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A bioassay for metals utilizing a human cell line
Toxicology in Vitro 22 (2008) 1025–1031
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A bioassay for metals utilizing a human cell line J. Shea a, T. Moran b, P.F. Dehn a,* a b
Department of Biology, Canisius College, Buffalo, NY 14208, USA Pollutech EnviroQuatics Ltd., Point Edward, ON, Canada
a r t i c l e
i n f o
Article history: Received 14 November 2005 Accepted 25 February 2008 Available online 4 March 2008 Keywords: HepG2 Human bioassay Metal mixtures Metallothionein Biomakers
a b s t r a c t The purpose of this study was to assess the ability of the HepG2 cell line to function as a bioassay for metal contamination in sediments, using metallothionein (MT) as a biomarker of exposure. Sediments were collected from the eastern and western ends of Lake Erie, extracted using EPA method 200.7, and analyzed for cadmium (Cd), mercury (Hg) and lead (Pb) levels using ICP-AES. Sediment extracts were neutralized then used at a 2.5% final concentration in the exposure medium. MT levels were measured using the cadmium–hemoglobin affinity assay after a 48 h exposure. Fortified blanks from the ICP protocol served as positive controls. Also, HepG2 cells were exposed to Cd, Pb or combinations of Cd and Pb to determine whether or not induction of MT observed in cells exposed to sediment extracts was due to a single metal, combinations of metals, pH, or some other factor. Additionally, cells were exposed to a range of Cd concentrations approximating the levels found in the extracts (0.0005–0.1 mg/L) to determine if a concentration-response occurred. Total metal levels ranged from 527 to 33.5 mg/kg with lead the predominant metal, accounting for 100–88.9% of the total quantifiable metals in the sediments. The biomarker response (MT induction) was strongly correlated (r2 = 0.9919, r2 = 0.990) with total metal and lead levels in the sediments, respectively, which supports recent field studies indicating the biomarker can discern differences in the strength of the inducing agent. Statistically significant MT induction was associated with sediments which contained measurable Cd concentrations and no significant differences were observed when comparing Cd only and Cd + Pb exposed cells indicating no interactions between Cd and Pb were occurring and supporting our finding that Cd was the main inducing agent in sediment extracts. MT levels also increased significantly in a concentration-dependent manner when cells were exposed only to Cd. Results suggest this human bioassay and the MT biomarker of exposure may be useful for monitoring complex metal mixtures in aquatic sediments. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction There is increasing concern of environmental contaminants impacting both human and wildlife health. Much of the monitoring done world-wide utilizes chemical analyses which indicate the presence or absence of contaminants in water, sediments and/or biota. Chemical analyses however, do not provide information on how biota responds to non-toxic, low level, chronic exposures, which are typically found at many contaminated sites. While toxicological testing for various endpoints other than lethality occurs, the majority examine single compound effects. Environmental contaminants do not occur singly, but rather exist as complex mixtures of both inorganic and organic forms, whose interactions have not been studied. Cell bioassays and biochemical indicators or biomarkers of exposure and/or effect offer potentially sensitive tools for inclusion into environmental monitoring, screening and assessment programs. They have the ability * Corresponding author. Tel.: +1 716 888 2555; fax: +1 716 888 3157. E-mail address: [email protected] (P.F. Dehn). 0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.02.014
to complement chemical analysis and can shed light on biotic responses. Several types of cell bioassays (both prokaryote and eukaryote, including human cell lines) have been utilized to monitor complex environmental mixtures (Fulladosa et al., 2002; Garrison et al., 1996; Giesy et al., 2002; Lee et al., 1999) although organic compounds that impact the estrogen receptor have been the focus of the majority of these studies (reviewed in Giesy et al., 2002). The HepG2, a human hepatoma, cell line has been well characterized and used routinely as a model for a variety of in vitro toxicological studies (see references in Dehn et al., 2004, 2005; Urani et al., 2003, 2005, 2007), particularly those involving biotransformations, since the major biotransformation reactions are intact. Of importance to this study is: (1) this cell line’s ability to induce metallothioneins (MTs) in response to metals (Dehn et al., 2004; Jahroudi et al., 1990; Jimenez et al., 2002; Miura et al., 1999; Perez and Cederbaum, 2003; Urani et al., 2003, 2005, 2007); (2) that HepG2 cells have both MT-1 and -2 isoforms, which are the most widely distributed isoforms in mammalian cells (Jahroudi et al., 1990; Perez and Cederbaum, 2003; Urani et al., 2003, 2007); and
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(3) it may serve as model that links toxicant exposure to anticipated responses in normal human hepatocytes (Urani et al., 2005). MTs are widely recognized as an important component of a cells defense against and recovery from environmental toxicant insult. MTs are a family of low-molecular weight (<7 kDa), cysteine-rich, intracellular compounds that are highly inducible. MTs have unique structures that produce potent metal-binding and redox capabilities (Urani et al., 2003). They are best known for their ability to bind metal ions, both those needed for normal metabolism, e.g., Zn and Cu, as well as heavy metal pollutants, e.g., Cd, Hg, and Pb (Kagi, 1993). Metal ions, cytokines, hormones, cytotoxic agents, organic chemicals, and stress have been shown to induce MTs (Haq et al., 2003; Klaassen and Liu, 1997). MT synthesis is controlled primarily at the transcriptional level (Haq et al., 2003; Klaassen et al., 1999). Post-transcriptional regulation also has been shown to be important to renal tissue MT regulation (Haq et al., 2003). MT induction has been identified and utilized as a biomarker of metal exposure in laboratory, field, and epidemiological studies (Bebianno et al., 2003; Carvalho et al., 2004; Domouhtsidou et al., 2004; Geret et al., 2002; Lecoeur et al., 2004; Lu et al., 2001; Lukkari et al., 2004; Perceval et al., 2002; Regoli et al., 2002). In field studies using sentinel animal species, MT induction seems to be affected by a variety of biotic and abiotic factors that may limit MTs use in quantifying the strength of the exposure (Domouhtsidou et al., 2004; Lecoeur et al., 2004; Perceval et al., 2002). Sediments represent vast sinks for contaminants. Several areas of concern (AOC) have been identified within the Lake Erie – St. Clair watershed under the Great Lakes water-quality agreement due to use impairments attributed in part to sediment contamination. Both the St. Clair and Niagara rivers have this designation. Extensive sediment contaminant data exists for the St. Clair River, which indicates a contaminant gradient occurs as one moves downstream from Lake Huron to Lake St. Clair (Moran et al., 1997; Pollutech EnviroQuatics and Ltd., 1999). At the Lake Erie end of the Niagara River, the Black Rock Lock is located immediately downstream of both the Times Beach Confined Disposal facility and the Buffalo River, an AOC, which serve as potential sources for contaminant inputs into the mouth of the Niagara River. Previous studies using zebra mussels as bioindicator organisms have shown significant levels of both organic and inorganic contaminants in tissues after 34 days at these sites (Roper et al., 1996). The purpose of this study was to expand preliminary studies in our laboratory indicating that the HepG2 cell line might function as a bioassay for metal contamination in sediments, using MT as a biomarker of exposure (Shea et al., 1999). To examine the validity of MT induction as a biomarker of exposure for contaminant mixtures contained within sediments, we measured MT levels in cells exposed to extracts of sediments taken from locations within the Great Lakes known to exhibit a gradient of metal contamination. In particular, we were interested in the heavy metals, cadmium, mercury and lead as they represent priority contaminants within the Great Lakes. We also were interested in discerning whether or not MT induction would respond to quantitative differences in metal mixtures in sediments. Or in other words, could this biomarker differentiate between a weak and a strong signal, which has been difficult to ascertain in field studies using sentinel animal species.
