ISSN 1607-6729, Doklady Biochemistry and Biophysics, 2017, Vol. 472, pp. 60–63. © Pleiades Publishing, Ltd., 2017. Original Russian Text © E.N. Esimbekova, E.V. Nemtseva, M.A. Kirillova, A.A. Asanova, V.A. Kratasyuk, 2017, published in Doklady Akademii Nauk, 2017, Vol. 472, No. 5, pp. 596–599.
BIOCHEMISTRY, BIOPHYSICS, AND MOLECULAR BIOLOGY
Bioluminescent Assay for Toxicological Assessment of Nanomaterials E. N. Esimbekovaa, b *, E. V. Nemtsevaa, b, M. A. Kirillova b, A. A. Asanovac, and V. A. Kratasyuka, b Presented by Academician I.I. Gitelson July 22, 2016 Received August 17, 2016
Abstract—A new method for assessing biotoxicity of nanomaterials, based on the use of soluble bioluminescent coupled enzyme system NAD(P)⋅H:FMN oxidoreductase and luciferase, is proposed. The results of this study indicate a significant adverse biological effect exerted by nanoparticles at the molecular level. It was found that the most toxic nanoparticles the nanoparticles are based on copper and copper oxide, as well as single-walled carbon nanotubes and multi-walled carbon nanofibers, which are referred to hazard class II. DOI: 10.1134/S1607672917010173
Increasing scope of production and use of numerous new nanomaterials in industrial and economic activity exacerbates the problems of assessing their safety for humans and the biosphere in general [1]. Currently, toxicological studies of nanomaterials are performed on cell lines and laboratory living organisms, including luminous bacteria [2, 3]. However, it should be taken into account that fundamental changes in living organisms under the influence of toxicants occur at the molecular level of organization of the living matter, corresponding to the nanoscale size. Recent published data indicate the importance of assessing the molecular mechanism of effect of nanomaterials [4, 5]. In this study, to assess the potential risks of the use of new materials, we propose to use the bacterial bioluminescent coupled enzyme system NAD(P)⋅H:FMN oxidoreductase + luciferase (R + L) as an object of influence instead of luminous bacteria themselves. The principle of this method of toxicological analysis consists in detecting the toxic properties of test substances by their effect on the bioluminescence parameters of the coupled enzyme system used [6]. The objects of this study were the commercially available carbon nanomaterials—single-walled carboxylated carbon nanotubes k-SWCNT-90A (SWCNT) manufactured at the Uglerod Chg Ltd. (Russia),
multi-walled carbon nanofiber “activated nanocarbon material” (MWCNF) manufactured at the Prankor Ltd. (Russia), and a solution of hydrated C60 fullerene (C60HyFn) manufactured at the Institute of Physiological Active Compounds (Ukraine). SWCNT and MWCNF suspensions were obtained as described in [7] using sodium lauryl sulfate. In addition, we investigated the toxic properties of nanoparticles based on metals and metal oxides, such as nanoparticles of silver, copper, and copper oxide (I), which were synthesized at the Department of Physical and Inorganic Chemistry, Institute of Nonferrous Metals and Materials of Siberian Federal University (Krasnoyarsk). Lyophilized preparations of high-purity enzymes luciferase (EC 1.14.14.3) and NAD(P)⋅H: FMN oxidoreductase (EC 1.5.1.29) were purchased by the Laboratory of Nanobiotechnology and Bioluminescence, Institute of Biophysics, Siberian Branch, Russian Academy of Sciences (Krasnoyarsk). One flask contained 0.5 mg of luciferase obtained from recombinant E. coli strain and 0.18 units of activity of NAD(P)⋅H:FMN oxidoreductase from Vibrio fischeri. Enzyme solutions were prepared in 0.05 M potassium phosphate buffer (pH 7.0). We also used the following reagents: FMN (Serva, Germany), NADH (Gerbu, Germany), and tetradecanal (Merck, Germany). The residual luminescence of the coupled enzyme system R + L was calculated by the formula It/Ic × 100%, where It and Ic are the maximum luminescence intensity of the coupled enzyme system R + L in the presence of the test sample and control solution, respectively. The influence of the test samples was evaluated by the parameters EC50 and EC20, which are the effective concentrations of the active substances
aInstitute of Biophysics, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk bSiberian Federal University, Krasnoyarsk cKrasnoyarsk State Agrarian University, * e-mail:
[email protected]
60
BIOLUMINESCENT ASSAY
BL
Optical density, normalized intensity
1.0
1.0
(a)
0.8
61
BL
(b)
0.8
0.6
0.6 [Ag] [MWCNF]
0.4
0.4 15.9 mg/L
2 mg/L 0.2 0 400
1 mg/L 450
500
550 600 650 Wavelength, nm
0.2 1.59 mg/L 0 400
500 600 Wavelength, nm
Fig. 1. Absorption spectra of (a) MWCNF suspensions and (b) silver nanoparticles. The normalized bacterial bioluminescence emission spectrum in vitro (BL) is shown as a dotted line.
