- Email: [email protected]
Is it possible to enhance the organism’s resistance to toxic effects of metallic nanoparticles?
Toxicology 337 (2015) 79–82
Contents lists available at ScienceDirect
Toxicology journal homepage: www.elsevier.com/locate/toxicol
Letter to the Editor Is it possible to enhance the organism’s resistance to toxic effects of metallic nanoparticles?
Metallic nanoparticles, both engineered and generated as a byproduct of metallurgical and welding technologies, can be listed among the most dangerous occupational and environmental hazards. Although our studies summarized by Katsnelson et al. (2014a) demonstrated a high efficiency of the organism’s defenses against such nanoparticles’ impact, safe levels of the latter should be very low if possible at all, due to these particles’ especially high toxicity and seemingly obligatory genotoxicity. Therefore we deemed worthwhile to try and enhance the natural resistance to their adverse health effects with the help of so called bio-protectors. This idea was based on our long-term experience in a successful bio-protection of the organism against many other toxics, including some mineral microparticles (Katsnelson et al., 2014b). The principle organism-level mechanisms of this bioprotection (as we see them based on this experience and theoretical premises) are schematically presented by a flow-chart (Fig. 1). In general, to protect a mammalian organism against occupational or environmental toxic impacts one can use: (a) bio-protectors aimed primarily at increasing the effectiveness of natural mechanisms of bio-transformation and elimination of toxics, and thus, at reducing the dose of a harmful substance retained in the organism and especially in the target organs (on our scheme designated as “toxicokinetic effects”); (b) bio-protectors aimed at enhancing the functional reserves at all levels of the organism affected by toxic substance; at increasing the effectiveness of repairing and compensatory processes; and at using physiological and toxicological antagonisms (on the scheme collectively designated as “toxicokodynamic effects”). However, these two modes of action are usually interrelated and interdependent, as it is schematically shown with the reciprocally directed arrows. Indeed, when reducing retention of a toxic substance in the organism and especially in target organs, a bio-protector inhibits the development of a pathological process (thus, a bio-protector of a primarily toxicokinetic action produces a beneficial toxicodynamic effect). On the other hand, a primary enhancement of the resistance to the damaging impact of a toxic on the cells and organs that control the processes of its elimination or detoxication (pulmonary macrophages, liver, kidneys) maintains the effectiveness of these processes and, thus, reduces the retention of this toxic in the organism (so we deal with a beneficial
http://dx.doi.org/10.1016/j.tox.2015.09.001 0300-483X/ ã 2015 Elsevier Ireland Ltd. All rights reserved.
toxicokinetic effect of a toxicodynamic bio-protector). Such bilateral interdependence of toxicokinetic and toxicodynamic effects is pronounced to a varying degree in response to the action of different harmful substances but, on the whole, can be considered as a consistent pattern. The flow-chart shows also that both toxicokinetic and toxicodynamic bio-protectors can be: more or less specific with regard to a particular toxic of a particular range of toxics if bioprotection interferes with the mechanisms of toxicokinetics and toxicodynamics pertaining just to these toxics; predominantly non-specific, if their effect is realized through such integral responses at the organism level as the Selye’s general adaptation syndrome or the related but still distinct concept of “non-specifically enhanced resistance” developed by the school of late Nikolay Lazarev, an outstanding Russian toxicologist and pharmacologist. However, one and the same bio-protector may in different cases either render a largely specific effect or help the organism mainly as an agent enhancing its nonspecific defenses and thus decreasing sensitivity or increasing resistance to harmful exposures (see respective blocks and links of the same scheme). Bio-protectors acting through not fully identical mechanisms proved, in our experiments, most effective when administered not separately but in combinations (“bio-protective complexes”, or BPCs). Keeping in mind the further possible usage of bio-protectors for humans, we test in animal experiments only those substances which, on their own, are innocuous when applied for a long time in preventively effective doses. During more than 30 years, on a lot of chronic or subchronic experimental intoxications with different inorganic and organic chemicals acting separately or in different combinations (modelling actual occupational and environmental combined chemical exposures) we demonstrated bio-protective efficacy of some amino acids; vitamins (such as A, B1, E, C) and essential trace elements (selenium, iodine, copper etc) or multivitamin-multimineral preparations; fish oils rich in polyunsaturated fatty acids predominantly of the omega 3 class; pectin enterosorbents; calcium and iron supplements. In many instances, the beneficial effects and intrinsic safety of the experimentally tested BPCs were then confirmed in field investigations on volunteers as a prerequisite to a wider prophylactic usage. (For an overview of all this scientific and medico-social activity of ours see Katsnelson et al., 2014b.) As concerns the bio-protection against adverse effects of metallic nanoparticles, up to now we chose on theoretical premises and tested experimentally three BPCs protecting from nano-silver (Katsnelson et al., 2013), nano-copper oxide (Privalova
80
Letter to the Editor / Toxicology 337 (2015) 79–82
Enhancing the organism’s general defensive reactivity
Non-specific action
Toxi cokinetic Toxicokine effects Decrea Decreasing sing the sensitivity or increasing the resistance to toxic(s)
Bio-protectors Specific action
Toxicodynamic effects
Fig. 1. A mechanistic flow-chart of the anti-toxic bioprotection.
