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A non-radioactive method for measuring Cu uptake in HepG2 cells
JOURNAL OF
Inorganic Biochemistry Journal of Inorganic Biochemistry 99 (2005) 1018–1022 www.elsevier.com/locate/jinorgbio
A non-radioactive method for measuring Cu uptake in HepG2 cells C. Fosset a, B.A. McGaw b, M.D. Reid a, H.J. McArdle a
a,*
The Rowett Research Institute, Bucksburn, Green Road, Aberdeen, Scotland AB21 9SB, UK b University of Lincoln, Brayford Pool, Lincoln LN6 7TS, UK
Received 17 August 2004; received in revised form 7 January 2005; accepted 10 January 2005 Available online 10 February 2005
Abstract At present, all data on Cu uptake and metabolism have been derived from radioactive uptake experiments. These experiments are limited by the availability of the radioactive isotopes 64Cu or 67Cu, and their short half-life (12.5 and 62 h, respectively). In this paper, we investigate an alternative method to study the uptake of Cu with natural isotopes in HepG2 cells, a liver cell line used extensively to study Cu metabolism. In nature, Cu occurs as two stable isotopes, 63Cu and 65Cu (63Cu/65Cu = 2.23). This ratio can be measured accurately using inductively coupled plasma mass spectrometry (ICP-MS). In initial experiments, we attempted to measure the time course of Cu uptake using 65Cu. The change in the 63Cu/65Cu ratio, however, was too small to allow measurement of Cu uptake by the cells. To overcome this difficulty, the natural 63Cu/65Cu ratio in HepG2 cells was altered using long-term incubation with 63Cu. This had a significant effect on Cu concentration in HepG2 cells, changing it from 81.9 ± 9.46 pmol lg DNA1 (week 1) to 155 ± 8.63 pmol lg DNA1 (week 2) and stabilising at 171 ± 4.82 pmol lg DNA1 (week 3). After three weeks of culture with 2 lM 63Cu the 63Cu/65Cu changed from 2.18 ± 0.05 to 15.3 ± 1.01. Cu uptake was then investigated as before using 65Cu. Uptake was linear over 60 min, temperature dependent and consistent with previous kinetics data. These observations suggest that stable isotope ICP-MS provides an alternative technique for the study of Cu uptake by HepG2 cells. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Inductively coupled plasma-mass spectrometry; Cu uptake; HepG2; Isotope dilution
1. Introduction Cu is an essential trace element, acting as a catalytic co-factor in many critical enzymatic reactions. Cu metalloenzymes are required for normal oxidative metabolism, haemoglobin, elastin and collagen synthesis, free radical detoxification and iron metabolism [1,2]. As well as being essential, Cu can be extremely toxic, generating oxygen radicals by Fenton type reactions [3]. Any change in the regulation of Cu, therefore, can have severe physiological consequences. Therefore, it is important to understand the mechanisms of action and
*
Corresponding author. Tel.: +44 1224 716628; fax: +44 1224 716622. E-mail address: [email protected] (H.J. McArdle). 0162-0134/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2005.01.005
regulation of cellular components responsible for the acquisition, distribution and detoxification of Cu. The liver plays a central role in Cu homeostasis and distribution. Hepatocytes, which are the primary site of Cu uptake and accumulation in the liver, utilise at least one pathway for Cu uptake. This pathway involves a reduction of Cu2+ to Cu1+ prior to uptake followed by transfer across the membrane through a classic carriermediated process [4]. Two candidates for such transporters have been identified as hCTR1 and hCTR2, which have a Km for Cu in the low micromolar range. hCTR1 has been shown to be ubiquitously expressed, with the highest levels being found in the liver and kidney [5]. In support of a major role for Ctr1 in mammalian Cu homeostasis, studies using human fibroblasts, transfected with hCTR1, showed a dramatic increase in capacity for Cu uptake. Ctr1 knockout homozygotes
C. Fosset et al. / Journal of Inorganic Biochemistry 99 (2005) 1018–1022
mice die in utero with severe developmental defects at the time of death, while heterozygotes have severely restricted tissue Cu level. At present, all data on Cu uptake and metabolism have been derived from experiments with radioactive isotopes (64Cu and 67Cu) [6–10]. However, these radioisotopes have inherent problems that include safety hazards, the legal requirements, storage and disposal. Further, their short half lives 64Cu or 67Cu (12.5 and 62 h, respectively) limit their use in long term experiments. Clearly, there is a need for a technique allowing the assessment of Cu uptake without the use of radioactive isotopes. We have developed a method using Cu stable isotope that circumvents the limitations of radiocopper studies and allows the accurate measurement of Cu uptake in HepG2 cells. The new approach, described here, takes advantage of the fact that the two stable isotopes of Cu naturally occur in a known ratio (63Cu/65Cu = 2.235). Thus, the cells and all cuprospecies within all kinetic compartments are uniformly labelled with this ratio. Experimentally, the 63Cu/65Cu ratio can be measured accurately using inductively coupled plasma mass spectrometry (ICP/MS). The benefit of the isotopic ratio analysis is the ability to determine results based on the ratio value rather than the total amount of the measured metal. Therefore, we used this powerful technique as a tool to measure Cu uptake in HepG2 cells, a liver cell line extensively used for the study of Cu metabolism.