that decreased the activity of the coupled enzyme system R + L by 50 and 20%, respectively. To prevent the bias of the bioluminescent signal by optical effects (scattering and absorption), we studied the absorption characteristics of nanomaterials and then corrected the results obtained in the bioassay in vitro. If the optical density of the test sample exceeded 0.1 in the range 450–600 nm, the bioluminescence intensity values obtained in the presence of nanomaterials were multiplied by the correction coefficient k, which was calculated by the formula [8]
k=
1
(1) , g(λ i ) ⎡ L ⎤ 1 − exp −Di (λ i ) L ⎣⎢ l ⎦⎥ λ ( ) D i =1 i i l where g(λi) is the ratio of the bioluminescence intensity at the wavelength λi to the total bioluminescence intensity at the optical path L and D(λi) is the absorption of nanomaterial solution at the wavelength λi at the optical path length l. The absorption spectra of analyzed samples were recorded with a Cary 5000i spectrophotometer (Agilent Technologies, United States), and the bioluminescence spectrum was recorded with an Aminco Bowman Series 2 spectrofluorometer (Thermo Spectronics, United States). Bioluminescence was measured with a Lumat LB 9507 luminometer (Berthold Technologies, Germany). Measurements of each experimental point were performed in five replicates. Statistical processing of the results was performed using Student’s t test. Differences were considered significant at p ≤ 0.05. The analysis of the optical characteristics of the materials showed that the absorption of nanotubes and nanofibers had no pronounced structure in the visible range and was determined by the light scattering n
∑
()
(
( ))
DOKLADY BIOCHEMISTRY AND BIOPHYSICS
Vol. 472
(Fig. 1a). The optical spectra of the copper nanoparticles had a characteristic surface plasmon resonance band with a maximum at 570 nm [9] and scattering in the range MWCNFs > C60HyFn, which is consis2017
62
ESIMBEKOVA et al.
It/Ic, % 120
It/Ic, % 140
(a)
(b)
120
100
SWCNT 100
MWCNF
80
80 60
*
40
*
60
20
40 *
*
20
0
0.25
0.50 0.75 Concentration, mg/L
0
50
100
150
200 250 [Ag], mg/L
Fig. 2. Dependence of residual luminescence of the soluble coupled enzyme system R + L on the concentration of (a) SWCNTs and MWCNFs and (b) silver nanoparticles. Data are represented as M ± m, n = 5, *p < 0.05 compared to the residual luminescence in the presence of SWNTs.
characteristics of obtained preparations, primarily on the quantity and physicochemical properties of admixtures contained in them. In this paper, the probability values of the toxicological parameter EC50 for C60HyFn were higher than the maximum tested concentration (7.5 mg/L); the EC20 value was 3.7 mg/L, which allowed these nanoparticles to be classified into the hazard class IV.
tent with the results obtained by other biological methods. However, the EC50 values obtained in our study when analyzing the effect of SWCNTs and MWCNFs on the coupled enzyme system L + R were two or three orders of magnitude lower than the values obtained using luminous bacteria as an object of exposure [7, 12]. Thus, the sensitivity of the coupled enzyme system R + L, used at the molecular level to assess the toxicity of carbon nanomaterials, is significantly higher than that of luminous bacteria (organismal level). The most toxic compound is MWCNF: its EC50 and EC20 values were 12 and 4 mg/L, respectively. Published data on the toxicity of fullerenes are most controversial. For example, it was shown in [13] that fullerene suspensions are toxic to fish brain cells at concentrations from 0.003 to 5 µM. At the same time, many authors indicate no adverse effects even at much higher concentrations of fullerenes. Most likely, the comments in [14] are most reasonable, according to which the toxic effect of fullerenes depends on the