et al., 2014), and a combination of nano-nickel oxide plus nanomanganese oxide (Minigalieva et al., 2015). Different in some important details depending on specific toxicodynamic and toxicokinetic mechanisms of different metals’ toxic action, the composition of all three BPCs still have much in common and, we believe, similar BPCs should comprise most of the same components when further looking for bioprotection against other metallic nanoparticles. These components of choice are: (1) Glutamate as an effective cell membrane stabilizer acting through the intensification of ATP synthesis under exposure to the damaging action of various cytotoxic particles e.g. (Morosova et al., 1984) and, at the same time, as one of the precursors of glutathione, which is a powerful cell protector against the oxidative stress, the latter being presumably one of the key mechanisms of virtually all metallic NPs’ cytotoxicity and genotoxicity (Fröhlich, 2013). In addition to these nonspecific and almost universal bio-protective effects of glutamate, we believed that its administration might specifically increase resistance to neurotoxicity of manganese, lead and some other metallic nanoparticles due to glutamate’s major role in transmitting excitatory signals in the mammalian central nervous system and thus involvement in most aspects of normal brain functioning. Indeed, it is known, for instance, that manganese impairs the expression and function of the main glutamate transporters in astrocytes (Karki et al., 2013) and that lead interferes with glutamate release in hippocampus (White et al., 2007). We thought that additional glutamate supply to the brain might partly compensate for these adverse effects.
(2) The other two glutathione precursors: glycine and cysteine (the latter in a highly active and metabolically well available form of N-acetylcysteine), taking into consideration both the above-mentioned general important role played by the oxidative stress as a mechanism of metallic nanoparticles toxicity and some experimental data demonstrating that glutathione deficiency potentiates metals toxicity – e.g. manganese-induced damage to the rat striatum and brainstem (Desole et al., 1994). (3) Other agents of the organism’s anti-oxidant system (vitamins A, E, and E, and selenium). (4) omega-3 polyunsaturated fatty acids whose intracellular derivatives are eicosanoids that activate DNA replication and thus play an important part in its repair. (5) Iodine, taking into consideration well-known disturbances of the thyroid function caused by lead, manganese and some other intoxication (6) Pectin enterosorbent as an agent that hinders the reabsorption of toxic metals excreted into the intestines with bile (which, again, is of special importance for manganese as it is excreted predominantly by this route). In all the studies we found that, as expected, the toxicity and even genotoxicity of metallic nanoparticles could be really attenuated against the background of adequately composed BPCs. It should be understood that we do not claim to be the first to show a possibility of inhibiting some metallic nanoparticles’ toxicity with this or that agent aimed at a certain mechanism of this toxicity. However, this possibility had been demonstrated by others, as a rule, in experiments in vitro and was used as an
Table 1 Some morphometric indices for tubular epithelium damage in the kidneys of rats after repeated intraperitoneal injections of some metallic oxides nanoparticles with or without background oral administration of a BPC (X s.e.). Groups of
Brush border loss (% lengthwise)
Epithelial desquamation
Rats given NiO nanoparticles + Mn3O4 nanoparticles Water (control) Nanoparticles Nanoparticles BPC
5.44 0.9 12.33 2.3a 7.08 1.7
0.00 0 2.43 1.0a 0.00 0b
CuO nanoparticles Water (control) Nanoparticles Nanoparticles BPC
5.39 0.42 8.36 0.76a 5.98 0.46b
0.33 0.13 1.16 0.38a 0.98 0.35
a b
(% Lengthwise)
Statistically significant difference from the control group. From the group given nanoparticles without the BPC (p < 0.05 by Student’s t-test).