um were obtained from GIBCO (Grand Island, NY); chemicals needed for the metallothionein assay, sediment extracts, and sediment analyses were purchased from Sigma (St. Louis, MO); protein assay materials were purchased from Bio-Rad (Melville, NY); the 109 Cd was purchased from DuPont NEN (Boston, MA). 2.2. Cell culture conditions HepG2 cells were maintained as described previously (Pieczonka and Dehn, 1993) in Dulbecco’s modified eagle’s medium (DMEM) with fungizone (125 lg/ml in 100 ml), penicillin–streptomycin (5000 U/ml), non-essential amino acids (0.1 mM), and 10% fetal bovine serum (maintenance DMEM), at 37 °C in 95% O2:5% CO2 conditions. The culture medium was changed three times each week and cells were passaged every 7–10 days. The cells were detached by trypsinization (0.05% trypsin, 0.53 mM EDTA). Cells were mycoplasma free, as determined by the MycoTect assay (GibcoBRL). All cells were plated at concentrations of 106 cells/ml, and allowed to attach for 24 h prior to testing. Volumes used for plating were 1.5 ml for 6 well plates. Only cells in passages 4–8 were used, as age of culture effects have been previously reported (Dehn et al., 2005). 2.3. Sediment collections Surface sediments were collected by hand using scuba on the Canadian side of the St. Clair River (Fig. 1A). Four sites were examined in the St. Clair River. At the upper end of the river, nearest Lake Huron, the Point Edward sample was above the 15 major industrial facilities which discharge directly or indirectly into the St. Clair River. The remaining three sites were below the heaviest industrial area, located on the Ontario side of the river. Surface sediments from the Black Rock Lock at the eastern end of Lake Erie (Fig. 1B) were hand shoveled from the rim of the lock gate when the locks were drained for repair in the middle of the winter. This location was chosen as sediments were still wet and not exposed to the atmosphere, which could compromise comparing sites (Shelton and Capel, 1994). All sediments were placed into plastic containers, stored on ice, returned to the laboratory and immediately frozen at 80 °C until thawed for preparation of the extracts and analysis. 2.4. Sediment extract preparation Sediments were thawed, mixed thoroughly, and any large stones or organic material removed. Sediments were extracted
2. Methods 2.1. Materials The human hepatoma cell line (HepG2, ATCC HB 8065) was obtained from the American Type Culture Collection (Rockville, MD). Culture medium, supplements, MycoTect kit, and fetal bovine ser-
Fig. 1A. Map of the St. Claire River sampling locations.
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Table 1 Analytical conditions and characteristics of the spectro analytical instruments ICPAES used for metal analyses Spectrometer
Spectroflame – EOP/CP Characteristics and conditions
Nebulizer RF power Argon flow
Meinhard pneumatic 1.2 kW Coolant – 14 L/min Auxiliary – 0.5 L/min Nebulizer gas flow – 0.8 L/min Total preflush – 20 s [Cd] 226.502 nm [Hg] 194.227 nm [Pb] 220.353 nm 3000 ms
Sample feed – manual Analyte lines
Sequential peak and background measurement times
2.6. Toxicological responses Fig. 1B. Map of the Black Rock Lock sampling station at the western end of Lake Erie in the entrance to the Niagara River.
according to EPA method 200.7 (Martin et al., 1994), which utilizes an acid extraction. Extracts were split for quantification of levels of cadmium (Cd), mercury (Hg) and lead (Pb) via inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and for cell culture exposures. Extraction blanks and fortified blanks containing known concentrations of the analytes were prepared and analyzed. The fortified blanks were utilized as positive controls. Reproducibility was initially determined using seven replicate extractions on subsamples of the Black Rock Lock sediment. All other sites used duplicate extractions, as reproducibility was within expected 95% confidence intervals. 2.5. HepG2 exposures For all sediment exposure studies, cells were plated for 24 h in maintenance DMEM. After 24 h the medium was removed and the cells were washed twice with sterile phosphate buffered saline (PBS). The maintenance DMEM was replaced with serum-free, protein free basal DMEM containing fungizone (125 lg/ml in 100 ml) and penicillin–streptomycin (5000 U/ml) (exposure DMEM). Cells were exposed for 48 h to exposure DMEM (controls), or exposure DMEM containing 2.5% (final volume in the exposure medium) of the sediment extract or the fortified blank (positive control). A 2.5% extract was used to minimize the effects of the acid and to reduce medium dilution effects. Previous work with organic solvents showed this to be an optimal concentration. All extracts used for HepG2 exposures were neutralized with 3 M NaOH to pH 7.4 after addition to the exposure DMEM and prior to being placed on the cells. Additionally, the exposure DMEM for the controls was acidified and neutralized, as above, prior to exposure to eliminate artifacts due to pH adjustments. Preliminary exposures of the sediment extracts or fortified blanks produced no cytotoxicity as measured by trypan blue dye exclusion (data not shown). HepG2 cells were exposed to cadmium (Cd), lead (Pb) or combinations of Cd and Pb to determine whether or not induction of MT observed in cells exposed to sediment extracts was due to a single metal, combinations of metals, pH, or some other factor. Also cells were exposed to a range of Cd concentrations approximating the levels found in the extracts (0.0005–0.1 mg/L) to determine if a concentration-response occurred, or if a Cd threshold existed. Cells were plated and exposed as above, with the exception that the neutralization step and the positive controls were not needed.
Metallothionein (MT) induction was measured by the cadmium–hemoglobin affinity assay (Eaton and Cherian, 1991) as modified in Dehn et al. (2004). A 100 ll aliquot of the MT assay supernatant was used for isotopic counting. Proteins were quantified by the Bio-Rad method as modified in Allen et al. (2001) using the sample homogenate. Metallothionein content was expressed as pmol Cd bound/mg protein. 2.7. ICP analysis of metals in the sediment extract Inductively coupled plasma (ICP-AES) spectroscopy was used to analyze extracts for levels of Cd, Hg, and Pb. Instrument characteristics and analytical conditions are listed in Table 1. Calibration was performed using single element standards, while a standard curve was generated for each analyte using mixed standards ranging from 1 mg/L to 1000 mg/L. Levels of metals are expressed as mg/L in the extract and in the exposure medium, and as mg/kg for the sediment. These levels are actual values measured and are not corrected to represent percent recoveries. All percent recoveries were within acceptable ranges for the method. 2.8. Statistical analysis Means and standard deviations were calculated for the individual metals and the HepG2 MT levels. The control MT levels for various replicate experiments using the same sediment extract or between replicate experiments using different sediment extracts yielded no significant differences using the Kruskal–Wallis ANOVA on Ranks test. Therefore, all control data were grouped so that a single comparison between the controls and positive controls could be made using the Mann–Whitney Rank Sum test (t-test). t-Tests were used to compare MT levels in HepG2 cells for each sediment extract and its appropriate control group, controls and Cd concentrations. ANOVA’s were used to compare MT levels in Cd only, Pb only, and Cd + Pb exposed cells with a Student–Newman–Keuls post-hoc test to determine which groups were different. All data were analyzed for significance at the alpha 0.05 level. All statistical analyses were done using SigmaStat (SPSS, Inc.). 3. Results Metal contamination was highest in the Black Rock Lock sediments, followed by the Corunna, Courtright, Mooretown and Point Edward sediments (Table 2). Total metal levels ranged from 527 to 37.5 mg/kg. Lead accounted for 100%, 100%, 99.7%, 98.2%, and 88.9% of the quantifiable metals in the Courtright, Mooretown,
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Table 2 Concentrations (mg/kg) of cadmium (Cd), mercury (Hg) and lead (Pb) in sediment from sites within the Lake Erie – St. Clair River watershed Treatment
Point Edward Corunna Mooretown Courtright Black Rock Lock
Extract (mg/kg) Cd
Hg
Pb
Total
BDL 0.3 ± 0.1 BDL BDL 8.4 ± 0.2
4.5 ± 1.4 BDL BDL BDL 1.2 ± 0.4
33 ± 1 87.2 ± 0.7 46.4 ± 0.8 50.2 ± 0.8 517.5 ± 6
37.5 87.5 46.4 50.2 527.1
Sites are listed in an upstream to downstream order. BDL signifies below detection limits. Data represent averages from duplicate extractions.
