Metallothionein and hsp70 expression in HepG2 cells after prolonged cadmium exposure

Toxicology in Vitro 21 (2007) 314–319 www.elsevier.com/locate/toxinvit

Metallothionein and hsp70 expression in HepG2 cells after prolonged cadmium exposure C. Urani a

a,¤

, P. Melchioretto a, C. Canevali b, F. Morazzoni b, L. Gribaldo

c

Dipartimento di Scienze dell’Ambiente e del Territorio, Università degli Studi di Milano Bicocca, piazza della Scienza, 1 20126 Milano, Italy b Dipartimento di Scienza dei Materiali, Università degli Studi di Milano Bicocca, via R. Cozzi, 53 20125 Milano, Italy c Institute for Health and Consumer Protection, European Centre for the Validation of Alternative Methods (ECVAM), Joint Research Centre, Ispra (VA), Italy Received 29 March 2006; accepted 31 August 2006 Available online 16 September 2006

Abstract Cadmium is a widely distributed industrial and environmental pollutant. Principle target organs are soft tissues such as the liver, where cadmium accumulates with a biological half-life of approximately 20–30 years causing a variety of toxic responses. In HepG2, CdCl2 exposure for short periods (from 1 to 24 h) induces diVerential expression of stress proteins, including MT and hsp70. However, less is known about the stress response during a prolonged exposure to this metal. MTT assay showed a low cytotoxicity of CdCl2 (0.1, 0.5, 1, 2, 5, 10 M), over a period of 72 h. Cadmium uptake by ICP–AES technique and the corresponding expression of stress proteins (MT, hsp70) during the same prolonged time were also analysed. Results show that Cd was continuously and increasingly accumulated, at the highest of the concentrations tested. Metallothionein expression was up-regulated with a saturation curve at 48 as well as 72 h after CdCl2 exposure. High levels of MT probably confer an acquired tolerance to the stress and protection against cell injury as demonstrated by low cytotoxicity values. On the contrary, the unchanged pattern of hsp70 expression suggests that this protective mechanism, unlike other members of the family, is less involved during CdCl2 prolonged exposure. © 2006 Elsevier Ltd. All rights reserved. Keywords: Cadmium; HepG2; hsp70; Metallothioneins

1. Introduction Cadmium is a biologically non-essential metal representing a widely distributed environmental and industrial pollutant. Apart from occupational exposure, food stuV, water and cigarette smoking are recognised as major sources of Cd for humans.

Abbreviations: BCIP, 5-bromo-4-chloro-3-indolyl phosphate; FBS, foetal bovine serum; Heat shock proteins 70 kDa, hsp70; ICP-AES, inductively coupled plasma-atomic emission spectrometry; MT, metallothioneins; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; NBT, nitro blue tetrazolium; OD, optical density; PBS, phosphate buVered saline; PMSF, phenyl–methylsulphonyl Xuoride; SDS, sodium dodecyl sulphate. * Corresponding author. Tel.: +39 02 64482923; fax: +39 02 64482996. E-mail address: [email protected] (C. Urani). 0887-2333/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2006.08.014

This metal is predominantly retained in soft tissues, such as the liver, with a long biological half-life (approximately 20–30 years in humans) leading to biomagniWcation and causing a variety of toxic responses (Beyersmann and Hechtenberg, 1997; Tchounwou et al., 2001). In addition to its many toxic eVects, Cd has also been classiWed as a human carcinogen (IARC, 1993). Metallothioneins (MT) and members of the heat shock protein family (e.g. hsp70, hsp90) are involved in protective mechanisms during short-term Cd exposure of target organs (Fatur et al., 2002; Somji et al., 2002; Urani et al., 2005). MT are small cysteine-rich proteins recognised as the most abundant metal-binding proteins. In mammals four MT isoforms have been identiWed (MT-1, to MT-4), but MT-1 and -2 are the most widely distributed. Metallothioneins are thought to play a critical role in cellular detoxiWcation of inorganic species by sequestering metal ions and