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medium (Gibco, Life technologies, Paisley, UK) supplemented with 10% fetal bovine serum (Gibco, Life technologies, Paisley, UK), penicillin and streptomycin (Gibco, Life technologies, Paisley, UK) using standard aseptic techniques. Cells were maintained in continuous culture at 37 °C in an atmosphere of 20% O2 and 5% CO2. The medium was changed every 3 days and the cells subcultured every week. Experiments were performed on 90% confluent cells. In experiments where the cells were pre-loaded with 63Cu, cells were incubated in culture medium with 2 lM 63CuHis2 for 1–3 weeks.
2. Materials and methods
2.2.2. Cu uptake experimental methods Culture medium was removed and cells were washed 3 times with ice-cold BSS (136 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 18 mM HEPES ((N-[2hydroxyethyl]piperazine-N 0 -[2-ethanesulfonic acid]), pH 7.4). Following washing, 8 mL of BSS supplemented with CuHis2 (final concentration as given in figures, 37 °C, pH 7.0) was added to each plate, incubated in a 37 °C water bath and removed after the required time. The cells were washed 3 times with ice-cold BSS prior the addition of pronase (1 mg mL1, 4 °C, 10 min). The cells were aspirated and centrifuged (1000g, 5 min at 4 °C). The pellet, representing surface bound Cu, was discarded, and the pellet, which contains internalised Cu, was resuspended in 8 mL of ultra-pure water. The pellet was disrupted by sonication and 0.5 mL of the disrupted cells was used for DNA quantification. The remaining cell homogenate was further spiked with 160 lL of a 1:50 dilution 65Cu (105 lM) and processed for the measurement of the 63Cu/65Cu ratio.
2.1. Stable isotopes
2.3. DNA quantification
65
Cu2NO3 (7.46 mg, 99.8% enrichment, AERE Harwell, Oxfordshire, UK) was dried in an oven (50 °C overnight) and dissolved in 2% v/v HNO3 (Primar grade, Fisher Scientific, Loughborough, UK). The isotope purity of the 65Cu solution was determined accurately by ICP-MS using a Cu standard solution (Spectrosol, BDH) and the concentration verified by reverse isotope dilution mass spectrometry. The Cu histidine ([65Cu]His2) stock solution was made by the addition of 11 mg histidine into 50 mL of 65Cu (105 lM [65Cu]His2 final concentration). The solution was adjusted to pH 7.0 with 1 M NaOH. The [63Cu]His2 (63Cu2NO3 99.7% enrichment AERE Harwell, Oxfordshire, UK) stock solution was made according to the same procedure with a final Cu concentration of 5 mM. 2.2. Uptake of
65
Cu by HepG2 cells
2.2.1. Cell culture HepG2 cells were obtained from the European Type Culture Collection. They were cultured in WilliamÕs E
DNA was quantified according to published methods [11] using Hoechst dye 33258 (Polysciences Ltd, Northampton, UK) and calf thymus DNA as a standard. Samples, standard and Hoechst Dye were diluted appropriately with TNE buffer (10 mM Tris (tris(hydroxymethyl)aminomethane)–HCl, 1 mM EDTA(ethylenediamine tetraacetic acid), 0.2 M NaCl, pH 7.4). Equal volume of sample and Hoechst dye (2 lg mL1) were mixed on a 96-well plate (Microfluor black plates, Dynatech, Billinghurst, UK) and read at extinction 356 nm/emission 458 nm on a Fluorlite 1000 plate reader (Dynatech, Billinghurst, UK.) 2.4. Preparation of samples for analysis by ICP-MS and AAS Cell homogenates were freeze-dried in a Savillex digestion vessel (Savillex Corporation, Minnesota, USA) and 1.8 mL of concentrated HNO3 and 0.2 mL H2O2 (AristaR grade, BDH, Poole, UK) were added to each vessel. They were sealed, incubated overnight
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at room temperature, and then subjected to microwave irradiation (10 s, 855 W) followed by cooling, until a clear solution was obtained. The sample digests were then dried on a hot plate; re-dissolved in 5 mL of 1 M ammonium acetate and adjusted to pH 8 using conc NH4OH (AristaR Grade, BDH, Poole, UK). Divalent cations, including Cu2+, were separated from monovalent cations and other contaminants, using Chelex-100 ion exchange chromatography (2 mL 200– 400 mesh, Na form Econocolumns, Bio-Rad, Hemel Hempstead, UK). The columns were prepared by washing with 20 mL of 2.5 M HNO3, followed by 20 mL of 2 M NH4OH and 20 mL of ultra-pure water. The samples were then added, followed by 20 mL of 1 M NH4Ac and 15 mL of purified water to wash out any remaining sample. The divalent metals including Cu2+ were eluted from the Chelex-100 columns using 2.5 M HNO3 (20 mL). These solutions were then analysed by ICP/ MS for 65Cu/63Cu isotope ratios and AAS for total intracellular Cu concentration. Standard solutions were prepared from a stock Cu atomic absorption standard solution containing 1000 mg Cu1L1 (Spectrosol, BDH; Poole, UK).