The results obtained in the toxicological analysis of nanoparticles formed by copper and copper oxide are consistent with the results of other bioassays [15]. The toxic effects of nanoparticles formed by metals and metal oxide semiconductors are explained by the appearance of metal ions in solution, which are desorbed from the surface of nanoparticles. The toxicological analysis based on bioluminescent enzyme system of luminous bacteria, presented in this work, has a high potential for the development of methods for safety assessment of new nanomaterials in
Values of toxicological parameters EC50 and EC20, determined by the influence of nanoparticles and nanomaterials on the activity of the coupled enzyme system R + L Carbon-based nanoparticles
Nanoparticles based on metals
Parameter EC50, mg/L EC20, mg/L *Hazard class
SWCNTs
MWCNFs
0.16 ± 0.3
0.012 ± 0.003
#
0.04 ± 0.01 II
0.004#
± 0.001 II
C60HyFn
Ag
–
16.7 ± 4.0
3.7 ± 0.7
11.2 ± 2.1
IV
IV
Cu2O
Cu 0.25 ± 0.015 0.064#
± 0.008 II
0.26 ± 0.05 0.086# ± 0.015 II
* Hazard class is specified in accordance with the order of the Ministry of Natural Resources and Ecology of the Russian Federation of April 12, 2014 no. 536 “About the statement of Criteria of reference of waste to the I–V hazard classes by the degree of adverse impact on the environment”, where I class includes very hazardous substances; class II, highly hazardous substances; class III, moderately hazardous substances; and class IV, low-hazard substances. Data are represented as M ± m, n = 5, #p < 0.05 compared to the EC50 parameter. Dash means that measurement was not performed. DOKLADY BIOCHEMISTRY AND BIOPHYSICS
Vol. 472
2017
BIOLUMINESCENT ASSAY
the course of their development, registration, production, trade, use, and disposal in the Russian Federation. In this case, parameters EC20 and EC50, which are commonly used in toxicology, are calculated as characteristics of the detected biological activity of nanomaterials. This method makes it possible to scan a large number of samples in a short time period (analysis takes only 2–3 min), is characterized by technical simplicity, and is not inferior in sensitivity to other standard methods used in toxicology. ACKNOWLEDGMENTS We are sincerely grateful to the staff of the Institute of Physiological Active Compounds (Kharkiv, Ukraine) for providing fullerene samples. This study was supported by the Russian Science Foundation (project no. 16-14-10115). REFERENCES 1. Nanotoxicity: From in vivo and in vitro Models to Health Risks, Sahu, S.C. and Casciano, D.A., Eds., London: Wiley, 2009. 2. Nanotoxicity: Methods and Protocols, Reineke, J., Ed., Meth. Mol. Biol., 2012, vol. 926, pp.1–414. 3. Maurer-Jones, M.A., Gunsolus, I.L., Murphy, C.J., and Haynes, C.L., Anal. Chem., 2013, vol. 85, pp. 3036–3049. 4. Wang, Z., Zhang, K., Zhao, J., Liu, X., and Xing, B., Chemosphere, 2010, vol. 79, pp. 86–92.
DOKLADY BIOCHEMISTRY AND BIOPHYSICS
Vol. 472
63
5. Chang, X.L., Yang, S.T., and Xing, G., J. Biomed. Nanotechnol., 2014, vol. 10, no. 10, pp. 2828–2851. 6. Kratasyuk, V. and Esimbekova, E., Combin. Chem. High Throughput Screening, 2015, vol. 18, no. 10, pp. 952–959. 7. Deryabin, D.G., Aleshina, E.S., and Efremova, L.V., Microbiology (Moscow), 2012, vol. 81, no. 4, pp. 492— 497. 8. Aleshina, E.S., Bolodurina, I.P., Deryabin, D.G., and Kucherenko, M.G., Vestn. OGU, 2010, no. 6, pp. 141– 145. 9. Ghodselahi, T., Vesaghi, M.A., and Shafiekhani, A., J. Phys. D: Appl. Phys., 2009, vol. 42, p. 015308. 10. Kelly, L.K., Coronado, E., Zhao, L.L., and Schatz, G.C., J. Phys. Chem. B, 2003, vol. 107, no. 3, pp. 668–677. 11. Lacowicz, J.R., Principles of Fluorescence Spectroscopy, New York: Springer, 2006. 12. Zheng, H., Liu, L., Lu, Y., Long, Y., Wang, L., Ho, K.P., and Wong, K.Y., Anal. Sci., 2010, vol. 26, no. 1, pp. 125–128. 13. Oberdorster, E., Environ. Health Perspect., 2004, vol. 112, no. 10, pp. 1058–1062. 14. Andrievsky, G.V., Klochkov, V.K., and Derevyanchenko, L.I., Fullerenes Nanotubes Carbon Nanostruct., 2005, vol. 13, no. 4, pp. 363–376. 15. Sizova, E.A., Miroshnikov, S.A., Polyakova, V.S., Lebedev, S.V., and Glushchenko, N.N., Morfologiya, 2013, no. 4, pp. 47–52.
Translated by M. Batrukova
2017