Corunna, Black Rock Lock, and Point Edward sediments, respectively. Cadmium was detectable only in the Black Rock Lock and Corunna sediments and accounted for 1.6% and 0.3% of the quantifiable metals, respectively. Mercury was detectable only in the Black Rock Lock and Point Edward samples and accounted for 0.2% and 19.2% of the quantifiable metals, respectively. Sediment extracts utilized in the bioassay contained variable levels of cadmium, mercury and lead (Table 3). All sediment extracts except those from the Black Rock Lock and Corunna had cadmium concentrations below the detection limit of 3.4 lg/L. All sediment extracts except those from the Black Rock Lock and Point Edward had mercury concentrations below the detection limit of 2.5 lg/L. Lead was the predominant metal in all sediments and lead levels ranged from 10.4 mg/L in the Black Rock Lock sediment extracts to 0.4 mg/L in the Point Edward sediment extracts. Actual levels used for exposures were much lower as the extract added was 2.5% of the total volume that covered the cells (Table 3). All percent recoveries for the fortified blanks were within acceptable ranges for the method (Table 3). Metallothionein (MT) levels in cells exposed only to exposure DMEM (control) varied between replicate experiments for a sediment extract. For example, three replicate experiments for a Black
Rock Lock extract produced MT levels in control cells of 41.6 ± 3.9, 34.0 ± 7.2, and 45.4 ± 3.9 with n = 3, 3 and 2, respectively. ANOVA’s to compare the variation, indicated no statistically significant differences (P = 0.145). Therefore, replicate runs were grouped together for analysis. Metallothionein (MT) levels in cells exposed to exposure DMEM (control), neutralized fortified blanks (positive control) varied over all experimental exposures (Table 4), however no statistically significant differences occurred between control groups for each sediment location (P = 0.291). Therefore, all control and positive control groups from each experiment were analyzed as single groups. Mean MT levels were 38.9 ± 7.7 and 178.5 ± 48.3 pmol Cd bound/mg protein in control (n = 22) and positive control (n = 15) exposed cells, respectively. Differences between these levels were statistically significant (P = <0.001) indicating an appropriately functioning bioassay response. Metallothionein (MT) levels were elevated in all HepG2 cells exposed to sediment extracts, although only MT levels in cells exposed to sediment extracts from the Black Rock Lock and Corunna were significantly higher (Table 4). In all cases the positive controls produced significant elevations in MT levels with respect to the controls (Table 4). When mean metallothionein level in HepG2 cells for each extract site was expressed as a percent of the mean metallothionein level for the control HepG2 cells for that site and regressed against total metal concentrations in the sediments for each site, there was a strong correlation (r2 = 0.9919) between these two variables (Fig. 2), indicating a dose-response relationship. Regressions between metallothionein expressed as a percent of the control and total lead levels also were strongly correlated (r2 = 0.990) (Fig. 3). Cells were exposed to Cd and Pb levels singly and in combination to determine whether MT induction observed in sediment extracts was due to a single metal or combinations of metals. Levels of MT ranged from 150 ± 63 to 3515 ± 446 pmol Cd bound/mg protein (Table 5). All exposures induced MT significantly when
Table 3 Mean (±SD) concentrations (mg/L) of cadmium (Cd), mercury (Hg) and lead (Pb) in neutralized fortified blanks (positive control), and neutralized sediment extracts from sites within the Lake Erie – St. Clair River watershed and their corresponding concentrations in the HepG2 bioassay Treatment
Point Edward Corunna Mooretown Courtright Black Rock Lock Positive control % Recovery
Extract (mg/L)
Bioassay (mg/L)
Cd
Hg
Pb
Cd
Hg
Pb
BDL 0.006 ± 0.002 BDL BDL 0.17 ± 0.003 15.2 ± 5.9 116 ± 47
0.09 ± 0.03 BDL BDL BDL 0.02 ± 0.01 13.1 ± 2.6 99.4 ± 21
0.4 ± 0.02 1.75 ± 0.01 0.93 ± 0.02 1.4 ± 0.4 10.4 ± 0.1 67.6 ± 11.3 104 ± 16
– 0.0003 – – 0.009 0.76 –
0.005 – – – 0.001 0.66 –
0.02 0.088 0.047 0.07 0.52 3.38 –
Mean (±SD) percent recoveries are given for the fortified blanks (positive controls). BDL signifies below detection limits. Duplicate extractions were done for all but the Black Rock Lock (n = 7) and positive controls (n = 10). A single extract was used for the bioassay.
Table 4 Mean (±SD) levels of metallothionein (pmol cadmium bound/mg protein) in HepG2 cells exposed to neutralized exposure medium (control), neutralized fortified blank (positive control), and neutralized sediment extract (extract) from within the Lake Erie – St. Clair River watershed Site
pmol Cd bound/mg protein Control
Point Edward Corunna Mooretown Courtright Black Rock Lock Total
44.2 ± 2.4 42.5 ± 2.7 47.7 ± 5.6 30.7 ± 6.4 39.7 ± 6.8 38.9 ± 7.7
(2) (4) (2) (6) (8) (22)
Positive control
Extract
226.8 ± 45.2 (3) P = 0.012 169.4 ± 12.3 (4) P = 0.029 176.1 ± 0.6 (2) P = 0.03 106.0 ± 11.2 (3) P < 0.001 216.3 ± 14.3 (3) P = 0.012 178.5 ± 48.3 (15) P < 0.001
56.7 ± 10.9 (9) P = 0.16 57.4 ± 2.8 (4) P < 0.001 58.3 ± 7.2 (6) P = 0.11 38.4 ± 3.8 (6) P = 0.065 198.9 ± 27.6 (21) P < 0.001
Respective sample sizes (n) and P values for t-test comparisons between controls and sediment extracts are indicated. Totals indicate the grouped data for the controls and positive controls.
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Metallothionein (% Control)
600
500
y = 0.779x + 87.75 r 2 = 0.9919
400
300
200
100
0 0
100
200
300
400
500
600
Total Sediment Metal Concentration (mg/kg)
Metallothionein (% Control)
600 y = 0.7915 x +88.24 r 2 = 0.99
400
300
200
100
0 0
100
200
300
400
500
6000 0. 479x
y = 130. 31e 2 R = 0.9544
5000
600
*
4000
* 3000
*
2000
1000
*
*
*
0 Con.
Fig. 2. Relationship between metallothionein content in HepG2 cells exposed to extracts from sediments expressed as a percent of the solvent control and the total metal concentration in the sediments from each sampling site within the Lake Erie – St. Claire River watershed.
500
MT Concentration (pmol Cd bound/mg Protein)
J. Shea et al. / Toxicology in Vitro 22 (2008) 1025–1031
0.0005
0.001
0.005
0.01
0.05
0.1
Concentration Cd (mg/L) Fig. 4. Cadmium concentration-metallothionein response relationship in HepG2 cells.
At higher exposure levels it appeared that additive effects might be occurring as adding mean MT levels of Cd only and Pb only exposed cells approximated those found in Cd + Pb exposed cells (3537 vs. 3515 pmol Cd bound/mg protein, respectively). ANOVA’s comparing MT levels in Cd only, Pb only, and Cd + Pb exposed cells at these higher exposure concentrations were significantly different (P = 0.001), but pairwise multiple comparisons showed no significant differences between 0.09 Cd only and 0.09 Cd + 0.52 Pb exposed cells, indicating Cd was the main inducing agent. To determine, if a threshold and or concentration-response relationship existed for MT, HepG2 cells were exposed to increasing concentrations of Cd whose range spanned those found in the sediment extracts (0–0.1 mg/L). MT levels exhibited a concentrationresponse relationship (Fig. 4). MT levels increased from 168 ± 73 to 4149 ± 678 with each concentration significantly different from the control.
Pb (mg/kg) Fig. 3. Relationship between metallothionein content in HepG2 cells exposed to extracts from sediments expressed as a percent of the solvent control and the lead (Pb) concentration in the sediments from each sampling site within the Lake Erie – St. Claire River watershed.
Table 5 Mean (±SD) levels of metallothionein (pmol cadmium bound/mg protein) in HepG2 cells exposed to neutralized exposure medium (control), neutralized exposure medium containing cadmium, lead, or cadmium and lead respective sample sizes and P values for t-test comparisons between controls and metal exposures are indicated (n = 4 unless indicated) Exposure concentration
MT levels (pmol Cd bound/mg protein)
Controls 0.0003 mg/L Cd 0.09 mg/L Cd 0.09 mg/L Pb 0.52 mg/L Pb 0.0003 mg/L Cd ± 0.09 mg/L Pb 0.09 mg/L Cd ± 0.052 mg/L Pb
150 ± 63 265 ± 53 P = 0.031 2984 ± 480 P = 0.029 324 ± 46 P = 0.004 553 ± 106 P = 0.001 (n = 3) 237 ± 32 P = 0.048 3515 ± 446 P = 0.029
Respective sample sizes and P values for t-test comparisons between controls and metal exposures are indicated (n = 4 unless indicated).
compared to the controls with highest induction responses seen in cells associated with higher exposure levels of Cd. ANOVA’s comparing MT levels in Cd only, Pb only, and Cd + Pb exposed cells were not significantly different at low exposure levels (P = 0.058).
4. Discussion MTs are induced by various physiological and toxicological stimuli, including metals, cytokines, hormones, cytotoxic agents, organic chemicals, and stress (Haq et al., 2003; Kagi, 1993; Klaassen et al., 1999). Both the MT-1 and -2 isoforms are basally expressed and highly inducible in virtually all mammalian cells (Haq et al., 2003). Recently, Urani et al. (2003) have shown basal levels of MT-1 and 2 not to be detectable in HepG2 cells using immunoblot techniques. In our study, as in most other studies measuring total MT levels via the cadmium-hemoglobin affinity assay, control cells exhibit basal levels of MT. This difference could be due to two factors. The media was changed daily in Urani’s et al. (2003) study which could have reduced the presence of other stressors, e.g., hormones or pH, which have the ability to induce MT synthesis. Secondly, the cadmium–hemoglobin affinity assay measures Cd-binding and not actual MT levels (Eaton and Toal, 1982). Therefore, elevation of MT levels in control cells may be an artifact due to the presence of other low-molecular weight proteins which have metal-binding characteristics, e.g., glutathione, although heat treatments are designed to reduce the impacts of these proteins (Eaton and Toal, 1982). For this reason it is imperative that control cells, which are not exposed to any metals but are exposed under the same conditions and in the same medium, be monitored since this is an indirect method that estimates MT content based on Cd-binding.