2.5. Analysis of copper isotope ratios by ICP-MS Isotope ratios were measured by ICP-MS using a VG PQ2+ instrument (VG Elemental, Winsford, UK), equipped with a quartz torch, a Meinhard glass expansion nebuliser and a Scott-type water-cooled double-pass spray chamber, cooled to 4 °C. Typical operating conditions were: RF power = 1350 W, nebuliser Ar flow rate = 0.8 L min1, cool Ar flow rate = 14 L min1 and auxiliary Ar flow rate = 0.7 L min1. Samples were introduced by natural aspiration for improved stability and a peristaltic pump (Gilson Medical Electronics, Middleton, WI) was used to drain the spray chamber with the flow rate set at 0.8 mL min1, a sample uptake time of 2 min and a wash time of 3 min. A blank value (for background subtraction) was acquired once, while samples and standards were acquired 5 times, all for 60 s. Data were collected using an m/z scan range of 60–68 amu, dwell time of 320 ls and 19 channels per amu. Isotope ratios obtained using Cu atomic absorption standards (Spectrosol, BDH; Poole, UK) were used to correct for mass bias. The amount of Cu in the HepG2 cells is proportional to the change in 63Cu/65Cu ratio caused by the 65Cu spiking solution and can be calculated using the Pickup and McPherson equation [12,13]. Therefore, Cu accumulation was calculated by subtracting the total amount of Cu in the HepG2 cells after 1 h incubation with 2 lM CuHis2, from the amount of Cu in HepG2 cells at the beginning of the incubation.
A¼
RðB Qh Þ ðB Ql Þ ; P l ðR P h Þ
where A is the amount of Cu to be determined, Pl is the light isotope natural abundance, Ph is the heavy isotope natural abundance, Ql is the light isotope in spike, Qh is the heavy isotope in spike, B is the amount of spike added, and R is the 63Cu/65Cu ratio measured. 2.6. Statistics Comparisons were carried out on individual measurements in separate experiments. Differences were tested for significance using StudentÕs t-test. Differences were taken as significant at p < 0.05. Fitting data to the Michaelis–Menten equation and linear regression analyses were carried out using non-linear iteration (GraphPad Prism 4.00. San Diego, CA). 3. Results We first determined whether the number of cells on a 57-mm diameter plate would be sufficient to measure the 63 Cu/65Cu ratio accurately and consistently. The natural ratio was determined as 2.185 ± 0.06 (n = 6), demonstrating that the cell number was adequate for accuracy and sensitivity (Figure not shown). Subsequently, we attempted to measure Cu uptake using [65Cu]His2 solution. However, the change in the 63Cu/65Cu ratio was too small to cause a significant alteration to the ratio after 1 h (data not shown). To increase sensitivity, therefore, we increased the natural 63Cu/65Cu ratio in HepG2 cells by incubating the cells with [63Cu]His2, which would allow us to detect small increases in 65Cu levels. After three weeks of culture with 2 lM [63Cu]His2 added to the culture medium, the 65Cu/63Cu ratio changed from 2.18 ± 0.05 to 15.3 ± 1.01 (Fig. 1). Thus, 93.6% of the intracellular Cu was now 63Cu. Long-term incubation with [63Cu]His2 also increased intracellular
Fig. 1. Loading HepG2 cells with [63Cu]His2 changes the 63Cu/65Cu ratio over time. cells were grown and supplemented with 2 lM [63Cu]His2 as described in Section 2. Data are expressed as isotopic ratios and are the means ± SEM of n = 6, combined from 3 experiments. Data with the same superscript do not differ significantly.
C. Fosset et al. / Journal of Inorganic Biochemistry 99 (2005) 1018–1022
Fig. 2. Cu concentration in [63Cu]His2 treated HepG2 cells is stable after 3 weeks. cells were grown and supplemented with 2 lM [63Cu]His2 before being treated as described in Section 2. Data are the means ± SEM of n = 6, combined from 2 experiments. Data with the same superscript do not differ significantly.
Cu over the first two weeks, but levelling by three weeks (Fig. 2). 65 Cu uptake was then measured. Uptake into the cells was linear over 60 min and temperature dependent (Fig. 3). To test whether 65Cu was acting as a tracer, total
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Fig. 5. 65Cu uptake is concentration dependent. [63Cu]His2 labelled cells were incubated with increasing concentration of [65Cu]His2. For 30 min as described in Section 2. Cu concentrations were calculated as described in Section 2. Data are presented as the means of 6 observations at each concentration. Curve is the line of best fit for the Michaelis–Menten equation.
intracellular Cu levels were measured at each time point. 65 Cu uptake did not change total Cu concentration over 50 min (Fig. 4). Uptake rates were measured at different [65Cu]His2 concentrations in HepG2 cells. Uptakes followed saturation kinetics and were fitted to the Michaelis–Menten equation as described (Fig. 5). The Vmax was calculated to be 33 ± 3 pmol lg DNA1 min1 and the apparent Km was 2.6 ± 0.8 lM (p < 0.01).
4. Discussion
Fig. 3. 65Cu uptake is time and temperature dependent. [63Cu]His2 labelled cells were incubated with 2 lM [65Cu]His2 as described in Section 2. Data are expressed as isotopic ratios and are the means ± SEM of n = 6, combined from 2 experiments.
