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Analysis of the distribution pattern of chromium species in single cells Xing Wei, Lin-Lin Hu, Ming-Li Chen, Ting Yang, and Jian-Hua Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03810 • Publication Date (Web): 22 Nov 2016 Downloaded from http://pubs.acs.org on November 22, 2016

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Analytical Chemistry

Analysis of the distribution pattern of chromium species in single cells

Xing Wei, Lin-Lin Hu, Ming-Li Chen*, Ting Yang, Jian-Hua Wang* Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Box 332, Shenyang 110819, China

AUTHOR INFORMATION *Corresponding Authors E-mail address: [email protected] (M.-L. Chen); [email protected] (J.-H. Wang). Tel: +86 24 83688944; Fax: +86 24 83687659 Notes The authors declare no competing financial interest.

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Abstract: The patterning of distribution models for specific heavy metal species in biological cells is highly important for elucidating their effect on single cells or on the lives. For this purpose, the variation of chromium levels and the distribution patterns of chromium species in single cell subjects are investigated by culturing two kinds of native cells, i.e., HeLa cells and MCF-7 cells, in the presence of either Cr(III) or Cr(VI). The analysis of single cells is performed with time-resolved inductively coupled plasma mass spectrometry. We found that total chromium level in the single cells after culturing in a Cr(VI) enriched medium is higher than that for those single cells cultured in a Cr(III) reinforced medium. It is interesting to see that at certain culturing conditions the chromium level in single individual cells increases linearly with Cr(III) concentration in the culture medium, while it increases exponentially with Cr(VI) concentration. This indicated that Cr(VI) is more prone to penetrate into the cells with respect to Cr(III), and after a concentration threshold, a lot more Cr(VI) enters into the interior of HeLa cells or MCF-7 cells.

Keywords: chromium distribution, patterning, single cell analysis, HeLa cells, MCF-7 cells, time resolved ICP-MS.

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INTRODUCTION It is known that a list of elements, including carbon, nitrogen, phosphorus, iodine, magnesium, calcium, potassium, iron, chrome, manganese, zinc, strontium and selenium, are essential for life1, 2. Mineral elements, even at a very low concentration, play a vital role in biological systems because of their deep involvement in various life processes, e.g., the formation of biomolecules, regulation of nucleic acids or gene expression, synthesis of polysaccharides or small organic ligands, anti-oxidation and cancer cell growth3-5. Among the above mentioned elements, chromium is a naturally occurring heavy metal that exhibit several oxidation states, i.e., II, III, IV, V and VI. The environmental chromium exists dominantly in inorganic forms, either as cationic chromium(III) or anionic oxochromium(VI)6, 7. Cr(III) is considered as a beneficial micronutrient in human diet, it promotes the binding of insulin to insulin-sensitive cells which improves their function, and exhibits impact on the metabolism of carbohydrates and lipids8, 9. However, Cr(III) may turn to toxic for living organisms at high levels in some evidences, where higher concentration of Cr(III) causes oxidative DNA damage and mutagenic damage in cell culture6, 10, 11, in addition to sterility, lethal mutation and cancer in experimentation animals12, 13. On the contrast, Cr(VI) is recognized as highly toxic species and carcinogenic to human beings by EPA and as Class I carcinogenic species by IARC (the International Agency for Research on Cancer) and regulated by the Dangerous Substances Directive (67/548/EEC) for being highly toxic14-16. The analysis of low level mineral elements in biological samples is generally performed with atomic spectrometry17-21 and inductively coupled plasma mass spectrometry (ICP-MS)22-24. The elemental analysis of single biological cell is nowadays a highly challenging subject4. In commonly used analytical strategies the biological cells are digested for measurement, and the results are presented in data of the average amounts of an element or several elements in cells. In this analysis mode the concentration information for the 3