Letter to the Editor / Toxicology 337 (2015) 79–82
evidence for the importance of the said mechanism rather than a foundation of a holistic bio-protective system. Meantime, it is just such a prevention-oriented system (a “biological prophylaxis” in our terminology) that is the goal of our mechanistically substantiated approach, and we believe we were the first indeed who began to investigate effects of bio-protectors against metallic nanoparticles on the whole mammalian organism in chronic or subchronic animal experiments. A high beneficial efficacy of this bioprotection was assessed in those experiments with a lot of indices, but here we propose not to repeat original publications referred to above but only to illustrate this efficacy with several examples. Thus, all the above-mentioned nano-metals proved markedly nephrotoxic exerting, in particular, a significant damage to epithelial cells of proximal convoluted renal tubules. On the PAS-stained histological preparations of kidneys In rats exposed intraperitoneally during 6–7 weeks to those nanoparticles we saw marked degenerative and necrotic changes of these cells up to their disappearance with partial destruction of the brush border, while in rats exposed to the same nanoparticles against the background BPC administration a marked alleviation of the tubular damage was manifest. In Table 1 we give respective morphometric results obtained in the experiment with nickel oxide and manganese oxide nanoparticles (Minigalieva et al., 2015) and similar results of the experiment with copper oxide nanoparticles (Privalova et al., 2014). Another adverse effect proved specific for the toxicity of copper oxide and of manganese oxide nanoparticles was a marked damage to some specialized structures of the brain (especially, to the striatum and the hippocampus), and this damage also was significantly attenuated by the BPC. Examples are given by Fig. 2 (Privalova et al., 2014) and Table 2 (Minigalieva et al., 2015).
81
Fig. 2. Number of cells without a nucleolus per 100 Golgi cells in nucleus caudatus of rats exposed (A) to water (Control); (B) to water suspension of copper oxide nanoparticles; (C) to the same against the background of bioprotective complex (BPC) administrations, (D) to the BPC only (Average values with 95% CI). Differences are statistically significant between (B) and (A); (C) and (B) (p < 0.05 by Student’s ttest).
As an illustration of the efficacy of bioprotection against less specific toxic effects of metallic nanoparticles we give some results of the same experiment with nickel plus manganese oxides nanoparticles (Table 3). This Table demonstrates as well that a BPC which has significantly attenuated the adverse shifts caused by nanoparticles has itself no effect on the respective indices. This is quite typical for all our experiments. It should be stressed also that a significant attenuation of toxic effects was not obligatorily associated with a decrease in target organs’ load with a toxic metal (including that in the form of nanoparticles), thus demonstrating an important role of predominantly toxicodynamic mechanisms of bio-protection. Nevertheless, a beneficial toxicokinetic effect of BPCs was also observed in some cases, as illustrated by Table 4 (Privalova et al., 2014). The last but not the least result we would like to stress is that, while all the nanoparticles studied by us up to now were more or
Table 2 Some morphometric indices for the state of rat’s brain after repeated intraperitoneal injections of NiO and Mn3O4 nanoparticles with or without background oral administration of a BPC (X s.e.). Golgi neurons (%%)
Rats injected with water (control)
Rats injected with nanoparticles
Rats injected with nanoparticles and administered a BPC
Nucleus caudatus Without a nucleolus With a distinct centrally located nucleolus
30,50 2,77 25,12 1,16
60,30 2,26a 12,35 0,95b
37,15 2,89b 23,28 1,09b
Hippocampus (CA 1) Without a nucleolus With a distinct centrally located nucleolus
30,50 2,30 46.4 2.92
70,40 3,75a 11.0 1,13a
41,30 2.14a,b 30.5 1.96a,b
a b
Statistically significant difference from the control group. From the group given nanoparticles without the BPC (p < 0.05 by Student’s t-test).
Table 3 Some functional indices for the condition of rat after repeated intraperitoneal injections of NiO and Mn3O4 nanoparticles and/or oral administration of a BPC (x s.e.). Index
Groups given: Water (control)
Nanoparticles
Nanoparticles and the BPC
BPC
Leukocytes, 103/ ml Bilirubin in blood serum, mmol/L Albumin in blood serum, g/L Diuresis, ml Urine relative density Creatinine in urine, mmol/L d–ALA in urine, mmol/day
4.3 0.4 2.02 0.4 46.6 0.8 32.7 1.8 1.017 0.001 1.09 0.1 0.23 0.07
6.1 0.5a 1.15 0.1a 38.6 0.8a 17.9 2.9a 1.023 0.001a 1.8 0.2a 0.54 0.13
5.7 0.6b 1.5 0.1b 41.8 1.1b 30.2 2.7b 1.019 0.001b 1.2 0.1b 0.22 0.02b
4.3 0.4 1.7 0.1 47.3 1.2 31.2 4.5 1.019 0.001 1.2 0.1 0.25 0.08
a b
Statistically significant difference from the control group. From the group given NiO-NPs + Mn3O4-NPs (without the BPC) (p < 0.05 by Student’s t-test with Bonferroni correction).