C. Urani et al. / Toxicology in Vitro 21 (2007) 314–319

by scavenging reactive oxygen species (Andrews, 2000; Nordberg, 1998). Heat shock proteins, among which the hsp70 is one of the most abundant and studied in mammals, are central components of the cellular network of molecular chaperones and folding catalysts. They assist a wide range of folding processes, are modulated under nonstressful conditions, and are up-regulated by a broad variety of toxic conditions which lead to the accumulation of non-native proteins (Mayer and Bukau, 2005; Santoro, 2000). Although Cd is a systemic poison known to cause hepatotoxicity in experimental animals and to aVect many cell functions (Wang and Shi, 2001), the cellular and molecular eVects of a prolonged exposure to non-cytotoxic concentrations of this metal are not completely understood. Since liver is one of the target organs of Cd toxicity, the human hepatoblastoma cell line (HepG2) was used to evaluate the eVects of a prolonged exposure to non-cytotoxic concentrations of the metal. Moreover, this model system is reported to retain many properties of primary hepatocytes and to display the classic heat shock response (Urani et al., 2001; Wilkening et al., 2003; Knasmüller et al., 2004). Therefore, the present work was designed to: (1) determine the cytotoxicity of cadmium to HepG2 cells after prolonged exposure; (2) measure the uptake of the metal by the cells and (3) analyse the pattern of expression of stress proteins (MT and hsp70) during prolonged Cd exposure.

315

Inc., NY, USA) for Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) analysis and biochemical preparations. CdCl2 was dissolved in ultra pure water (Milli-Q, Millipore, Bedford, MA, USA) at a concentration of 1 mM, Wltered (0.22 m), and stored at 4 °C. Working solutions were freshly prepared in complete culture medium. 2.3. Cytotoxicity assay Twenty four hours after seeding, HepG2 cells were exposed to 0.1, 0.5, 1, 2, 5, 10 M of CdCl2 and the cell viability was determined after 24, 48 and 72 h. The reduction of MTT by the mitochondrial dehydrogenase of viable cells to a blue formazan product (Carmichael et al., 1987) was measured (560 nm absorbance) at indicated times after CdCl2 addition. Viability of treated samples is expressed as percentage of controls (untreated). Data represent the mean § S.D. of at least three independent cultures (experiments). 2.4. Intracellular Cd uptake

Chemicals were purchased from the following suppliers: CdCl2, Cd standard solutions, 3-(4,5-dimethylthiazole-2yl)-2,5-diphenyltetrazolium bromide (MTT), and secondary antibodies from Sigma (Sigma Chemical Co., St. Louis, MO, USA); Opti-MEM medium, antibiotics, and foetal bovine serum from invitrogen, Carlsbad, CA, USA; mouse anti-metallothionein antibody from Zymed Laboratories, Inc. San Francisco, CA, USA; mouse monoclonal antibody anti-hsp70 from StressGen Biotechnologies, Victoria, Canada. All other chemicals were of reagent grade.

Twenty four hour after seeding, HepG2 cells were grown in complete culture medium containing 10 M CdCl2 for intracellular accumulation analysis by ICP-AES (Instruments SA, Jobin-Yvon 38 Sequential, France). Cells grown in complete culture medium were used as control for comparisons with physiological conditions. The presence of Cd was measured at 228.802 nm line and then normalised to 106 cells (ppm/106 cells). To this purpose, at the end of the exposure times (24, 48, and 72 h), cells were harvested and processed as previously detailed (Urani et al., 2001). BrieXy, after washing in PBS, the cells were counted in a Bürker chamber to normalise Cd concentrations (ppm) to a Wxed cell number (106 cells). The cells were then collected by centrifugation for resuspension in ultrapure water (Milli-Q, Millipore) and sonicated for 3 min at 10 m amplitude (Soniprep 150, MSE). All solutions used along with samples of control and CdCl2 treated cells were analysed by ICPAES. Calibration was performed using Cd standard solutions in Milli-Q water.