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element of interest in some particular single cells is lost25, while such information is definitely much more important for elucidating the biological impact of the element. Recently, ICP-MS had been introduced to analyze the intracellular elements at single cell level23, 26, 27. With time resolved ICP-MS, the frequency of signal is proportional to the density of cells in cell suspension, and the signal intensity is directly related to the intracellular level of the element of interest28. The theoretical study of single particle analysis by ICP-MS revealed a close relationship between the signal intensity and the time-resolved signal frequency with the particle size and its concentration in the initial colloidal suspension29. This has been further demonstrated by the analysis of intracellular quantum dots in a single cell level28. The simultaneous cell counting and analysis of constituent metals in single cells has also been carried out with time-resolved ICP-MS26, where106 atoms per cell for the selected isotope can be detected, and the quantification of metals in biological cells was performed using polydisperse metal oxide particles for calibration. So far, the information on heavy metal distribution patterning in biological cells is generally unavailable, although such information is highly desired and of importance. In the present study, two types of single cells, i.e., HeLa cells and MCF-7 cells, were chosen for the investigation of chromium level and their uptake behaviors for chromium(III) and chromium(VI), as well as the distribution patterning of the two chromium species in these cells. The cell suspension is directly mobilized into aerosols followed by detection with timed resolved ICP-MS (TR-ICP-MS). It revealed significant difference for the chromium levels and distribution patternings of Cr(III) and Cr(VI) in HeLa cells and MCF-7 cells by culturing in either Cr(III) or Cr(VI) enriched medium. This observation provides very useful information for further elucidating the biological impact of heavy metal species.

EXPERIMENTAL SECTION Materials. High-glucose DMEM media and Trypsin-EDTA solution were purchased 4


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from Thermo Fisher Scientific (USA). Fetal bovine serum (FBS) was obtained from CLARK Bioscience (USA). Dulbecco’s phosphate buffered saline (PBS), nitric acid and hydrogen peroxide were the products of Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Chromic nitrate and potassium dichromate standards were achieved from National Research Center for Certified Reference Materials (Beijing, China). DMSO was provided by Sigma-Aldrich Co., LLC. (USA). De-ionized water of 18 MΩcm-1 from Heal Force Bio-Meditech Holdings Limited (Shanghai, China) was used throughout, and all the reagents used are at least of analytical reagent grade. Preparation of cells. Living HeLa cells were maintained in a DMEM medium containing 10% fetal bovine serum, 100 units mL-1 penicillin and 100 µg mL-1 streptomycin. Cell culture was performed in a complete medium at 37°C under 5% CO2. MCF-7 cells were cultured in the same medium, including fetal bovine serum, penicillin and streptomycin. For the investigation of chromium uptake behaviors, various concentrations of trivalent and hexavalent chromium solutions at 50, 100, 250 and 500 µg/L in DMEM media were used for cell culture. After culturing for 12 h, the cells were digested with trypsin, centrifuged at 800 rpm for 3 min and washed with PBS saline solution for three times to prepare cell suspension. For the purpose of single cell analysis, cell suspension was prepared in PBS saline solution. Afterwards, the cell suspension was sprayed into aerosol by using a V-groove nebulizer and introduced into ICP for time-resolved ICP-MS measurement. For the measurement of total chromium in cells, the fixed cells were digested with 4 mL of nitric acid and 1 mL of hydrogen peroxide (30%) at 180°C for 4 min. The digest was then diluted with 2% HNO3 and determined by ICP-MS. At the same time, a blank control solution was prepared similarly for comparison and calibration. Cell number was counted by haemocytometer (Shanghai Qiujing Biochemical Reagents Instrument Co., Shanghai, China). The cell suspension was firstly diluted to different cell 5



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number density with PBS saline solution in a 15 mL centrifuge tube, which was then allowed to count cell number under an inverted biological microscope (Nikon Eclipse TS100, Tokyo, Japan) at 100-fold magnification. The error in cell counting was controlled within an error range of 5×105 mL-1, the increase of counts intensity and the overlap of cell signals indicated that multiple cells were detected. For further investigations, a cell number density of 3×105 mL-1 was employed. 5000

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Figure 3. Determination of chromium level in Hela cells. (a) the response of signal events from PBS blank solution; (b) the response of signal events from native HeLa cells cultured in PBS saline solution; (c) the response of signal events from HeLa cells cultured in the medium containing 100 µg L-1 Cr(III) and cleaned by PBS saline solution; (d) the frequency histograms of 53Cr in Hela cells after treatment with 100 µg L-1 Cr(III) for12 h. Histograms are fitted to lognormal distribution.

