/sex/dose/time point). *P < 0.05, control male rats versus lead-exposed male rats; #P < 0.05, control female rats versus lead-exposed female rats; eP < 0.05, dose-dependent effects in male rats; /P < 0.05, dose-dependents in female rats; uP < 0.05, time-dependent effects in male rats in 0.5%; rP < 0.05, time-dependent effects in male rats in 1%; $P < 0.05, timedependent effects in female rats in 0.5% groups; dP < 0.05, time-dependent effects in female rats in 1% dose groups; u P < 0.05, male rats versus female rats.

on 15d and 36d. It appears that sex-dependent physiological differences in lead metabolism machinery in the liver may be responsible for the sex-dependent effects observed in p38-MAPKa/b activities. In untreated control females, the protein activities were less than that in male rats indicating the possibility of inherent sex-dependent differences related to differences in physiological changes. The p38-MAPKa/b expression decreased in males and increased in females in correlation with increased oxidative stress and apoptosis. The increased p38-MAPKa/b expression in females may be related to cell death induction. The reduced p38-MAPKa/b expression in males may be either an anti-apoptotic response or may not be related to apoptosis at all, upon lead exposure. The JNKs (JNK1 and JNK3) are involved in both cell proliferation and cell death (Seki et al., 2012). A variety of factors ranging from cytokines to free radicals activate JNKs by phosphorylating upstream MAP3Ks and MAP2Ks, and

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the JNKs, in turn, activate numerous downstream proteins including components of activator protein-1 (Haeusgen et al., 2011). Thus, lead-induced oxidative stress might be the stimulus that triggered upregulation of JNK1/3 in the present study. An increase in JNK1/3 in merged groups on 4d and in 1% dose groups on 15d and 36d indicate a correlation with apoptosis. Initial transient JNK1/3 activation on 4d may be related to cell survival and proliferation, a process mediated through its downstream transcription factors, whereas a prolonged oxidative stress and JNK1/3 activation seen in 1% groups on 15d and 36d may be related to apoptosis (Seki et al., 2012). The control female rat livers showed less JNK1/3 than that of the control male rats, may be due to sex-dependent physiological differences. But, among leadexposed groups, the female rats showed more JNK1/3 expression in 1% dose group on 36d indicating a shift in sex-dependent expression of the protein. As there are no reports on sex-dependent expression of MAPKs in livers of even normal animals, it is difficult to provide any suitable explanation for these differences, but we believe that discrete dissimilarities in sex hormone physiology may be a reason for the differences in the expression of the protein. Lead exposure is known to upregulate other pro-apoptotic proteins such as Bax and downregulate Bcl-2, indicating activation of intrinsic apoptotic pathway (Xu et al., 2008). That there are numerous proteins involved in execution of cell death, it may be difficult to ascertain which other protein expressions are regulated by lead. In vitro experiments in C6 rat glioma cells exposed to lead indicated decreased cell viability and an increased activation of both p38-MAPKa/b and JNKs (Posser et al., 2007). In bovine chromaffin and human neuroblastoma cells, Leal et al. (2002) found a leadinduced upregulation of p38-MAPK and its substrate HSP27. In human non-small cell lung adenocarcinoma CL3 cells, lead induced the activation of ERK1/2 but not that of p38-MAPK and JNKs and the authors reported that through the ERK1/2 activation, lead enhanced nucleotide excision repair synthesis (Lin et al., 2003). Moreover, inhibition of ERK1/2 greatly increased the cytotoxic effects of lead in the CL3 cells (Lin et al., 2003). Thus, in our study, besides the activation of p38-MAPKa/b and JNK1/3, the increased hepatocyte death may be related to inhibition of ERK1/2 expression. Nevertheless, the results obtained from in vitro studies on different cell lines cannot be simply extrapolated to interpret the results of the current study as in vivo effects of lead may or may not be in consensus with that of in vitro experiments. These in vitro experiments indicate a role for lead in alteration of intracellular MAPKs expression, but at the same time, the reports are conflicting. However, the in vivo effects of lead on MAPKs signaling in the liver have not previously been investigated. Another interesting observation of this study is upregulation of ERK1/2, p38MAPKa/b, and JNK1/3 indicating the prevalence of both pro-cell survival and pro-cell death pathways indicating the earlier finding of coexistence of cell proliferation and death