2.2. Cell cultures and treatment

2.5. MT extraction and analysis

The HepG2 cells, a human hepatoma cell line, were obtained from the American Type Culture Collection (ATCC). HepG2 were routinely grown in a monolayer culture in the presence of Opti-MEM medium supplemented with 10% heat inactivated foetal bovine serum and antibiotics (100 IU/ml penicillin and 100 mg/ml streptomycin) at 37 °C in a humidiWed atmosphere of 5% CO2. The medium was replaced twice a week, the cells were trypsinised and split every 7 days at 1:3 ratio, and used between passage 90 and 140. The cells were transferred either into 8 cm2 plastic plates (10000 cells/cm2) (Corning Inc., NY, USA), for cytotoxicity assay, or into 165 cm2 Xasks (5 £ 106 cells) (Corning

HepG2 cells were exposed 24 h after seeding to diVerent CdCl2 concentrations (0.1, 0.5, 1, 2, 5, 10 M) for 48, or 72 h. Controls were represented by untreated samples. At the end of treatments, the cells were harvested, collected by centrifugation (200g, 10 min, 4 °C), and washed by an additional centrifugation with PBS to remove CdCl2 and FBS excess. The cell pellets were homogenised and centrifuged according to published protocols (Mizzen et al., 1996) to obtain suspensions containing the low molecular weight proteins. Protein concentration was determined by Lowry method (Lowry et al., 1951), using BSA as a standard. These samples were resuspended 1:1 in sample buVer (0.25 M

2. Materials and methods 2.1. Chemicals

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2.6. Hsp70 extraction and analysis HepG2 cells were exposed 24 h after seeding to diVerent CdCl2 concentrations (0.1, 0.5, 1, 2, 5, 10 M) for 48 or 72 h. Cells grown in complete medium represented the controls. After treatments the cells were collected, homogenised in sample buVer (0.05 M Tris–HCl, pH 6.8, containing 2% SDS, 10% glycerol, 10% -mercaptoethanol, 1 mM PMSF), boiled for 5 min, and passed 3–4 times through a syringe needle (22 ga Ø). Total protein concentration of homogenates was determined by Lowry method (Lowry et al., 1951), using BSA as a standard. Hsp70 expression of samples stored at ¡80 °C was evaluated by immunochemical analysis separating 30 g of total proteins on 7% Tris-acetate NuPage gels (invitrogen, Carlsbad, CA, USA) for 1 h at 150 V. The protein bands were transferred (1 h at 30 V) onto nitrocellulose membranes using a transfer buVer (invitrogen, Carlsbad, CA, USA). Membranes were blocked for 1 h with Tween buVer (0.1% Tween-20, 8 mM NaN3 in PBS) containing 5% BSA. For the detection of hsp70, a mouse monoclonal antibody anti-hsp70 (1:1000 in blocking solution) was used. The secondary antibody (1:15000) was a goat anti-mouse alkaline phosphatase-conjugate. The binding was visualised by addition of the colorimetric substrate BCIP/NBT. 2.7. Optical densitometry and statistical analysis The relative protein expression of immunochemical results was measured by the Gel Doc image system (BioRad Laboratories, Milano, Italy) followed by quantitative analysis with Quantity One dedicated software. Data are expressed as mean § S.D. of at least three independent cultures (experiments).

Comparisons among groups were performed by means of one-way analysis of variance (ANOVA). The Multiple Range Test and the Kruskall–Wallis test methods (Statgraphics Plus version 5.0) were used for multiple comparisons. SigniWcance was considered at P < 0.05, unless otherwise stated. Regression curves were obtained by SPSS package version 11. 3. Results 3.1. Cell viability Exposure of HepG2 to diVerent CdCl2 concentrations (0.1, 0.5, 1, 2, 5, 10 M) for 24, 48 and 72 h did not aVect cell survival (data not shown). The mean cell viability was never under 80% of controls even after prolonged (72 h) CdCl2 exposure, as measured by MTT assay. 3.2. Cd accumulation Exposure to 10 M CdCl2 concentration leaded to a kinetic of continuous uptake within the cells at diVerent time points (24, 48 and 72 h), as measured by ICP-AES (Fig. 1). The calculated Cd concentrations, normalised to a Wxed cell number (106) for comparisons, revealed an increasing accumulation (0.048 § 0.005, 0.093 § 0.008, and 0.22 § 0.05 ppm/106 cells at 24, 48 and 72 h, respectively) with signiWcant diVerences among all samples. As expected, considering that Cd is a non-essential element, Cd levels in control cells were below the detectable limit of the instrument (0.003 § 0.002 ppm/106 cells). 3.3. MT and hsp70 expression To investigate the involvement of MT during prolonged CdCl2 exposure (48 and 72 h) and at increasing concentrations (0.1, 0.5, 1, 2, 5, 10 M), Western blot analysis was performed and results are shown in Fig. 2. Unexposed control cells (C) show undetectable MT levels. Metallothionein * #