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Metallothioneins are widely recognized as an important component of a cell’s defense against and recovery from environmental insult. Many metals, including Ag, Bi, Cd, Co, Cu, Hg, Ni, Pb, Zn, induce MT synthesis (Klaassen and Liu, 1997), although only Zn, Cd, Hg, Pb, Cu and Bi bind to MT (Haq et al., 2003; Rhee and Huang, 1989). In HepG2 cells, as in many other cells and/or organisms studied, cadmium is the most potent inducer when administered singly (Dehn et al., 2004). In this study, total MT levels increased in all cells exposed to sediment extracts. However, these increases were significant only for those extracts containing quantifiable levels of cadmium. It is impossible to determine whether or not cadmium was responsible for this significant increase; thresholds for cadmium induction of MT synthesis have not been determined, and below detection limits do not signify the absence of this compound. However, in our cadmium concentration-response study, Cd at a concentration very near that found in the sediment extract (0.0005 vs. 0.0003 mg/L, respectively) did significantly increase MT levels. So it is likely, even though we did not monitor other potential known inducers of MT, e.g., zinc or copper, which have been found within the St. Clair River (Moran et al., 1997; Moran et al., 2003) and most of which have been shown to induce MTs in HepG2 cells (Dehn et al., 2004; Jahroudi et al., 1990; Jimenez et al., 2002; Miura et al., 1999; Perez and Cederbaum, 2003; Urani et al., 2003) that cadmium was responsible for the significant increase in MT levels in sediment extract exposed cells. It is of interest to note the strong correlation (r2 = 0.990) between levels of lead in the sediments and MT expressed as a percentage of the control. Lead has been shown to induce MTs (Bae et al., 2001; Klaassen and Liu, 1997; Rhee and Huang, 1989), although it is not as potent of an inducer as cadmium or zinc. Lead only exposures at 0.09 and 0.52 mg/L did significantly increase MT levels in HepG2 cells. Our results show that HepG2 cells are a viable bioassay and MT is a viable biomarker for lead exposure, supporting those of Campana et al. (2003) who found MT to be a viable biomarker for lead exposure in the toadfish, and those of Rhee and Huang (1989) who showed a correlation between lead concentration and both MT content and MT mRNA levels in CHO (Chinese hamster ovarian) Cdr cells. Metal–metal interactions can occur, which have the potential to either increase or decrease the impact of the inducing agents. Very few metal mixture studies have been done to examine these impacts under controlled conditions. Using cytotoxicity responses in human keratinocytes exposed to a four metal mixture of As, Cd, Cr, and Pb, Bae et al. (2001) found hormetic, additive, synergistic and then antagonistic interactions as dose levels increased. MT levels were found to increase in a dose-dependent manner in three of the four keratinocyte cell strains exposed to the mixture and glutathione levels were elevated at the mixture levels where antagonistic interactions were observed (Bae et al., 2001). In this study we found no indication of metal–metal interactions on MT induction in HepG2 cells. Several laboratory and in situ field studies have examined MT levels as a biomarker of exposure for metal mixtures and contaminated sites containing metal mixtures as well as other organic contaminants. Within both laboratory and field studies, concentration gradients have been defined by the biomarker response (Domouhtsidou et al., 2004; Fulladosa et al., 2002; Lecoeur et al., 2004; Lukkari et al., 2004; Perceval et al., 2002; Regoli et al., 2002). Likewise levels of MT gene expression have been correlated with levels of occupational exposure in human epidemiological studies (Lu et al., 2001). In this study we found a strong correlation between total metal levels in the sediment and increased MT production, when expressed as a percentage of the control; indicating the ability of the bioassay to discern quantitative differences in metal mixtures in sediments. This study supports the human bio-
assay and the MT biomarker of exposure as useful tools for monitoring complex metal mixtures in aquatic sediments. Acknowledgements We would like to acknowledge our colleague Larry Tassini for his technical expertise in maintaining the HepG2 cell line and his willingness to be available for students, which made the completion of this work possible. Also we thank Sara Morris for her assistance in creating the GIS maps used in Figs. 1 and 2. This project was supported, in part, by a Howard Hughes Medical Institute Undergraduate Science Education Program grant (1996) to Canisius College for undergraduate researcher J.S. References Allen, S., Shea, J.M., Felmet, T., Gadra, J., Dehn, P.F., 2001. A kinetic microassay for glutathione in cells plated on 96-well microtiter plates. Meth. Cell Sci. 22, 305– 312. Bae, D.S., Gennings, C., Carter, W.H., Yang, R.S.H., Campain, J.A., 2001. Toxicological interactions among arsenic, cadmium, chromium, and lead in human keratinocytes. Toxicol. Sci. 63, 132–142. Bebianno, M.J., Cravo, A., Miguel, C., Morais, S., 2003. Metallothionein concentrations in a population of Patella aspera: variation with size. Sci. Total Environ. 301, 151–161. Campana, O., Sarasquete, C., Blasco, J., 2003. Effect of lead on ALA-D activity, metallothionein levels, and lipid peroxidation in blood, kidney, and liver of the toadfish Halobatrachus didactylus. Ecotoxicol. Environ. Saf. 55, 116–125. Carvalho, C.S., de Araujo, H.S.S., Fernandes, M.N., 2004. Hepatic metallothionein in a teleost (Prochilodus scrofa) exposed to copper at pH 4.5 and pH 8.0. Comp. Biochem. Physiol. 137B, 225–234. Dehn, P.F., White, C.M., Conners, D.E., Shipkey, G., Cumbo, T.A., 2004. Characterization of the human hepatocellular carcinoma (HepG2) cell line as an in vitro model for cadmium toxicity studies. In Vitro Cell. Devlop. Biol. 40, 172–182. Dehn, P.F., Allen-Mocherie, S., Karek, J., Thenappan, A., 2005. Organochlorine insecticides: impacts on human HepG2 cytochrome P4501A, 2B activities and glutathione levels. Toxicol. In Vitro 19, 261–273. Domouhtsidou, G.P., Dailianis, S., Kaloyianni, M., Dimitriadis, V.K., 2004. Lysosomal membrane stability and metallothionein content in Mytilllus galloprovincialis (L), as biomarkers – combination with trace metal concentrations. Mar. Pollut. Bull. 48, 572–586. Eaton, D.L., Cherian, M.G., 1991. Determination of metallothionein in tissues by cadmium–hemoglobin affinity assay. In: Riordan, J.F., Vallee, B.L. (Eds.), Methods in Enzymology, vol. 205. Academic Press, New York, pp. 83–88. Eaton, D.L., Toal, B.F., 1982. Evaluation of the Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues. Toxicol. Appl. Pharmacol. 66, 134–142. Fulladosa, E., Delmas, F., Jun, L., Villaescusa, I., Murat, J.C., 2002. Cellular stress induced in cultured human cells by exposure to sludge extracts from water treatment plants. Ecotox. Environ. Saf. 53, 134–140. Garrison, P.M., Tullis, K., Aarts, J.M.M.J.G., Brouwer, A., Giesy, J.P., Denison, M.S., 1996. Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. Fund. Appl. Toxicol. 30, 194–203. Geret, F., Jouan, A., Turpin, V., Bebianno, M.J., Cosson, R.P., 2002. Influence of metal exposure on metallothionein synthesis and lipid peroxidation in two bivalve mollusks: the oyster (Crassostrea gigas) and the mussel (Mytilus edulis). Aqaut. Living Resour. 15, 61–66. Giesy, J.P., Hilscherova, K., Jones, P.D., Kannan, K., Machala, M., 2002. Cell bioassays for detection of aryl hydrocarbon (AhR) and estrogen receptor (ER) mediated activity in environmental samples. Mar. Pollut. Bull. 45, 3–16. Haq, F., Mahoney, M., Koropatnick, J., 2003. Signaling events for metallothionein induction. Mutat. Res. 533, 211–226. Jahroudi, N., Foster, R., Price-Haughey, J., Beitel, G., Gedamu, L., 1990. Cell-type specific and differential regulation of human metallothionein genes. J. Biol. Chem. 265, 6506–6511. Jimenez, I., Aracena, P., Letelier, M.E., Navarro, P., Speisky, H., 2002. Chronic exposure of HepG2 cells to excess copper results in depletion of glutathione and induction of metallothionein. Toxicol. In Vitro 16, 167–175. Kagi, J.H.R., 1993. Evolution, structure and chemical activity of class I metallothioneins: an overview. In: Suzuki, K.T., Imura, N., Kimura, M. (Eds.), Metallothionein III. Biological Roles and Medical Implications. Birkhauser Verlag, Berlin, pp. 29–56. Klaassen, C.D., Liu, J., 1997. Role of metallothionein in cadmium-induced hepatotoxicity and nephrotoxicity. Drug Metab. Rev. 29, 79–102. Klaassen, C.D., Liu, J., Choudhuri, S., 1999. Metallothionein: an intracellular protein to protect against cadmium toxicity. Annu. Rev. Pharmacol. Toxicol. 39, 267–294. Lecoeur, S., Videmann, B., Berny, Ph., 2004. Evaluation of metallothionein as a biomarker of single and combined Cd/Cu exposure in Dreissena polymorpha. Environ. Res. 94, 184–191.