82
Letter to the Editor / Toxicology 337 (2015) 79–82
Table 4 Copper content of some organs (mcg/g of dry-frozen tissue) in rats after repeated intraperitoneal injections of copper oxide nanoparticles and/or oral administration of a BPC (x s.e.) Group of rats given
Kidneys
Liver
Spleen
Brain
Water (control) Nanoparticles Nanoparticles and BPC BPC
42.4 2.9 62.5 7.1a 59.4 10.0 50.4 5.6
12.2 2.4 28.8 6.3a 22.1 3.5a 10.6 0.3
22.5 2.1 24.2 1.5 18.0 2.5b 25.3 2.2
18.9 0.7 21.5 1.7 18.8 1.4 20.8 1.5
a b
Statistically significant difference from the control group. from the group given nanoparticles without the BPC (p < 0.05 by Student’s t-test).
Table 5 Coefficients of the genomic DNA fragmentation in rats exposed to subchronic administration of silver nanoparticles with or without BPC based on the results of RAPD test (x s.e). Group of given:
Water (controls) Nano-particles Nanopar-ticles and BPC a b
Tissues Liver
Bone marrow
Spleen
Kidney
Nuclea-ted cells of blood
0.40 0.001 0.46 0.002a 0.41 0.011b
0.39 0.003 0.46 0.032a 0.37 0.003a,b
0.38 0.002 0.46 0.001a 0.42 0.003a,b
0.39 0.003 0.42 0.008a 0.40 0.006a,b
0.38 0.001 0.41 0.012a 0.39 0.007
Statistically significant difference from the control group. From the group given nanoparticles without the BPC (p < 0.05 by Student’s t-test).
less genotoxic, all the three tested BPCs significantly attenuated this most worrying effect. Table 5 demonstrates it by the example of silver nanoparticles (Katsnelson et al., 2013). To conclude, we maintain that the notoriously high toxicity of metallic nanoparticles make it necessary not only to try and keep respective dangerous exposures as low as possible but also to look for ways of increasing the organism’s resistance to them. As we believe, we have demonstrated that against the background of adequately composed combinations of some bioactive agents used in innocuous doses, the integral and specific toxicity of metallic nanoparticles and even their genotoxicity could be markedly attenuated. Therefore we strongly recommend to further develop this vector of nanotoxicological research. Our previous positive experience in organizing first a selective and then a large-scale “biological prophylaxis” of adverse health effects of other toxicants makes us expect that it would be no less practicable and effective in the field of nanotechnologies as well. References Desole, M.S., Miele, M., Esposito, G., Migheli, R., Fresu, L., De, N., atale, G., Miele, E., 1994. Dopaminergic system activity and cellular defense mechanisms in the striatum and striatal synaptosomes of the rat subchronically exposed to manganese. Arch. Toxicol. 68, 566–570. Fröhlich, E., 2013. Cellular targets and mechanisms in the cytotoxic action of nonbiodegradable engineered nanoparticles. J. Curr. Drug Metab. 14, 976–988. Karki, P., Lee, E., Aschner, M., 2013. Manganese Neurotoxicity: a Focus on Glutamate Transporters. Ann. Occup. Environ. Med. 25, 4. doi:http://dx.doi.org/10.1186/ 2052-4374-25-4. Katsnelson, B.A., Privalova, L.I., Gurvich, V.B., Makeyev, O.H., Shur, V., Ya Beikin YaB. Sutunkova, M.P., Kireyeva, E.P., Minigalieva, I.A., Loginova, N.V., 2013. Comparative in vivo assessment of some adverse bio-effects of equidimensional gold and silver nanoparticles and the attenuation of nanosilver’s effects with a complex of innocuous bioprotectors. Int. J. Mol. Sci. 14, 2449–2483. Katsnelson, B.A., Privalova, L.I., Gurvich, V.B., Kuzmin, S.V., Kireyeva, E.P., Minigalieva, I.A., Sutunkova, M.P., Loginova, N.V., Malykh, O.L., Yarushin, S.V., Soloboyeva, J.I., Kochneva, N.I., 2014a. Enhancing population’s resistance to toxic exposures as an auxilliary tool of decreasing environmental and occupational health risks (a self-overview). J. Environ. Protect. 5, 1435–1449. Katsnelson, B.A., Privalova, L.I., Sutunkova, M.P., Gurvich, V.B., Loginova, N.V., Minigalieva, I.A., Kireyeva, E.P., Shur, V.Y., a, Shishkina, E.V., 2014b. Some inferences frm in vivo experiments with metal and metal oxide nanoparticles: the pulmonary phagocytosis response, subchronic systemic toxicity and genotoxicity, regulatory proposals, searching for bioprot ectors (a selfoverview). Int. J. Nanomed. 10, 3013–3029.