Fig. 4. Incubating with 65Cu does not increase Cu levels within HepG2 cells 63Cu]His2 labelled cells were incubated with 2 lM [65Cu]His2 as described in Section 2. The left y-axis represents isotopic ratios and the right y axis represents the intracellular Cu concentration. The 63 Cu/65Cu ratio is presented as closed circles while intracellular Cu levels are open circles. Data are the means ± SEM of n = 3. Lines are calculated as described in Section 2.
This paper has demonstrated a new method for measuring Cu uptake into cells in culture. Until recently, virtually all studies have used the radioisotopes 64Cu and 67 Cu [6,14–17]. 67Cu, with its longer half life, has been particularly useful in examining kinetics of Cu in both cell culture and in animal studies. With these methods, uptake was demonstrated to be a carrier-mediated process, in a wide variety of cell types, including hepatocytes, fibroblasts, CHO and K562 cells, and in trophoblast cells from placenta. In animals, the isotope was also used to measure transfer across the placenta and to implicate ceruloplasmin in this process [18–20]. However, as described in the introduction, there are also problems with the use of the labels, and alternative methods have been tested at different times. Solioz and colleagues [21,22] have used Ag as also a substrate for the bacterial homologue of the Menkes transport protein and it has also been used for the diagnosis of the disease. The approach is limited, however, since the isotope is not commercially available. Phen Green FL has been used for detecting divalent metals such as Fe2+, Hg2+, Cd2+, Pb2+ and Ni2+ at submicromolar concentrations [23–25] but can also be used for Cu2+. For example, Chavez-Crooker et al. [26] have used the dye to differentiate between intracellular Cu levels for four types of lobster hepatopancreatic epithelial cells. However, the approach suffers from poor spec-
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ificity and data become complicated when other metals, such as Fe, are present. In this paper, an alternative method of studying Cu uptake has been developed and evaluated. To date, it is the first report describing a stable isotope technique applied to a cell culture model. ICP-MS is one of the widely used contemporary techniques to measure isotope ratio, because it is the most sensitive, accurate and has a high dynamic range. The use of isotope dilution mass spectrometry (IDMS) to measure Cu content in HepG2 cells has several advantages. Partial loss of the analyte after equilibrium of the spike and the sample will not influence the accuracy of the determination. This is of great importance since the samples have to be digested and purified before analysis. Further, physical and chemical interferences are less likely to affect the measurements since they have similar effects on each isotope of the same element. Previous workers have realised that the natural ratio of 63Cu and 65Cu presents problems in using stable isotopes to measure uptake and our own data confirm that, to measure uptake and kinetics, it is first necessary to alter the balance of the two isotopes. This leads to some problems with increased Cu concentrations within the cell, but it seems clear that a stable state is reached by three weeks of incubation. The increase in the ratio of 63 Cu/65Cu (to about 15 within three weeks) far outweighs any disadvantages this may have. As with radioisotope uptake studies, we have shown that Cu uptake was temperature and time dependent [27–29]. Uptake was linear over the time period, demonstrating, as confirmed by actual measurements, that 65 Cu was acting as a tracer and we could derive kinetic parameters for uptake. These values were similar to those already published using radioactive Cu. This method, therefore, though somewhat cumbersome, represents a real option for studying Cu metabolism in different systems. It also introduces the possibility for studying long term metabolism in animals, since it is feasible one could feed them 63Cu as we have done with the cells and then measure uptake and metabolism using 65 Cu as the tracer. Acknowledgement This work was supported by the International Copper Association.