0.30 0.25 0.20

6

Tris–HCl, pH 6.8, 2% SDS, 30% glycerol, 10% -mercaptoethanol, 0.01% Bromophenol blue) and stored at ¡20 °C until use. Proteins (20 g) were separated by SDS-polyacrylamide gel electrophoresis using 12% NuPage gels (invitrogen, Carlsbad, CA, USA) as previously detailed (Urani et al., 2005). Gels of separated proteins were equilibrated for 20 min in transfer buVer (invitrogen, Carlsbad, CA, USA) prior to Western blotting. After blotting (1 h at 30 V), the nitrocellulose membranes were Wxed in 2.5% glutaraldehyde in water for 1 h. The membranes were next washed in PBS followed by washing in PBS+50 mM monoethanolamine. To saturate the non-speciWc sites, the membranes were incubated with a blocking buVer (Tween buVer: 0.1% Tween-20, 8 mM NaN3 in PBS containing 5% BSA), and incubated overnight with mouse anti-metallothionein antibody (1:1000 in blocking solution) that reacts with both MT-1 and -2 isoforms. The membranes were then incubated with an anti-mouse alkaline phosphatase conjugate antibody at 1:1000 dilution, and the protein binding localised by addition of the colorimetric substrate BCIP/NBT.

Cd ppm/10 cells

316

** ##

0.15 0.10 * 0.05 0.00 Contr

Cd 24h

Cd 48h

Cd 72h

Fig. 1. Accumulation of 10 M CdCl2 in HepG2 cells after 24, 48 and 72 h of exposure. Concentrations are normalised as ppm/106 cells and represent the mean § S.D. of at least three independent experiments. ¤SigniWcantly diVerent from control (¤P < 0.05, ¤¤P < 0.01); ##signiWcantly diVerent from 24 h treatment (P < 0.01), and #from 48 h treatment (P < 0.05).

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Fig. 2. Representative immunochemical detection of MT-1, -2 in HepG2 samples (20 g/lane) treated with increasing CdCl2 concentrations (0.1, 0.5, 1, 2, 5, 10 M) for 48 and 72 h. Cells grown in complete medium were used as controls (C) and show no detectable basal levels of the proteins. CdCl2 treated cells at indicated concentrations show increasing levels of MT.

expression resulted to be up-regulated even at very low CdCl2 concentrations (0.1 and 0.5 M), as revealed by the presence of reaction bands of apparent molecular weight between 8 and 6 kDa after both 48 and 72 h of exposure to the metal. The exposure to higher CdCl2 concentrations (1– 10 M) revealed the presence of a stronger signal related to increased levels of the proteins at both 48 and 72 h. The course of MT induction after 48 and 72 h CdCl2 treatment is shown by curves of Fig. 3. Interpolation of OD data obtained by densitometry analysis strongly correlate in both cases (R2 D 0.97) with a logistic interpolation. Both logistic curves (Fig. 3a and b) show that between 0.5 and 2 M Cd the induction of MT is not proportional to the concentration and follows a sudden increase typical of a sigmoid. Moreover, 2 M Cd seems to be the highest concentration able to increment MT expression, as revealed by the presence of a plateau. In order to quantify the concentration- and time-related changes of MT levels, the percentage increments from control OD (OD) were calculated and compared by multiple range test (ANOVA). At low concentrations (0.1, 0.5, 1 M) CdCl2 is able to continuously induce MT, as revealed by statistically diVerent (P < 0.05) OD between 48 (16.4% § 2.9, 5.3% § 4.9, 198% § 12.4, at 0.1, 0.5 and 1 M CdCl2, respectively) and 72 h (46.1% § 6.3, 63.1% § 17.3, 321.9% § 14.1 at 0.1, 0.5 and 1 M CdCl2, respectively). On the contrary, comparing the OD of MT at highest CdCl2 levels of 48 h (560.7% § 22.5, 614.6% § 30.1,