J. Shea et al. / Toxicology in Vitro 22 (2008) 1025–1031 Lee, K., Nagler, J.J., Fournier, M., Lebeuf, M., Cyr, D.G., 1999. Toxicological characterization of sediments from Baie Des Anglais on the St. Lawrence estuary. Chemosphere 39, 1019–1035. Lu, J., Jin, T., Nordberg, G., Nordberg, M., 2001. Metallothionein gene expression in peripheral lymphocytes from cadmium-exposed workers. Cell Stress Chaperon. 6, 97–104. Lukkari, T., Taavitsainen, M., Soimasuo, M., Oikari, A., Haimi, J., 2004. Biomarker responses of the earthworm Aporrectodea tuberculata to copper and zinc exposure: differences between populations with and without earlier metal exposure. Environ. Pollut. 129, 377–386. Martin, T.D., Brockhoff, C.A., Creed, J.T., EMMC methods workgroup, 1994. Method 200.7, determination of metals and trace elements in water and wastes by inductively coupled plasma-atomic emission spectroscopy. Revision 4.4. US EPA. Miura, N., Matsumoto, Y., Miyairi, S., Nishiyama, S., Naganuma, A., 1999. Protective effects of triterpene compounds against cytotoxicity of cadmium in HepG2 cells. Mol. Pharmacol. 56, 1324–1328. Moran, T., Adams, D., Walker, K., Zajdlik, B., 1997. Lambton Industrial Society 1994/ 1995 St. Clair River sediment program defining spatial extent and environmental conditions. Final Report, Pollutech EnviroQuatics Limited, Point Edward, ON. Moran, T., Houtby, J., Zajdlik, B., Kierstead, T., 2003. Sarnia Lambton Environmental Association 2001/2002 St. Clair River biological program – assessment of bioaccumulation/biomagnification potential and trends. Final Report, Pollutech EnviroQuatics Limited, Point Edward, ON, p. 112. Perceval, O., Pinel-Alloul, B., Methot, G., Couillard, Y., Giguere, A., Campbell, P.G.C., Hare, L., 2002. Cadmium accumulation and metallothionein synthesis in freshwater bivalves (Pyganodon grandis): relative influence of the metal exposure gradient vs. limnological variability. Environ. Pollut. 118, 5–17. Perez, M.J., Cederbaum, A.I., 2003. Metallothionein 2A induction by zinc protects HepG2 cells against CYP2E1-dependent toxicity. Free Rad. Biol. Med. 34, 443– 455.
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Pieczonka, C.M., Dehn, P.F., 1993. An automated end-point microassay for adenosine-50 -triphosphate using a microplate reader. J. Tissue Cult. Meth. 15, 210–215. Pollutech EnviroQuatics Ltd., 1999. St. Clair River sediment program: establishment of a reference condition. Regoli, F., Pellegrini, D., Winston, G.W., Gorbi, S., Giuliani, S., Virno-Lamberti, C., Bompadre, S., 2002. Application of biomarkers for assessing the biological impact of dredged materials in the Mediterranean: the relationship between antioxidant responses and susceptibility to oxidative stress in the red mullet (Mullus barbatus). Mar. Pollu. Bull. 44, 912–922. Rhee, S.J., Huang, P.C., 1989. Metallothionein accumulation in CHO Cdr cells in response to lead treatment. Chem. Biol. Interact. 72, 347–361. Roper, J.M., Cherry, D.S., Simmers, J.W., Tatem, H.E., 1996. Bioaccumulation of toxicants in the zebra mussel, Dreissena polymorpha, at the Times Beach Confined Disposal Facility, Buffalo, NY. Environ. Pollut. 94, 117–129. Shea, J., Moran, T., Dehn, P.F., 1999. HepG2 Bioassay for metals. Canadian Tech. Rep. of Fisheries and Aquatic Sciences No. 2293, 29. Shelton, L.R., Capel, P.D., 1994. Guidelines for collecting and processing samples of stream bed sediment for analysis of trace elements and organic contaminants for the national water-quality assessment program. USGS Open-File Rep. 94458, p. 20. Urani, C., Calini, V., Melchioretto, P., Morazzoni, F., Canevali, C., Camatini, M., 2003. Different induction of metallothioneins and Hsp70 and presence of the membrane transporter ZnT-1 in HepG2 cells exposed to copper and zinc. Toxicol. In Vitro 17, 553–559. Urani, C., Melchioretto, P., Canevali, C., Crosta, G.F., 2005. Cytotoxicity and induction of protective mechanisms in HepG2 cells exposed to cadmium. Toxicol. In Vitro 19, 887–892. Urani, C., Melchioretto, P., Canevali, C., Morazzoni, F., Gribaldo, L., 2007. Metallothionein and hsp70 expression in HepG2 cells after prolonged cadmium exposure. Toxicol. In Vitro 21, 314–319.
Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
A bioassay for metals utilizing a human cell line J. Shea a, T. Moran b, P.F. Dehn a,* a b
Department of Biology, Canisius College, Buffalo, NY 14208, USA Pollutech EnviroQuatics Ltd., Point Edward, ON, Canada
a r t i c l e
i n f o
Article history: Received 14 November 2005 Accepted 25 February 2008 Available online 4 March 2008 Keywords: HepG2 Human bioassay Metal mixtures Metallothionein Biomakers
a b s t r a c t The purpose of this study was to assess the ability of the HepG2 cell line to function as a bioassay for metal contamination in sediments, using metallothionein (MT) as a biomarker of exposure. Sediments were collected from the eastern and western ends of Lake Erie, extracted using EPA method 200.7, and analyzed for cadmium (Cd), mercury (Hg) and lead (Pb) levels using ICP-AES. Sediment extracts were neutralized then used at a 2.5% final concentration in the exposure medium. MT levels were measured using the cadmium–hemoglobin affinity assay after a 48 h exposure. Fortified blanks from the ICP protocol served as positive controls. Also, HepG2 cells were exposed to Cd, Pb or combinations of Cd and Pb to determine whether or not induction of MT observed in cells exposed to sediment extracts was due to a single metal, combinations of metals, pH, or some other factor. Additionally, cells were exposed to a range of Cd concentrations approximating the levels found in the extracts (0.0005–0.1 mg/L) to determine if a concentration-response occurred. Total metal levels ranged from 527 to 33.5 mg/kg with lead the predominant metal, accounting for 100–88.9% of the total quantifiable metals in the sediments. The biomarker response (MT induction) was strongly correlated (r2 = 0.9919, r2 = 0.990) with total metal and lead levels in the sediments, respectively, which supports recent field studies indicating the biomarker can discern differences in the strength of the inducing agent. Statistically significant MT induction was associated with sediments which contained measurable Cd concentrations and no significant differences were observed when comparing Cd only and Cd + Pb exposed cells indicating no interactions between Cd and Pb were occurring and supporting our finding that Cd was the main inducing agent in sediment extracts. MT levels also increased significantly in a concentration-dependent manner when cells were exposed only to Cd. Results suggest this human bioassay and the MT biomarker of exposure may be useful for monitoring complex metal mixtures in aquatic sediments. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction There is increasing concern of environmental contaminants impacting both human and wildlife health. Much of the monitoring done world-wide utilizes chemical analyses which indicate the presence or absence of contaminants in water, sediments and/or biota. Chemical analyses however, do not provide information on how biota responds to non-toxic, low level, chronic exposures, which are typically found at many contaminated sites. While toxicological testing for various endpoints other than lethality occurs, the majority examine single compound effects. Environmental contaminants do not occur singly, but rather exist as complex mixtures of both inorganic and organic forms, whose interactions have not been studied. Cell bioassays and biochemical indicators or biomarkers of exposure and/or effect offer potentially sensitive tools for inclusion into environmental monitoring, screening and assessment programs. They have the ability * Corresponding author. Tel.: +1 716 888 2555; fax: +1 716 888 3157. E-mail address: [email protected] (P.F. Dehn). 0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.02.014
to complement chemical analysis and can shed light on biotic responses. Several types of cell bioassays (both prokaryote and eukaryote, including human cell lines) have been utilized to monitor complex environmental mixtures (Fulladosa et al., 2002; Garrison et al., 1996; Giesy et al., 2002; Lee et al., 1999) although organic compounds that impact the estrogen receptor have been the focus of the majority of these studies (reviewed in Giesy et al., 2002). The HepG2, a human hepatoma, cell line has been well characterized and used routinely as a model for a variety of in vitro toxicological studies (see references in Dehn et al., 2004, 2005; Urani et al., 2003, 2005, 2007), particularly those involving biotransformations, since the major biotransformation reactions are intact. Of importance to this study is: (1) this cell line’s ability to induce metallothioneins (MTs) in response to metals (Dehn et al., 2004; Jahroudi et al., 1990; Jimenez et al., 2002; Miura et al., 1999; Perez and Cederbaum, 2003; Urani et al., 2003, 2005, 2007); (2) that HepG2 cells have both MT-1 and -2 isoforms, which are the most widely distributed isoforms in mammalian cells (Jahroudi et al., 1990; Perez and Cederbaum, 2003; Urani et al., 2003, 2007); and