Analysis of chromium in single cells. The contents of chromium in single HeLa cells were analyzed by time-resolved ICP-MS. Figure 3a-c showed the temporal profiles of chromium in the blank (PBS saline solution), untreated HeLa cells cultured in PBS saline solution and HeLa cells cultured in a medium with chromium(III) spiked. Figure 3a-b illustrated slightly different signal events for blank and native Hela cells, which indicated that there seems to be very low level of endogenous chromium in HeLa cells. It was regrettable that it was too low to be quantifiable by the present system. On the other hand, the signal events of HeLa cells in Figure 3c showed a logarithmic growth after culturing in a medium with 100 µg L-1 chromium (III). By picking up the red marked signal in Figure 3c as an example for performing single cell analysis, the chromium content in this particular cell was calculated to be 0.095 pg (details on the calculations were given in the following section). Figure 3d showed an approximately lognormal distribution of chromium content in Hela cells cultured in a medium containing 100 µg L-1 chromium (III). This observation well illustrated the difference of chromium distribution in the single cells.

Chromium distribution pattern in single HeLa cells and MCF-7 cells. By time-resolved ICP-MS with a 10 ms dwell time, spike signals corresponding to individual HeLa cell events were successfully detected for chromium on introduction of the cell 11



suspension, where a single signal event corresponds to a single cell, and the signal intensity is directly related to chromium level in the individual cells. Figure 4 illustrated the temporal profile of chromium in HeLa cells after cultured with various concentrations of chromium(III). It could be seen that a clear increase on the signal intensity was observed with the increase of chromium concentration in the cell culture medium. Meanwhile the intensity of mass spectrum varies widely even when the injected cell suspension was cultured in the same medium and with identical concentration of chromium, and the signal intensity from one particular single cell was very different from that for another cell. That well indicated the specificity of single cell behavior. 5000

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100, 250, 500 µg L-1 chromium (III) for 12 h (a-d), and the variation of signal with chromium concentration in the culturing medium (e) (Fitting curve: y=3.099x+2439, R2=0.9909).

By choosing the maximum response of one particular cell event in the temporal profile as marked with the red triangular , the response from single cells versus chromium concentration in the culture medium was plotted (Figure 4f), where a linear relationship was achieved between the response from single cells and the concentration of chromium within a range of 50-500 µg L-1. The analytical data obtained by time resolved ICP-MS can provide the distribution patterns of chromium in individual cells, which closely related to the cell variability32. According to the corresponding chromium ion current chromatogram, the frequency histograms of chromium in Hela cells after culturing in a trivalent chromium enriched medium could be readily achieved (Figure 5), that was the plot of number of events versus log(intensity of 53Cr)28. An increase of the chromium contents in single cells is observed after cultured with trivalent chromium, and the chromium levels in single cells cultured with different concentrations of trivalent chromium were also profoundly different. By plotting the maximum value of lognormal distribution, i.e., log(intensity of 53Cr), versus chromium concentration in the culture medium (Figure 5e), a linear relationship was observed which was consistent with that discussed in the response of one particular cell event. 1.0

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Figure 5. The frequency histograms of chromium in Hela cells after treatment with trivalent chromium at 50 µg L-1 (a), 100 µg L-1 (b), 250 µg L-1 (c), 500 µg L-1 (d) for 12 h, and the variation of maximum distribution with Cr(III) concentration in the culturing medium (e) (Fitting curve: y=2.300×10-5x+2.301, R2=0.9826).

Chromium(VI) was further employed for performing similar studies as those for chromium(III). Hela cells were first cultured in a Cr(VI) enriched medium for 12 h, and then 53