References [1] H.J. McArdle, A. Ralph, Copper Metabolism and Requirements in the Pregnant Mother, Her Fetus and Children: A Critical Review, International Copper Association, Ltd., New York, 2001. [2] R. Uauy, M. Olivares, M. Gonzalez, Am. J. Clin. Nutr. 67 (1998) 952S–959S. [3] B. Halliwell, J.M. Gutteridge, Biochem. J. 219 (1984) 1–14. [4] G.J. van den Berg, H.J. McArdle, Biochim. Biophys. Acta 1195 (1994) 276–280. [5] B. Zhou, J. Gitschier, Proc. Natl. Acad. Sci. USA 94 (1997) 7481– 7486. [6] K. Gibbs, J.M. Walshe, S.B. Osborn, Clin. Sci. 37 (1969) 877–878. [7] H.J. McArdle, R. Erlich, J. Nutr. 121 (1991) 208–214. [8] A. Crowe, E.H. Morgan, Biochim. Biophys. Acta 1291 (1996) 53– 59. [9] J.R. Subramaniam, W.E. Lyons, J. Liu, T.B. Bartnikas, J. Rothstein, D.L. Price, D.W. Cleveland, J.D. Gitlin, P.C. Wong, Nat. Neurosci. 5 (2002) 301–307. [10] H. Speisky, P. Navarro, M.G. Cherian, I. Jimenez, Biometals 16 (2003) 113–123. [11] R. Danzeisen, C. Fosset, Z. Chariana, K. Page, S. David, H.J. McArdle, Am. J. Physiol. Cell. Physiol. 282 (2002) C472– C478. [12] K.E. Jarvis, A.L. Gray, R.S. Houk, Handbook of Inductively Coupled Plasma Mass Spectrometry, Blackie Academic and Professional, An Imprint of Chapman & Hall, 1995. [13] V. Ducros, A. Favier, J. Chromatogr. 583 (1992) 35–44. [14] T. Terao, C.A. Owen Jr., Am. J. Physiol. 232 (1977) E172–E179. [15] N.G. Beratis, P. Price, G. Labadie, K. Hirschhorn, Pediatr. Res. 12 (1978) 699–702. [16] S. Iwanska, D. Strusinska, Acta Physiol. Pol. 29 (1978) 465– 474. [17] J.R. Mann, J. Camakaris, D.M. Danks, Biochem. J. 180 (1979) 613–619. [18] S.S. Percival, E.D. Harris, Am. J. Physiol. 258 (1990) C140–C146. [19] S.J. Orena, C.A. Goode, M.C. Linder, Biochem. Biophys. Res. Commun. 139 (1986) 822–829. [20] A. Mas, B. Sarkar, Biochim. Biophys. Acta 1135 (2) (1992) 123– 128. [21] F.W. Verheijen, C.E. Beerens, A.C. Havelaar, W.J. Kleijer, G.M. Mancini, J. Med. Genet. 35 (1998) 849–851. [22] M. Solioz, A. Odermatt, J. Biol. Chem. 270 (1995) 9217–9221. [23] F. Petrat, H. Rauen de Groot, Hepatology 29 (1999) 1171–1179. [24] L. Escriche, J.A. Munoz, J. Rosell, R. Kivekas, R. Sillanpaa, J. Casabo, Inorg. Chem. 37 (1998) 4807–4813. [25] O. Kakhlon, Z.I. Cabantchik, Free Radic. Biol. Med. 33 (2002) 1037–1046. [26] P. Chavez-Crooker, N. Garrido, G.A. Ahearn, J. Exp. Biol. 204 (2001) 1433–1444. [27] M.J. Bingham, H.J. McArdle, Hepatology 20 (1994) 1024– 1031. [28] R.J. Stockert, P.S. Grushoff, A.G. Morell, G.E. Bentley, H.A. OÕBrien, I.H. Scheinberg, I. Sternlieb, Hepatology 6 (1986) 60–64. [29] K.K. Tong, H.J. McArdle, Biochim. Biophys. Acta 1269 (1995) 233–236.
Inorganic Biochemistry Journal of Inorganic Biochemistry 99 (2005) 1018–1022 www.elsevier.com/locate/jinorgbio
A non-radioactive method for measuring Cu uptake in HepG2 cells C. Fosset a, B.A. McGaw b, M.D. Reid a, H.J. McArdle a
a,*
The Rowett Research Institute, Bucksburn, Green Road, Aberdeen, Scotland AB21 9SB, UK b University of Lincoln, Brayford Pool, Lincoln LN6 7TS, UK
Received 17 August 2004; received in revised form 7 January 2005; accepted 10 January 2005 Available online 10 February 2005
Abstract At present, all data on Cu uptake and metabolism have been derived from radioactive uptake experiments. These experiments are limited by the availability of the radioactive isotopes 64Cu or 67Cu, and their short half-life (12.5 and 62 h, respectively). In this paper, we investigate an alternative method to study the uptake of Cu with natural isotopes in HepG2 cells, a liver cell line used extensively to study Cu metabolism. In nature, Cu occurs as two stable isotopes, 63Cu and 65Cu (63Cu/65Cu = 2.23). This ratio can be measured accurately using inductively coupled plasma mass spectrometry (ICP-MS). In initial experiments, we attempted to measure the time course of Cu uptake using 65Cu. The change in the 63Cu/65Cu ratio, however, was too small to allow measurement of Cu uptake by the cells. To overcome this difficulty, the natural 63Cu/65Cu ratio in HepG2 cells was altered using long-term incubation with 63Cu. This had a significant effect on Cu concentration in HepG2 cells, changing it from 81.9 ± 9.46 pmol lg DNA1 (week 1) to 155 ± 8.63 pmol lg DNA1 (week 2) and stabilising at 171 ± 4.82 pmol lg DNA1 (week 3). After three weeks of culture with 2 lM 63Cu the 63Cu/65Cu changed from 2.18 ± 0.05 to 15.3 ± 1.01. Cu uptake was then investigated as before using 65Cu. Uptake was linear over 60 min, temperature dependent and consistent with previous kinetics data. These observations suggest that stable isotope ICP-MS provides an alternative technique for the study of Cu uptake by HepG2 cells. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Inductively coupled plasma-mass spectrometry; Cu uptake; HepG2; Isotope dilution
1. Introduction Cu is an essential trace element, acting as a catalytic co-factor in many critical enzymatic reactions. Cu metalloenzymes are required for normal oxidative metabolism, haemoglobin, elastin and collagen synthesis, free radical detoxification and iron metabolism [1,2]. As well as being essential, Cu can be extremely toxic, generating oxygen radicals by Fenton type reactions [3]. Any change in the regulation of Cu, therefore, can have severe physiological consequences. Therefore, it is important to understand the mechanisms of action and