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Fig. 11. Effects of lead exposure on expression of JNK1/3 in the liver. Merged images of hepatocytes showing the immunolocalization of the JNKs in the cytoplasm (green, FITC). The nuclei were counterstained with DAPI. Note that nuclear translocation of the proteins was also observed. Original magnification, 6003. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

in the liver after lead exposure (Narayana and Raghupathy, 2012). For our knowledge, there are no studies on lead-induced sex differences in the parameters described in this study. However, lead exposure led to earlier onset of liver hyperplasia and more upregulated hepatocyte divisions in male rats than that in female rats (Tessitore et al., 2000). The pronounced hyperplasia in males showed an association with elevated total cholesterol, cholesterol esters, and reduced plasma high density lipoprotein (Tessitore et al., 2000). During regression of lead-induced hyperplasia, apoptosis also occurs earlier and in a more upregulated pattern in males than that in females (Tessitore et al., 1995). Conversely, we observed apoptosis in female livers than that in males in lead-exposed groups up to 15d, but by 36d, the males

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showed more apoptosis than the females. This may be related to hormonal differences in both sexes, although there are no studies indicating that this is the case. However, the involvement of sex hormones has been implicated in genesis of sex differences in cadmium-induced hepatotoxicity in which progesterone and b-estradiol enhanced the heavy metal-induced liver damage (Shimada et al., 2012). Thus, the upregulated apoptosis in female rats in 0.5% dose groups up to 15d and the p38-MAPKa/b expression compared to that in male rats may be related to progesterone and estradiol actions. In conclusion, a correlation exists between oxidative stress, apoptosis and MAPKs in lead-exposed hepatocytes. Long-term exposures result in dose-dependent effects, while time-dependent increases in lipid peroxidation is only

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ATSDR reviewed-Agency for Toxic Substances and Disease Registry Division of Toxicology and Environmental Medicine/ Applied Toxicology Branch. 2007. Toxicological profile for lead. ATSDR 1–582. Begriche K, Massart J, Robin MA, Borgne-Sanchez A, Fromenty B. 2011. Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver. J Hepatol 54:773–794. Bogoyevitch MA, Ngoei KRW, Zhao TT, Yeap YYC, Ng DCH. 2009. c-Jun N-terminal kinase (JNK) signaling: recent advances and challenges. Biochem Biophys Acta 1804:463–475. Chang L, Karin M. 2001. Mammalian MAP kinase signalling cascades. Nature 410:37–40. Chen YR, Tan TH. 2000. The c-Jun N-terminal kinase pathway and apoptotic signaling. Int J Oncol 16:651–662. Chu CT, Levinthal DJ, Kulich SM. 2004. Oxidative neuronal injury. The dark side of ERK1/2. Eur J Biochem 271:2060– 2066.

Fig. 12. Effects of lead exposure on JNK1/3 in livers. (A) Western blotting showed a band for the JNKs at 55 kDa level. (B) Densitometric data for the JNKs in merged groups (N 5 8; mean 6 SD). *P < 0.05, control rats versus leadexposed rats. (C) Sex-dependent effects of lead-exposure on the JNKs. *P < 0.05, control male rats versus leadexposed male rats; #P < 0.05, control female rats versus lead-exposed female rats; eP < 0.05, dose-dependent effects in male rats; /P < 0.05, dose-dependent effects in female rats; $P < 0.05, time-dependent effects in female rats in 0.5% dose groups; dP < 0.05, time-dependent effects in female rats in 1% dose groups; uP < 0.05, male rats versus female rats.

correlated with increases in ERK1/2 at higher dose in both sexes. The sex-dependent effects may be due to differences in hormonal or other physiological mechanisms. Cell death in lead-exposed hepatocytes may be induced via oxidative stress-mediated alterations in MAPKs.

The authors thank the Animal Resource Center for providing experimental animals. The authors gratefully acknowledge Ms. Susan Verghese, Ms. Jeena Prashanth, Dr. Saju Jacob, and Ms. Amal for their technical assistance.

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Environmental Toxicology DOI 10.1002/tox