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(3) it may serve as model that links toxicant exposure to anticipated responses in normal human hepatocytes (Urani et al., 2005). MTs are widely recognized as an important component of a cells defense against and recovery from environmental toxicant insult. MTs are a family of low-molecular weight (<7 kDa), cysteine-rich, intracellular compounds that are highly inducible. MTs have unique structures that produce potent metal-binding and redox capabilities (Urani et al., 2003). They are best known for their ability to bind metal ions, both those needed for normal metabolism, e.g., Zn and Cu, as well as heavy metal pollutants, e.g., Cd, Hg, and Pb (Kagi, 1993). Metal ions, cytokines, hormones, cytotoxic agents, organic chemicals, and stress have been shown to induce MTs (Haq et al., 2003; Klaassen and Liu, 1997). MT synthesis is controlled primarily at the transcriptional level (Haq et al., 2003; Klaassen et al., 1999). Post-transcriptional regulation also has been shown to be important to renal tissue MT regulation (Haq et al., 2003). MT induction has been identified and utilized as a biomarker of metal exposure in laboratory, field, and epidemiological studies (Bebianno et al., 2003; Carvalho et al., 2004; Domouhtsidou et al., 2004; Geret et al., 2002; Lecoeur et al., 2004; Lu et al., 2001; Lukkari et al., 2004; Perceval et al., 2002; Regoli et al., 2002). In field studies using sentinel animal species, MT induction seems to be affected by a variety of biotic and abiotic factors that may limit MTs use in quantifying the strength of the exposure (Domouhtsidou et al., 2004; Lecoeur et al., 2004; Perceval et al., 2002). Sediments represent vast sinks for contaminants. Several areas of concern (AOC) have been identified within the Lake Erie – St. Clair watershed under the Great Lakes water-quality agreement due to use impairments attributed in part to sediment contamination. Both the St. Clair and Niagara rivers have this designation. Extensive sediment contaminant data exists for the St. Clair River, which indicates a contaminant gradient occurs as one moves downstream from Lake Huron to Lake St. Clair (Moran et al., 1997; Pollutech EnviroQuatics and Ltd., 1999). At the Lake Erie end of the Niagara River, the Black Rock Lock is located immediately downstream of both the Times Beach Confined Disposal facility and the Buffalo River, an AOC, which serve as potential sources for contaminant inputs into the mouth of the Niagara River. Previous studies using zebra mussels as bioindicator organisms have shown significant levels of both organic and inorganic contaminants in tissues after 34 days at these sites (Roper et al., 1996). The purpose of this study was to expand preliminary studies in our laboratory indicating that the HepG2 cell line might function as a bioassay for metal contamination in sediments, using MT as a biomarker of exposure (Shea et al., 1999). To examine the validity of MT induction as a biomarker of exposure for contaminant mixtures contained within sediments, we measured MT levels in cells exposed to extracts of sediments taken from locations within the Great Lakes known to exhibit a gradient of metal contamination. In particular, we were interested in the heavy metals, cadmium, mercury and lead as they represent priority contaminants within the Great Lakes. We also were interested in discerning whether or not MT induction would respond to quantitative differences in metal mixtures in sediments. Or in other words, could this biomarker differentiate between a weak and a strong signal, which has been difficult to ascertain in field studies using sentinel animal species.
um were obtained from GIBCO (Grand Island, NY); chemicals needed for the metallothionein assay, sediment extracts, and sediment analyses were purchased from Sigma (St. Louis, MO); protein assay materials were purchased from Bio-Rad (Melville, NY); the 109 Cd was purchased from DuPont NEN (Boston, MA). 2.2. Cell culture conditions HepG2 cells were maintained as described previously (Pieczonka and Dehn, 1993) in Dulbecco’s modified eagle’s medium (DMEM) with fungizone (125 lg/ml in 100 ml), penicillin–streptomycin (5000 U/ml), non-essential amino acids (0.1 mM), and 10% fetal bovine serum (maintenance DMEM), at 37 °C in 95% O2:5% CO2 conditions. The culture medium was changed three times each week and cells were passaged every 7–10 days. The cells were detached by trypsinization (0.05% trypsin, 0.53 mM EDTA). Cells were mycoplasma free, as determined by the MycoTect assay (GibcoBRL). All cells were plated at concentrations of 106 cells/ml, and allowed to attach for 24 h prior to testing. Volumes used for plating were 1.5 ml for 6 well plates. Only cells in passages 4–8 were used, as age of culture effects have been previously reported (Dehn et al., 2005). 2.3. Sediment collections Surface sediments were collected by hand using scuba on the Canadian side of the St. Clair River (Fig. 1A). Four sites were examined in the St. Clair River. At the upper end of the river, nearest Lake Huron, the Point Edward sample was above the 15 major industrial facilities which discharge directly or indirectly into the St. Clair River. The remaining three sites were below the heaviest industrial area, located on the Ontario side of the river. Surface sediments from the Black Rock Lock at the eastern end of Lake Erie (Fig. 1B) were hand shoveled from the rim of the lock gate when the locks were drained for repair in the middle of the winter. This location was chosen as sediments were still wet and not exposed to the atmosphere, which could compromise comparing sites (Shelton and Capel, 1994). All sediments were placed into plastic containers, stored on ice, returned to the laboratory and immediately frozen at 80 °C until thawed for preparation of the extracts and analysis. 2.4. Sediment extract preparation Sediments were thawed, mixed thoroughly, and any large stones or organic material removed. Sediments were extracted
2. Methods 2.1. Materials The human hepatoma cell line (HepG2, ATCC HB 8065) was obtained from the American Type Culture Collection (Rockville, MD). Culture medium, supplements, MycoTect kit, and fetal bovine ser-
Fig. 1A. Map of the St. Claire River sampling locations.
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Table 1 Analytical conditions and characteristics of the spectro analytical instruments ICPAES used for metal analyses Spectrometer
Spectroflame – EOP/CP Characteristics and conditions
Nebulizer RF power Argon flow
Meinhard pneumatic 1.2 kW Coolant – 14 L/min Auxiliary – 0.5 L/min Nebulizer gas flow – 0.8 L/min Total preflush – 20 s [Cd] 226.502 nm [Hg] 194.227 nm [Pb] 220.353 nm 3000 ms
Sample feed – manual Analyte lines
Sequential peak and background measurement times
2.6. Toxicological responses Fig. 1B. Map of the Black Rock Lock sampling station at the western end of Lake Erie in the entrance to the Niagara River.
according to EPA method 200.7 (Martin et al., 1994), which utilizes an acid extraction. Extracts were split for quantification of levels of cadmium (Cd), mercury (Hg) and lead (Pb) via inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and for cell culture exposures. Extraction blanks and fortified blanks containing known concentrations of the analytes were prepared and analyzed. The fortified blanks were utilized as positive controls. Reproducibility was initially determined using seven replicate extractions on subsamples of the Black Rock Lock sediment. All other sites used duplicate extractions, as reproducibility was within expected 95% confidence intervals. 2.5. HepG2 exposures For all sediment exposure studies, cells were plated for 24 h in maintenance DMEM. After 24 h the medium was removed and the cells were washed twice with sterile phosphate buffered saline (PBS). The maintenance DMEM was replaced with serum-free, protein free basal DMEM containing fungizone (125 lg/ml in 100 ml) and penicillin–streptomycin (5000 U/ml) (exposure DMEM). Cells were exposed for 48 h to exposure DMEM (controls), or exposure DMEM containing 2.5% (final volume in the exposure medium) of the sediment extract or the fortified blank (positive control). A 2.5% extract was used to minimize the effects of the acid and to reduce medium dilution effects. Previous work with organic solvents showed this to be an optimal concentration. All extracts used for HepG2 exposures were neutralized with 3 M NaOH to pH 7.4 after addition to the exposure DMEM and prior to being placed on the cells. Additionally, the exposure DMEM for the controls was acidified and neutralized, as above, prior to exposure to eliminate artifacts due to pH adjustments. Preliminary exposures of the sediment extracts or fortified blanks produced no cytotoxicity as measured by trypan blue dye exclusion (data not shown). HepG2 cells were exposed to cadmium (Cd), lead (Pb) or combinations of Cd and Pb to determine whether or not induction of MT observed in cells exposed to sediment extracts was due to a single metal, combinations of metals, pH, or some other factor. Also cells were exposed to a range of Cd concentrations approximating the levels found in the extracts (0.0005–0.1 mg/L) to determine if a concentration-response occurred, or if a Cd threshold existed. Cells were plated and exposed as above, with the exception that the neutralization step and the positive controls were not needed.