*
Corresponding author. Tel.: +44 1224 716628; fax: +44 1224 716622. E-mail address: [email protected] (H.J. McArdle). 0162-0134/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2005.01.005
regulation of cellular components responsible for the acquisition, distribution and detoxification of Cu. The liver plays a central role in Cu homeostasis and distribution. Hepatocytes, which are the primary site of Cu uptake and accumulation in the liver, utilise at least one pathway for Cu uptake. This pathway involves a reduction of Cu2+ to Cu1+ prior to uptake followed by transfer across the membrane through a classic carriermediated process [4]. Two candidates for such transporters have been identified as hCTR1 and hCTR2, which have a Km for Cu in the low micromolar range. hCTR1 has been shown to be ubiquitously expressed, with the highest levels being found in the liver and kidney [5]. In support of a major role for Ctr1 in mammalian Cu homeostasis, studies using human fibroblasts, transfected with hCTR1, showed a dramatic increase in capacity for Cu uptake. Ctr1 knockout homozygotes
C. Fosset et al. / Journal of Inorganic Biochemistry 99 (2005) 1018–1022
mice die in utero with severe developmental defects at the time of death, while heterozygotes have severely restricted tissue Cu level. At present, all data on Cu uptake and metabolism have been derived from experiments with radioactive isotopes (64Cu and 67Cu) [6–10]. However, these radioisotopes have inherent problems that include safety hazards, the legal requirements, storage and disposal. Further, their short half lives 64Cu or 67Cu (12.5 and 62 h, respectively) limit their use in long term experiments. Clearly, there is a need for a technique allowing the assessment of Cu uptake without the use of radioactive isotopes. We have developed a method using Cu stable isotope that circumvents the limitations of radiocopper studies and allows the accurate measurement of Cu uptake in HepG2 cells. The new approach, described here, takes advantage of the fact that the two stable isotopes of Cu naturally occur in a known ratio (63Cu/65Cu = 2.235). Thus, the cells and all cuprospecies within all kinetic compartments are uniformly labelled with this ratio. Experimentally, the 63Cu/65Cu ratio can be measured accurately using inductively coupled plasma mass spectrometry (ICP/MS). The benefit of the isotopic ratio analysis is the ability to determine results based on the ratio value rather than the total amount of the measured metal. Therefore, we used this powerful technique as a tool to measure Cu uptake in HepG2 cells, a liver cell line extensively used for the study of Cu metabolism.
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medium (Gibco, Life technologies, Paisley, UK) supplemented with 10% fetal bovine serum (Gibco, Life technologies, Paisley, UK), penicillin and streptomycin (Gibco, Life technologies, Paisley, UK) using standard aseptic techniques. Cells were maintained in continuous culture at 37 °C in an atmosphere of 20% O2 and 5% CO2. The medium was changed every 3 days and the cells subcultured every week. Experiments were performed on 90% confluent cells. In experiments where the cells were pre-loaded with 63Cu, cells were incubated in culture medium with 2 lM 63CuHis2 for 1–3 weeks.
2. Materials and methods
2.2.2. Cu uptake experimental methods Culture medium was removed and cells were washed 3 times with ice-cold BSS (136 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 18 mM HEPES ((N-[2hydroxyethyl]piperazine-N 0 -[2-ethanesulfonic acid]), pH 7.4). Following washing, 8 mL of BSS supplemented with CuHis2 (final concentration as given in figures, 37 °C, pH 7.0) was added to each plate, incubated in a 37 °C water bath and removed after the required time. The cells were washed 3 times with ice-cold BSS prior the addition of pronase (1 mg mL1, 4 °C, 10 min). The cells were aspirated and centrifuged (1000g, 5 min at 4 °C). The pellet, representing surface bound Cu, was discarded, and the pellet, which contains internalised Cu, was resuspended in 8 mL of ultra-pure water. The pellet was disrupted by sonication and 0.5 mL of the disrupted cells was used for DNA quantification. The remaining cell homogenate was further spiked with 160 lL of a 1:50 dilution 65Cu (105 lM) and processed for the measurement of the 63Cu/65Cu ratio.