317

Fig. 4. Representative immunochemical detection of hsp70 in HepG2 samples (30 g/lane) treated with increasing CdCl2 concentrations for 48 and 72 h. Constitutive levels in untreated control samples are shown in lane (C); from 0.1 to 10 (M) are CdCl2 treated samples.

543.4% § 27.2 at 2, 5, 10 M CdCl2, respectively) and 72 h (495.6% § 18.7, 615.7% § 33.2, 549.2% § 7.9 at 2, 5, 10 M CdCl2, respectively) no further increment of MT was observed. The expression of hsp70 was analysed at the same treatment conditions as above to evaluate the involvement of this protective mechanism during stress conditions. Fig. 4 shows the constitutive level of hsp70 in untreated samples (C), that is apparently the same after 48 and 72 h Cd treatment. The densitometry analysis and the statistical comparison of OD conWrmed that CdCl2 treatment, even after prolonged exposure times at all concentrations used (0.1, 0.5, 1, 2, 5, 10 M), had no eVect on the levels of hsp70 (data not shown). 4. Discussion We have examined the eVects of a prolonged exposure to CdCl2 on cell viability and stress protein expression in the human hepatoma cell line (HepG2), a model deriving from one of the primary target organs. Our analytical measurements, by ICP-AES technique, demonstrated that CdCl2 is continuously uptaken and increasingly accumulated when HepG2 cells were exposed to the highest of tested concentrations (10 M) over a period of 72 h. Cadmium uptake in hepatocytes does not occur by passive diVusion but rather by an active transport mechanism that involves, among others, Ca2+ channels (Beyersmann and Hechtenberg, 1997). The kinetics of cadmium uptake is complex and is inXuenced by factors such as the in vitro experimental

Fig. 3. Course of MT expression after 48 (a) and 72 h (b) of exposure to increasing CdCl2 concentrations (0.1, 0.5, 1, 2, 5, 10 M). Logistic regressions were obtained by interpolation of optical densities (OD) data of MT expression measured by scanning densitometry. Correlation coeYcients (R2) and equations are indicated.

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conditions. The presence of albumin in the culture medium, to which cadmium can bind, may inXuence the transport and uptake of this metal leading to reduced cytotoxicity (DelRaso et al., 2003). The estimated IC50 value in HepG2 cells exposed to CdCl2 in the presence of albumin in the culture medium was previously reported to be 25 M (Urani et al., 2005) or 30 M (Tchounwou et al., 2001). In the present work, HepG2 cells were exposed to a range of concentrations (0.1, 0.5, 1, 2, 5, 10 M) below the estimated IC50 value for a prolonged period (72 h). The cell viability, measured by MTT test, in those samples was comparable, and a low cytotoxicity percentage was observed even at 10 M, the highest CdCl2 concentration tested (data not shown). The induction of MT genes represents one of the most intensely studied eVects of cadmium exposure. Transcription of these genes is rapidly and dramatically up-regulated in response to cadmium, as well as in response to agents which cause oxidative stress and/or inXammation (Andrews, 2000). In whole animals, cadmium is the stronger MT inducer followed by metals such as zinc and copper (Beyersmann and Hechtenberg, 1997). The concentrations of cadmium required for the induction of MT are remarkably low in the sub toxic range, i.e. a few M (Beyersmann, 2002). A short period exposure (12–24 h) to CdCl2 is able to up-regulate the expression of MT in a dose-dependent manner in HepG2, as we have previously reported (Fatur et al., 2002; Urani et al., 2005). In contrast to these experimental conditions, the present study demonstrates that a prolonged exposure over a period of 72 h yielded to a continuous up-regulation of MT only at low (0.1, 0.5, 1 M) CdCl2 levels and to saturation at the highest concentrations used (2, 5, 10 M). This course was conWrmed by the strong correlation (R2 D 0.97) of interpolation of OD data from immunochemical results with a sigmoid curve at both 48 and 72 h, suggesting as well that 2 M CdCl2 is the highest concentration able to elicit MT over-expression in HepG2 cells. The induction of this protective mechanism probably confers an acquired relative tolerance to the stress by binding the metal and thus reducing its reactivity with cell components (Klaassen et al., 1999), as demonstrated in our experiments by low cytotoxicity levels during prolonged CdCl2 exposure. Other eVectors of cellular stress response are the heat shock proteins (hsps), among which the hsp70 family represents the prototype of molecular chaperons and folding catalysts. The hsps are a large superfamily of proteins that confer protection against and recovery from environmental insults both physical and chemical (Mayer and Bukau, 2005). These proteins are a major gene product induced by stress in numerous cell systems. Protein perturbation either by direct protein damage or disruption of nascent chain elongation or folding are signals that up-regulate hsp70 gene transcription. Even though up-regulation of hsp70 expression has been previously demonstrated in rat and human hepatoma cells after short CdCl2 treatment (from 1 h to 24 h) (Tchounwou et al., 2001; Farzaneh et al., 2005; Sousa et al., 2004; Urani et al., 2005), we observed a diVer-