Metallothionein (MT) induction was measured by the cadmium–hemoglobin affinity assay (Eaton and Cherian, 1991) as modified in Dehn et al. (2004). A 100 ll aliquot of the MT assay supernatant was used for isotopic counting. Proteins were quantified by the Bio-Rad method as modified in Allen et al. (2001) using the sample homogenate. Metallothionein content was expressed as pmol Cd bound/mg protein. 2.7. ICP analysis of metals in the sediment extract Inductively coupled plasma (ICP-AES) spectroscopy was used to analyze extracts for levels of Cd, Hg, and Pb. Instrument characteristics and analytical conditions are listed in Table 1. Calibration was performed using single element standards, while a standard curve was generated for each analyte using mixed standards ranging from 1 mg/L to 1000 mg/L. Levels of metals are expressed as mg/L in the extract and in the exposure medium, and as mg/kg for the sediment. These levels are actual values measured and are not corrected to represent percent recoveries. All percent recoveries were within acceptable ranges for the method. 2.8. Statistical analysis Means and standard deviations were calculated for the individual metals and the HepG2 MT levels. The control MT levels for various replicate experiments using the same sediment extract or between replicate experiments using different sediment extracts yielded no significant differences using the Kruskal–Wallis ANOVA on Ranks test. Therefore, all control data were grouped so that a single comparison between the controls and positive controls could be made using the Mann–Whitney Rank Sum test (t-test). t-Tests were used to compare MT levels in HepG2 cells for each sediment extract and its appropriate control group, controls and Cd concentrations. ANOVA’s were used to compare MT levels in Cd only, Pb only, and Cd + Pb exposed cells with a Student–Newman–Keuls post-hoc test to determine which groups were different. All data were analyzed for significance at the alpha 0.05 level. All statistical analyses were done using SigmaStat (SPSS, Inc.). 3. Results Metal contamination was highest in the Black Rock Lock sediments, followed by the Corunna, Courtright, Mooretown and Point Edward sediments (Table 2). Total metal levels ranged from 527 to 37.5 mg/kg. Lead accounted for 100%, 100%, 99.7%, 98.2%, and 88.9% of the quantifiable metals in the Courtright, Mooretown,
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Table 2 Concentrations (mg/kg) of cadmium (Cd), mercury (Hg) and lead (Pb) in sediment from sites within the Lake Erie – St. Clair River watershed Treatment
Point Edward Corunna Mooretown Courtright Black Rock Lock
Extract (mg/kg) Cd
Hg
Pb
Total
BDL 0.3 ± 0.1 BDL BDL 8.4 ± 0.2
4.5 ± 1.4 BDL BDL BDL 1.2 ± 0.4
33 ± 1 87.2 ± 0.7 46.4 ± 0.8 50.2 ± 0.8 517.5 ± 6
37.5 87.5 46.4 50.2 527.1
Sites are listed in an upstream to downstream order. BDL signifies below detection limits. Data represent averages from duplicate extractions.
Corunna, Black Rock Lock, and Point Edward sediments, respectively. Cadmium was detectable only in the Black Rock Lock and Corunna sediments and accounted for 1.6% and 0.3% of the quantifiable metals, respectively. Mercury was detectable only in the Black Rock Lock and Point Edward samples and accounted for 0.2% and 19.2% of the quantifiable metals, respectively. Sediment extracts utilized in the bioassay contained variable levels of cadmium, mercury and lead (Table 3). All sediment extracts except those from the Black Rock Lock and Corunna had cadmium concentrations below the detection limit of 3.4 lg/L. All sediment extracts except those from the Black Rock Lock and Point Edward had mercury concentrations below the detection limit of 2.5 lg/L. Lead was the predominant metal in all sediments and lead levels ranged from 10.4 mg/L in the Black Rock Lock sediment extracts to 0.4 mg/L in the Point Edward sediment extracts. Actual levels used for exposures were much lower as the extract added was 2.5% of the total volume that covered the cells (Table 3). All percent recoveries for the fortified blanks were within acceptable ranges for the method (Table 3). Metallothionein (MT) levels in cells exposed only to exposure DMEM (control) varied between replicate experiments for a sediment extract. For example, three replicate experiments for a Black
Rock Lock extract produced MT levels in control cells of 41.6 ± 3.9, 34.0 ± 7.2, and 45.4 ± 3.9 with n = 3, 3 and 2, respectively. ANOVA’s to compare the variation, indicated no statistically significant differences (P = 0.145). Therefore, replicate runs were grouped together for analysis. Metallothionein (MT) levels in cells exposed to exposure DMEM (control), neutralized fortified blanks (positive control) varied over all experimental exposures (Table 4), however no statistically significant differences occurred between control groups for each sediment location (P = 0.291). Therefore, all control and positive control groups from each experiment were analyzed as single groups. Mean MT levels were 38.9 ± 7.7 and 178.5 ± 48.3 pmol Cd bound/mg protein in control (n = 22) and positive control (n = 15) exposed cells, respectively. Differences between these levels were statistically significant (P = <0.001) indicating an appropriately functioning bioassay response. Metallothionein (MT) levels were elevated in all HepG2 cells exposed to sediment extracts, although only MT levels in cells exposed to sediment extracts from the Black Rock Lock and Corunna were significantly higher (Table 4). In all cases the positive controls produced significant elevations in MT levels with respect to the controls (Table 4). When mean metallothionein level in HepG2 cells for each extract site was expressed as a percent of the mean metallothionein level for the control HepG2 cells for that site and regressed against total metal concentrations in the sediments for each site, there was a strong correlation (r2 = 0.9919) between these two variables (Fig. 2), indicating a dose-response relationship. Regressions between metallothionein expressed as a percent of the control and total lead levels also were strongly correlated (r2 = 0.990) (Fig. 3). Cells were exposed to Cd and Pb levels singly and in combination to determine whether MT induction observed in sediment extracts was due to a single metal or combinations of metals. Levels of MT ranged from 150 ± 63 to 3515 ± 446 pmol Cd bound/mg protein (Table 5). All exposures induced MT significantly when
Table 3 Mean (±SD) concentrations (mg/L) of cadmium (Cd), mercury (Hg) and lead (Pb) in neutralized fortified blanks (positive control), and neutralized sediment extracts from sites within the Lake Erie – St. Clair River watershed and their corresponding concentrations in the HepG2 bioassay Treatment
Point Edward Corunna Mooretown Courtright Black Rock Lock Positive control % Recovery
Extract (mg/L)
Bioassay (mg/L)
Cd
Hg
Pb
Cd
Hg
Pb
BDL 0.006 ± 0.002 BDL BDL 0.17 ± 0.003 15.2 ± 5.9 116 ± 47
0.09 ± 0.03 BDL BDL BDL 0.02 ± 0.01 13.1 ± 2.6 99.4 ± 21
0.4 ± 0.02 1.75 ± 0.01 0.93 ± 0.02 1.4 ± 0.4 10.4 ± 0.1 67.6 ± 11.3 104 ± 16
– 0.0003 – – 0.009 0.76 –
0.005 – – – 0.001 0.66 –
0.02 0.088 0.047 0.07 0.52 3.38 –
Mean (±SD) percent recoveries are given for the fortified blanks (positive controls). BDL signifies below detection limits. Duplicate extractions were done for all but the Black Rock Lock (n = 7) and positive controls (n = 10). A single extract was used for the bioassay.
Table 4 Mean (±SD) levels of metallothionein (pmol cadmium bound/mg protein) in HepG2 cells exposed to neutralized exposure medium (control), neutralized fortified blank (positive control), and neutralized sediment extract (extract) from within the Lake Erie – St. Clair River watershed Site
pmol Cd bound/mg protein Control
Point Edward Corunna Mooretown Courtright Black Rock Lock Total
44.2 ± 2.4 42.5 ± 2.7 47.7 ± 5.6 30.7 ± 6.4 39.7 ± 6.8 38.9 ± 7.7
(2) (4) (2) (6) (8) (22)
Positive control
Extract
226.8 ± 45.2 (3) P = 0.012 169.4 ± 12.3 (4) P = 0.029 176.1 ± 0.6 (2) P = 0.03 106.0 ± 11.2 (3) P < 0.001 216.3 ± 14.3 (3) P = 0.012 178.5 ± 48.3 (15) P < 0.001
56.7 ± 10.9 (9) P = 0.16 57.4 ± 2.8 (4) P < 0.001 58.3 ± 7.2 (6) P = 0.11 38.4 ± 3.8 (6) P = 0.065 198.9 ± 27.6 (21) P < 0.001
Respective sample sizes (n) and P values for t-test comparisons between controls and sediment extracts are indicated. Totals indicate the grouped data for the controls and positive controls.