2.1. Stable isotopes
2.3. DNA quantification
65
Cu2NO3 (7.46 mg, 99.8% enrichment, AERE Harwell, Oxfordshire, UK) was dried in an oven (50 °C overnight) and dissolved in 2% v/v HNO3 (Primar grade, Fisher Scientific, Loughborough, UK). The isotope purity of the 65Cu solution was determined accurately by ICP-MS using a Cu standard solution (Spectrosol, BDH) and the concentration verified by reverse isotope dilution mass spectrometry. The Cu histidine ([65Cu]His2) stock solution was made by the addition of 11 mg histidine into 50 mL of 65Cu (105 lM [65Cu]His2 final concentration). The solution was adjusted to pH 7.0 with 1 M NaOH. The [63Cu]His2 (63Cu2NO3 99.7% enrichment AERE Harwell, Oxfordshire, UK) stock solution was made according to the same procedure with a final Cu concentration of 5 mM. 2.2. Uptake of
65
Cu by HepG2 cells
2.2.1. Cell culture HepG2 cells were obtained from the European Type Culture Collection. They were cultured in WilliamÕs E
DNA was quantified according to published methods [11] using Hoechst dye 33258 (Polysciences Ltd, Northampton, UK) and calf thymus DNA as a standard. Samples, standard and Hoechst Dye were diluted appropriately with TNE buffer (10 mM Tris (tris(hydroxymethyl)aminomethane)–HCl, 1 mM EDTA(ethylenediamine tetraacetic acid), 0.2 M NaCl, pH 7.4). Equal volume of sample and Hoechst dye (2 lg mL1) were mixed on a 96-well plate (Microfluor black plates, Dynatech, Billinghurst, UK) and read at extinction 356 nm/emission 458 nm on a Fluorlite 1000 plate reader (Dynatech, Billinghurst, UK.) 2.4. Preparation of samples for analysis by ICP-MS and AAS Cell homogenates were freeze-dried in a Savillex digestion vessel (Savillex Corporation, Minnesota, USA) and 1.8 mL of concentrated HNO3 and 0.2 mL H2O2 (AristaR grade, BDH, Poole, UK) were added to each vessel. They were sealed, incubated overnight
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at room temperature, and then subjected to microwave irradiation (10 s, 855 W) followed by cooling, until a clear solution was obtained. The sample digests were then dried on a hot plate; re-dissolved in 5 mL of 1 M ammonium acetate and adjusted to pH 8 using conc NH4OH (AristaR Grade, BDH, Poole, UK). Divalent cations, including Cu2+, were separated from monovalent cations and other contaminants, using Chelex-100 ion exchange chromatography (2 mL 200– 400 mesh, Na form Econocolumns, Bio-Rad, Hemel Hempstead, UK). The columns were prepared by washing with 20 mL of 2.5 M HNO3, followed by 20 mL of 2 M NH4OH and 20 mL of ultra-pure water. The samples were then added, followed by 20 mL of 1 M NH4Ac and 15 mL of purified water to wash out any remaining sample. The divalent metals including Cu2+ were eluted from the Chelex-100 columns using 2.5 M HNO3 (20 mL). These solutions were then analysed by ICP/ MS for 65Cu/63Cu isotope ratios and AAS for total intracellular Cu concentration. Standard solutions were prepared from a stock Cu atomic absorption standard solution containing 1000 mg Cu1L1 (Spectrosol, BDH; Poole, UK).
2.5. Analysis of copper isotope ratios by ICP-MS Isotope ratios were measured by ICP-MS using a VG PQ2+ instrument (VG Elemental, Winsford, UK), equipped with a quartz torch, a Meinhard glass expansion nebuliser and a Scott-type water-cooled double-pass spray chamber, cooled to 4 °C. Typical operating conditions were: RF power = 1350 W, nebuliser Ar flow rate = 0.8 L min1, cool Ar flow rate = 14 L min1 and auxiliary Ar flow rate = 0.7 L min1. Samples were introduced by natural aspiration for improved stability and a peristaltic pump (Gilson Medical Electronics, Middleton, WI) was used to drain the spray chamber with the flow rate set at 0.8 mL min1, a sample uptake time of 2 min and a wash time of 3 min. A blank value (for background subtraction) was acquired once, while samples and standards were acquired 5 times, all for 60 s. Data were collected using an m/z scan range of 60–68 amu, dwell time of 320 ls and 19 channels per amu. Isotope ratios obtained using Cu atomic absorption standards (Spectrosol, BDH; Poole, UK) were used to correct for mass bias. The amount of Cu in the HepG2 cells is proportional to the change in 63Cu/65Cu ratio caused by the 65Cu spiking solution and can be calculated using the Pickup and McPherson equation [12,13]. Therefore, Cu accumulation was calculated by subtracting the total amount of Cu in the HepG2 cells after 1 h incubation with 2 lM CuHis2, from the amount of Cu in HepG2 cells at the beginning of the incubation.
A¼
RðB Qh Þ ðB Ql Þ ; P l ðR P h Þ
where A is the amount of Cu to be determined, Pl is the light isotope natural abundance, Ph is the heavy isotope natural abundance, Ql is the light isotope in spike, Qh is the heavy isotope in spike, B is the amount of spike added, and R is the 63Cu/65Cu ratio measured. 2.6. Statistics Comparisons were carried out on individual measurements in separate experiments. Differences were tested for significance using StudentÕs t-test. Differences were taken as significant at p < 0.05. Fitting data to the Michaelis–Menten equation and linear regression analyses were carried out using non-linear iteration (GraphPad Prism 4.00. San Diego, CA). 3. Results We first determined whether the number of cells on a 57-mm diameter plate would be sufficient to measure the 63 Cu/65Cu ratio accurately and consistently. The natural ratio was determined as 2.185 ± 0.06 (n = 6), demonstrating that the cell number was adequate for accuracy and sensitivity (Figure not shown). Subsequently, we attempted to measure Cu uptake using [65Cu]His2 solution. However, the change in the 63Cu/65Cu ratio was too small to cause a significant alteration to the ratio after 1 h (data not shown). To increase sensitivity, therefore, we increased the natural 63Cu/65Cu ratio in HepG2 cells by incubating the cells with [63Cu]His2, which would allow us to detect small increases in 65Cu levels. After three weeks of culture with 2 lM [63Cu]His2 added to the culture medium, the 65Cu/63Cu ratio changed from 2.18 ± 0.05 to 15.3 ± 1.01 (Fig. 1). Thus, 93.6% of the intracellular Cu was now 63Cu. Long-term incubation with [63Cu]His2 also increased intracellular
Fig. 1. Loading HepG2 cells with [63Cu]His2 changes the 63Cu/65Cu ratio over time. cells were grown and supplemented with 2 lM [63Cu]His2 as described in Section 2. Data are expressed as isotopic ratios and are the means ± SEM of n = 6, combined from 3 experiments. Data with the same superscript do not differ significantly.