Cr isotope in single HeLa cells was measured by time reserved ICP-MS system with a cell

number density of 3×105 mL-1. The results were illustrated in Figure 6. It indicated that an increase of chromium level was observed in the HeLa cells with the increase of Cr(VI) concentration in the culture medium. It was also seen that with a same concentration of chromium in the culture medium, either Cr(III) or Cr(VI), the chromium level in HeLa cells cultured from Cr(VI) medium was much higher than that for those cells cultured from a Cr(III) medium. In Figure 4a, the chromium level in HeLa cell cultured in 50µg L-1 Cr(III) medium (as marked by the red triangle) was 0.116 pg, while a chromium level is 0.304 pg was observed for the cell cultured in Cr(VI) medium of a same concentration (red triangle marked in Figure 6a). Unlike the HeLa cells cultured in Cr(III) medium, where the response from single cells is increased linearly with Cr(III) concentration in the culture medium, while the response for the Hela cells cultured in Cr(VI) medium exhibited an exponential increase with Cr(VI) concentration in the culture medium within 50-500 µg L-1. This indicated that Cr(VI) was more readily to penetrate into the cells with respect to Cr(III), and after a concentration threshold, a lot more Cr(VI) enters into the interior of HeLa cells.

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Figure 6. Temporal profile of 53Cr in HeLa cells after cultured at Cr(VI) concentration of 50, 100, 250, 500 µg L-1 for 12 h (a-d), and the variation of signal with chromium (VI) concentration in the culturing medium (e) (Fitting curve: y=-1716.178+6465.215×exp(0.422×10-2x), R2=0.9999). . Similarly, distribution patterns of chromium in individual cells after cultured in Cr(VI) medium was investigated, by plotting the number of events versus log(intensity of 53Cr), the corresponding log(intensity of 53Cr) versus chromium(VI) concentration in the culture medium (Figure 7). In comparison with the distribution patterns of chromium in Hela cells cultured in Cr(III) (Figure 5), a difference was encountered in the chromium levels in single 15



Hela cells cultured in different concentrations of Cr(VI), and the log(intensity of 53Cr) of cells was higher than those cultured in Cr(III) medium. Another difference was that the plotting of the maximum value of lognormal distribution versus chromium concentration in culture medium was not linear, it was an exponential function, which was consistent with that discussed in the response of one particular cell event. 1.0

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Figure 7. The frequency histograms of chromium in Hela cells after treatment with hexavalent chromium at 50 µg L-1 (a), 100 µg L-1 (b), 250 µg L-1 (c), 500 µg L-1 (d) for 12 h, and the variation of maximum distribution with Cr(VI) concentration in the culturing medium (e) (Fitting curve: y=2.275+0.079exp(0.433×10-2x), R2=0.9997).

At a number density of 3×105 mL-1 and a dwell time of 10 ms, the temporal profiles of chromium in another cell line, i.e., MCF-7 cells, were investigated after cultured in either Cr(III) or Cr(VI) enriched medium, and the results were illustrated in Figure S1 and Figure S2. For both cases, an increase on the signal intensity was observed with the increase of Cr(III) or Cr(VI) concentration in the culture medium. In addition, very different mass spectrum was recorded even for cell suspension cultured with same concentration of chromium in the culture medium, and the signal intensity from one particular single cell varies from the others. Similar to Hela cells, when plotting the response from single cells versus Cr(III) or Cr(VI) concentration in the culture medium, a linear relationship was achieved for Cr(III), while an 16


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exponential increase was observed for Cr(VI). The distribution patterns of chromium level in individual MCF-7 cells after cultured in Cr(III) or Cr(VI) enriched medium were illustrated in Figure S3 and Figure S4. The results clearly indicated the similar variation trends of chromium level in the individual cells were observed, e.g., chromium level increased linearly with the increase of Cr(III) concentration in the culture medium, while an exponential increase of chromium level was observed for those cells cultured in a Cr(VI) enriched culture medium. The above observations illustrated that Hela cells and MCF-7 cells exhibited similar behaviors for chromium uptake, and similar distribution patterns of chromium in both cell lines were identified. It is known that Cr(III) plays an indispensable role in lipid, glucose and protein metabolism, and it is identified as an active component of glucose tolerance factor (GTF), which could bind insulin to receptor sites and improves the efficacy of insulin as a cofactor36, 37