ent response after prolonged exposure. Actually, CdCl2 did not alter the pattern of hsp70 expression over the 72 h exposure that remained at basal levels at all tested concentrations, as demonstrated by immunochemical results. Our data are consistent with those of other authors (Bonham et al., 2003) that demonstrated the up-regulation of hsp70 in renal epithelial cells after prolonged exposure (72 h) to cadmium only at concentrations suYcient to induce cell death. These data support the idea that CdCl2 is a poor inducer of hsp70 expression in HepG2 cells in conditions of prolonged exposure, in agreement with previous reports in other cell targets (Somji et al., 1999; Bonham et al., 2003). These data suggest that, in contrast to the relevance during the acute stress response after CdCl2 exposure, hsp70 are less involved as protective mechanism during prolonged chronic conditions in comparison to other proteins of the same family (i.e. hsp27) that are up-regulated even after prolonged CdCl2 exposure (Bonham et al., 2003). In summary, our results show that CdCl2 accumulates within HepG2 cells continuously and increasingly over the prolonged period of exposure. As a consequence, MT are up-regulated and probably confer resistance to the cells, as demonstrated by low cytotoxicity values. The increased MT expression can be correlated with exposure to CdCl2 only over a limited range of concentrations, represented by the lowest used in the present study (0.1, 0.5, 1 M), and reach a saturation curve at the highest concentrations used (2, 5, 10 M). Moreover, the increased MT levels are poorly correlated with other markers of cellular injury such as the increase of hsp70 that follow a diVerent pathway of expression. Acknowledgements C.U. gratefully acknowledges the partial support by Fondo di Ateneo per la Ricerca, granted by Università di Milano Bicocca (F.A.R. 2004) and the European Centre for the Validation of Alternative Methods (ECVAM) of the European Commission. References Andrews, G.K., 2000. Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochemical Pharmacology 59, 95–104. Beyersmann, D., 2002. EVects of carcinogenic metals on gene expression. Toxicology Letters 127, 63–68. Beyersmann, D., Hechtenberg, S., 1997. Cadmium, gene regulation and cellular signalling in mammalian cells. Toxicology and Applied Pharmacology 144, 247–261. Bonham, R.T., Fine, M.R., Pollock, F.M., Shelden, E.A., 2003. Hsp27, hsp70, and metallothionein in MDCK and LLC-PK1 renal epithelial cells: eVects of prolonged exposure to cadmium. Toxicology and Applied Pharmacology 191, 63–73. Carmichael, J., DeGraV, W.G., Gadzar, A.F., Minna, J.D., Mitchell, J., 1987. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Research 47, 936–942. DelRaso, N.J., Foy, B.D., Gearhart, J.M., Frazier, J.M., 2003. Cadmium uptake kinetics in rat hepatocytes: correction for albumin binding. Toxicological Sciences 72, 19–30.

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