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Fig. 2. Cu concentration in [63Cu]His2 treated HepG2 cells is stable after 3 weeks. cells were grown and supplemented with 2 lM [63Cu]His2 before being treated as described in Section 2. Data are the means ± SEM of n = 6, combined from 2 experiments. Data with the same superscript do not differ significantly.
Cu over the first two weeks, but levelling by three weeks (Fig. 2). 65 Cu uptake was then measured. Uptake into the cells was linear over 60 min and temperature dependent (Fig. 3). To test whether 65Cu was acting as a tracer, total
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Fig. 5. 65Cu uptake is concentration dependent. [63Cu]His2 labelled cells were incubated with increasing concentration of [65Cu]His2. For 30 min as described in Section 2. Cu concentrations were calculated as described in Section 2. Data are presented as the means of 6 observations at each concentration. Curve is the line of best fit for the Michaelis–Menten equation.
intracellular Cu levels were measured at each time point. 65 Cu uptake did not change total Cu concentration over 50 min (Fig. 4). Uptake rates were measured at different [65Cu]His2 concentrations in HepG2 cells. Uptakes followed saturation kinetics and were fitted to the Michaelis–Menten equation as described (Fig. 5). The Vmax was calculated to be 33 ± 3 pmol lg DNA1 min1 and the apparent Km was 2.6 ± 0.8 lM (p < 0.01).
4. Discussion
Fig. 3. 65Cu uptake is time and temperature dependent. [63Cu]His2 labelled cells were incubated with 2 lM [65Cu]His2 as described in Section 2. Data are expressed as isotopic ratios and are the means ± SEM of n = 6, combined from 2 experiments.
Fig. 4. Incubating with 65Cu does not increase Cu levels within HepG2 cells 63Cu]His2 labelled cells were incubated with 2 lM [65Cu]His2 as described in Section 2. The left y-axis represents isotopic ratios and the right y axis represents the intracellular Cu concentration. The 63 Cu/65Cu ratio is presented as closed circles while intracellular Cu levels are open circles. Data are the means ± SEM of n = 3. Lines are calculated as described in Section 2.
This paper has demonstrated a new method for measuring Cu uptake into cells in culture. Until recently, virtually all studies have used the radioisotopes 64Cu and 67 Cu [6,14–17]. 67Cu, with its longer half life, has been particularly useful in examining kinetics of Cu in both cell culture and in animal studies. With these methods, uptake was demonstrated to be a carrier-mediated process, in a wide variety of cell types, including hepatocytes, fibroblasts, CHO and K562 cells, and in trophoblast cells from placenta. In animals, the isotope was also used to measure transfer across the placenta and to implicate ceruloplasmin in this process [18–20]. However, as described in the introduction, there are also problems with the use of the labels, and alternative methods have been tested at different times. Solioz and colleagues [21,22] have used Ag as also a substrate for the bacterial homologue of the Menkes transport protein and it has also been used for the diagnosis of the disease. The approach is limited, however, since the isotope is not commercially available. Phen Green FL has been used for detecting divalent metals such as Fe2+, Hg2+, Cd2+, Pb2+ and Ni2+ at submicromolar concentrations [23–25] but can also be used for Cu2+. For example, Chavez-Crooker et al. [26] have used the dye to differentiate between intracellular Cu levels for four types of lobster hepatopancreatic epithelial cells. However, the approach suffers from poor spec-
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ificity and data become complicated when other metals, such as Fe, are present. In this paper, an alternative method of studying Cu uptake has been developed and evaluated. To date, it is the first report describing a stable isotope technique applied to a cell culture model. ICP-MS is one of the widely used contemporary techniques to measure isotope ratio, because it is the most sensitive, accurate and has a high dynamic range. The use of isotope dilution mass spectrometry (IDMS) to measure Cu content in HepG2 cells has several advantages. Partial loss of the analyte after equilibrium of the spike and the sample will not influence the accuracy of the determination. This is of great importance since the samples have to be digested and purified before analysis. Further, physical and chemical interferences are less likely to affect the measurements since they have similar effects on each isotope of the same element. Previous workers have realised that the natural ratio of 63Cu and 65Cu presents problems in using stable isotopes to measure uptake and our own data confirm that, to measure uptake and kinetics, it is first necessary to alter the balance of the two isotopes. This leads to some problems with increased Cu concentrations within the cell, but it seems clear that a stable state is reached by three weeks of incubation. The increase in the ratio of 63 Cu/65Cu (to about 15 within three weeks) far outweighs any disadvantages this may have. As with radioisotope uptake studies, we have shown that Cu uptake was temperature and time dependent [27–29]. Uptake was linear over the time period, demonstrating, as confirmed by actual measurements, that 65 Cu was acting as a tracer and we could derive kinetic parameters for uptake. These values were similar to those already published using radioactive Cu. This method, therefore, though somewhat cumbersome, represents a real option for studying Cu metabolism in different systems. It also introduces the possibility for studying long term metabolism in animals, since it is feasible one could feed them 63Cu as we have done with the cells and then measure uptake and metabolism using 65 Cu as the tracer. Acknowledgement This work was supported by the International Copper Association.
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