. Cr(VI) can readily permeate through the sulfate transport system followed by interaction

with protein and nucleic acid38, 39, which leads to rapid penetration into the cell interior and therein causes adverse effect to the cells. This is also defined as the source of various cancer diseases36. This indicated that different existing states and biological interaction mechanisms of Cr(III) and Cr(VI) in cells correspond to different behaviors of the cells. With respect to Cr(III), Cr(VI) is more likely to enter into the cell interior. On the other hand, many reduction sites in the cell may reduce Cr(VI) to Cr(III) or other low valence chromium, and causing further accumulation of Cr in cells. 3.5 Mapping of chromium in Hela cells by culturing in Cr (III) or Cr (VI) enriched media It was hypothesized that the signal intensity in ICP-MS detection was proportional with the mass of the element of interest in a single cell26 by assuming that the element in the cells 17



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and in aqueous solution go through a same process of atomization and ionization in the high temperature plasma1, 30. It means the signal intensity of mass spectra depends only on the mass of the elements introduced into the plasma. Thus standard calibration curve could be used for the quantification of chromium in the individual cells with detection by time resolved ICP-MS. For the purpose of comparison, acid digestion was also adopted for acquiring the average chromium level in cells, by following identical cell culture conditions. The mass of chromium in single cells was calculated by the following equation, with mc as the mass of chromium in a single cell, c as chromium concentration in the single cell obtained by calibration curve, and V as the volume of a single cell. The cells were assumed to be single spheres, with diameter of 15 µm. mc = c × V The results Table 1 indicated that after culturing with chromium(III) or chromium(VI) enriched medium, a clear increase of chromium level was observed in single cells in comparison with native Hela cells, and a large span of chromium level was encountered for single individual cells. On the other hand, however, the average chromium level obtained by single cell analysis with time resolved ICP-MS was consistent with that of the total average level as obtained by analyzing the cell digest. However, the single cell average chromium level is significantly different from that achieved by acid digest. The digestion average reflects the average level of a whole cells population, while the single cell average reflects the differences between individual cells. Table 1. Chromium levels in HeLa cells by culturing with Cr(III) or Cr(VI) enriched culture media 53

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(con. span)

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(0.026-0.304) 0.112±0.012

(0.035-0.126) 250

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(0.072-0.397) 0.136±0.011

(0.029-0.150) 500

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(0.027-0.182)

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(0.174-2.536)

ND: chromium level in native Hela cells is not quantifiable

CONCLUSIONS In the present study, the level of chromium in HeLa cells and MCF-7 cells subject to culture in Cr(III) or Cr(VI) spiked culture medium were thoroughly investigated with time resolved ICP-MS, and based on which the distribution patterning of chromium in both kinds of cell lines were elucidated. It indicated that the chromium level in individual cells increased linearly with the concentration of Cr(III) in the culture medium, while an exponential increase trend was observed for those single cells cultured in a Cr(VI) enriched culture medium. The present procedure has the potential for the measurement the metal species in single cells, for the purpose of inspecting specific behavior of single cells and the biological impact of metal species on the behavior or biological function of the single cells. The observations herein provide very important information for further studies in the fields of metallomics, disease control drug treatment and environmental toxicity assessment for metal species.

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ACKNOWLEDGEMENTS Financial support from the Natural Science Foundation of China (21675019, 21375013, 21235001, 21475017), and Fundamental Research Funds for the Central Universities (N140505003, N141008001) are highly appreciated.

Supporting Information Available: ICP-MS operating parameters; Temporal profile of 53Cr in MCF-7 cells after cultured in medium containing 50, 100, 250, 500 µg L-1 chromium (III) for 12 h, and the variation of signal with chromium concentration in the culture medium; Temporal profile of 53Cr in MCF-7 cells after cultured at Cr(VI) concentration of 50, 100, 250, 500 µg L-1 for 12 h, and the variation of signal with chromium(VI) concentration in the culture medium; The frequency histograms of chromium in MCF-7 cells after treatment with trivalent chromium at 50 µg L-1, 100 µg L-1, 250 µg L-1, 500 µg L-1 for 12 h, and the variation of maximum distribution with Cr(III) concentration in the culture medium; The frequency histograms of chromium in MCF-7 cells after treatment with hexavalent chromium at 50 µg L-1, 100 µg L-1, 250 µg L-1, 500 µg L-1 for 12 h, and the variation of maximum distribution with Cr(VI) concentration in the culture medium.

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