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Metallothionein (% Control)
600
500
y = 0.779x + 87.75 r 2 = 0.9919
400
300
200
100
0 0
100
200
300
400
500
600
Total Sediment Metal Concentration (mg/kg)
Metallothionein (% Control)
600 y = 0.7915 x +88.24 r 2 = 0.99
400
300
200
100
0 0
100
200
300
400
500
6000 0. 479x
y = 130. 31e 2 R = 0.9544
5000
600
*
4000
* 3000
*
2000
1000
*
*
*
0 Con.
Fig. 2. Relationship between metallothionein content in HepG2 cells exposed to extracts from sediments expressed as a percent of the solvent control and the total metal concentration in the sediments from each sampling site within the Lake Erie – St. Claire River watershed.
500
MT Concentration (pmol Cd bound/mg Protein)
J. Shea et al. / Toxicology in Vitro 22 (2008) 1025–1031
0.0005
0.001
0.005
0.01
0.05
0.1
Concentration Cd (mg/L) Fig. 4. Cadmium concentration-metallothionein response relationship in HepG2 cells.
At higher exposure levels it appeared that additive effects might be occurring as adding mean MT levels of Cd only and Pb only exposed cells approximated those found in Cd + Pb exposed cells (3537 vs. 3515 pmol Cd bound/mg protein, respectively). ANOVA’s comparing MT levels in Cd only, Pb only, and Cd + Pb exposed cells at these higher exposure concentrations were significantly different (P = 0.001), but pairwise multiple comparisons showed no significant differences between 0.09 Cd only and 0.09 Cd + 0.52 Pb exposed cells, indicating Cd was the main inducing agent. To determine, if a threshold and or concentration-response relationship existed for MT, HepG2 cells were exposed to increasing concentrations of Cd whose range spanned those found in the sediment extracts (0–0.1 mg/L). MT levels exhibited a concentrationresponse relationship (Fig. 4). MT levels increased from 168 ± 73 to 4149 ± 678 with each concentration significantly different from the control.
Pb (mg/kg) Fig. 3. Relationship between metallothionein content in HepG2 cells exposed to extracts from sediments expressed as a percent of the solvent control and the lead (Pb) concentration in the sediments from each sampling site within the Lake Erie – St. Claire River watershed.
Table 5 Mean (±SD) levels of metallothionein (pmol cadmium bound/mg protein) in HepG2 cells exposed to neutralized exposure medium (control), neutralized exposure medium containing cadmium, lead, or cadmium and lead respective sample sizes and P values for t-test comparisons between controls and metal exposures are indicated (n = 4 unless indicated) Exposure concentration
MT levels (pmol Cd bound/mg protein)
Controls 0.0003 mg/L Cd 0.09 mg/L Cd 0.09 mg/L Pb 0.52 mg/L Pb 0.0003 mg/L Cd ± 0.09 mg/L Pb 0.09 mg/L Cd ± 0.052 mg/L Pb
150 ± 63 265 ± 53 P = 0.031 2984 ± 480 P = 0.029 324 ± 46 P = 0.004 553 ± 106 P = 0.001 (n = 3) 237 ± 32 P = 0.048 3515 ± 446 P = 0.029
Respective sample sizes and P values for t-test comparisons between controls and metal exposures are indicated (n = 4 unless indicated).
compared to the controls with highest induction responses seen in cells associated with higher exposure levels of Cd. ANOVA’s comparing MT levels in Cd only, Pb only, and Cd + Pb exposed cells were not significantly different at low exposure levels (P = 0.058).
4. Discussion MTs are induced by various physiological and toxicological stimuli, including metals, cytokines, hormones, cytotoxic agents, organic chemicals, and stress (Haq et al., 2003; Kagi, 1993; Klaassen et al., 1999). Both the MT-1 and -2 isoforms are basally expressed and highly inducible in virtually all mammalian cells (Haq et al., 2003). Recently, Urani et al. (2003) have shown basal levels of MT-1 and 2 not to be detectable in HepG2 cells using immunoblot techniques. In our study, as in most other studies measuring total MT levels via the cadmium-hemoglobin affinity assay, control cells exhibit basal levels of MT. This difference could be due to two factors. The media was changed daily in Urani’s et al. (2003) study which could have reduced the presence of other stressors, e.g., hormones or pH, which have the ability to induce MT synthesis. Secondly, the cadmium–hemoglobin affinity assay measures Cd-binding and not actual MT levels (Eaton and Toal, 1982). Therefore, elevation of MT levels in control cells may be an artifact due to the presence of other low-molecular weight proteins which have metal-binding characteristics, e.g., glutathione, although heat treatments are designed to reduce the impacts of these proteins (Eaton and Toal, 1982). For this reason it is imperative that control cells, which are not exposed to any metals but are exposed under the same conditions and in the same medium, be monitored since this is an indirect method that estimates MT content based on Cd-binding.
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Metallothioneins are widely recognized as an important component of a cell’s defense against and recovery from environmental insult. Many metals, including Ag, Bi, Cd, Co, Cu, Hg, Ni, Pb, Zn, induce MT synthesis (Klaassen and Liu, 1997), although only Zn, Cd, Hg, Pb, Cu and Bi bind to MT (Haq et al., 2003; Rhee and Huang, 1989). In HepG2 cells, as in many other cells and/or organisms studied, cadmium is the most potent inducer when administered singly (Dehn et al., 2004). In this study, total MT levels increased in all cells exposed to sediment extracts. However, these increases were significant only for those extracts containing quantifiable levels of cadmium. It is impossible to determine whether or not cadmium was responsible for this significant increase; thresholds for cadmium induction of MT synthesis have not been determined, and below detection limits do not signify the absence of this compound. However, in our cadmium concentration-response study, Cd at a concentration very near that found in the sediment extract (0.0005 vs. 0.0003 mg/L, respectively) did significantly increase MT levels. So it is likely, even though we did not monitor other potential known inducers of MT, e.g., zinc or copper, which have been found within the St. Clair River (Moran et al., 1997; Moran et al., 2003) and most of which have been shown to induce MTs in HepG2 cells (Dehn et al., 2004; Jahroudi et al., 1990; Jimenez et al., 2002; Miura et al., 1999; Perez and Cederbaum, 2003; Urani et al., 2003) that cadmium was responsible for the significant increase in MT levels in sediment extract exposed cells. It is of interest to note the strong correlation (r2 = 0.990) between levels of lead in the sediments and MT expressed as a percentage of the control. Lead has been shown to induce MTs (Bae et al., 2001; Klaassen and Liu, 1997; Rhee and Huang, 1989), although it is not as potent of an inducer as cadmium or zinc. Lead only exposures at 0.09 and 0.52 mg/L did significantly increase MT levels in HepG2 cells. Our results show that HepG2 cells are a viable bioassay and MT is a viable biomarker for lead exposure, supporting those of Campana et al. (2003) who found MT to be a viable biomarker for lead exposure in the toadfish, and those of Rhee and Huang (1989) who showed a correlation between lead concentration and both MT content and MT mRNA levels in CHO (Chinese hamster ovarian) Cdr cells. Metal–metal interactions can occur, which have the potential to either increase or decrease the impact of the inducing agents. Very few metal mixture studies have been done to examine these impacts under controlled conditions. Using cytotoxicity responses in human keratinocytes exposed to a four metal mixture of As, Cd, Cr, and Pb, Bae et al. (2001) found hormetic, additive, synergistic and then antagonistic interactions as dose levels increased. MT levels were found to increase in a dose-dependent manner in three of the four keratinocyte cell strains exposed to the mixture and glutathione levels were elevated at the mixture levels where antagonistic interactions were observed (Bae et al., 2001). In this study we found no indication of metal–metal interactions on MT induction in HepG2 cells. Several laboratory and in situ field studies have examined MT levels as a biomarker of exposure for metal mixtures and contaminated sites containing metal mixtures as well as other organic contaminants. Within both laboratory and field studies, concentration gradients have been defined by the biomarker response (Domouhtsidou et al., 2004; Fulladosa et al., 2002; Lecoeur et al., 2004; Lukkari et al., 2004; Perceval et al., 2002; Regoli et al., 2002). Likewise levels of MT gene expression have been correlated with levels of occupational exposure in human epidemiological studies (Lu et al., 2001). In this study we found a strong correlation between total metal levels in the sediment and increased MT production, when expressed as a percentage of the control; indicating the ability of the bioassay to discern quantitative differences in metal mixtures in sediments. This study supports the human bio-
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