Minigalieva, I.A., Katsnelson, B.A., Privalova, L.I., 2015. Attenuation of combined nickel (II) oxide and manganese (II,III) oxide nanoparticles’ adverse effects with a complex of bioprotectors. Int. J. Mol. Sci. 16, 22555–22583. Morosova, K.I., Katsnelson, B.A., Rotenberg, Y., u, Belobragina, S., GV, 1984. A further experimental study of the antisilicotic effect of glutamate. Br. J. Ind. Med. 41 (4), 518–525. Privalova, L.I., Katsnelson, B.A., Loginova, N.V., Gurvich, V.B., Shur, V.Y., a, Valamina, I. E., Makeyev, O.H., Sutunkova, M.P., Minigalieva, I.A., 2014. Subchronic toxicity of copper oxide nanoparticles and its attenuation with the help of a combination of bioprotectors. Int. J. Mol. Sci. 15, 12379–12406. White, L.D., Cory-Slechta, D.A., Gilbert, M.E., Tiffany-Castiglioni, E., Zawia, N.H., Virgolini, M., Rossi-George, A., Lasley, S.M., Qian, Y.C., 2007. New and evolving concepts of the neurotoxicology of lead. Toxicol. Appl. Pharmacol. 225, 1–27.
Boris A. Katsnelson* Larisa I. Privalova Marina P. Sutunkova Ilzira A. Minigalieva Vladimir B. Gurvich Vladimir Y. Shur Oleg H. Makeyev Irina E. Valamina Ekaterina V. Grigoryeva The Ekaterinburg Medical Research Center for Prophylaxis and Health Protection in Industrial Workers, Ekaterinburg, Russia The Institute of Natural Sciences, the Ural Federal University, Ekaterinburg, Russia The Central Research Laboratory, the Ural State Medical University, Ekaterinburg, Russia The Ekaterinburg Medical Research Center for Prophylaxis and Health Protection in Industrial Workers, Ekaterinburg, Russia * Corresponding author. E-mail address: [email protected] (B. Katsnelson). Received 8 September 2015 Accepted 8 September 2015 Available online 10 September 2015
Contents lists available at ScienceDirect
Toxicology journal homepage: www.elsevier.com/locate/toxicol
Letter to the Editor Is it possible to enhance the organism’s resistance to toxic effects of metallic nanoparticles?
Metallic nanoparticles, both engineered and generated as a byproduct of metallurgical and welding technologies, can be listed among the most dangerous occupational and environmental hazards. Although our studies summarized by Katsnelson et al. (2014a) demonstrated a high efficiency of the organism’s defenses against such nanoparticles’ impact, safe levels of the latter should be very low if possible at all, due to these particles’ especially high toxicity and seemingly obligatory genotoxicity. Therefore we deemed worthwhile to try and enhance the natural resistance to their adverse health effects with the help of so called bio-protectors. This idea was based on our long-term experience in a successful bio-protection of the organism against many other toxics, including some mineral microparticles (Katsnelson et al., 2014b). The principle organism-level mechanisms of this bioprotection (as we see them based on this experience and theoretical premises) are schematically presented by a flow-chart (Fig. 1). In general, to protect a mammalian organism against occupational or environmental toxic impacts one can use: (a) bio-protectors aimed primarily at increasing the effectiveness of natural mechanisms of bio-transformation and elimination of toxics, and thus, at reducing the dose of a harmful substance retained in the organism and especially in the target organs (on our scheme designated as “toxicokinetic effects”); (b) bio-protectors aimed at enhancing the functional reserves at all levels of the organism affected by toxic substance; at increasing the effectiveness of repairing and compensatory processes; and at using physiological and toxicological antagonisms (on the scheme collectively designated as “toxicokodynamic effects”). However, these two modes of action are usually interrelated and interdependent, as it is schematically shown with the reciprocally directed arrows. Indeed, when reducing retention of a toxic substance in the organism and especially in target organs, a bio-protector inhibits the development of a pathological process (thus, a bio-protector of a primarily toxicokinetic action produces a beneficial toxicodynamic effect). On the other hand, a primary enhancement of the resistance to the damaging impact of a toxic on the cells and organs that control the processes of its elimination or detoxication (pulmonary macrophages, liver, kidneys) maintains the effectiveness of these processes and, thus, reduces the retention of this toxic in the organism (so we deal with a beneficial
http://dx.doi.org/10.1016/j.tox.2015.09.001 0300-483X/ ã 2015 Elsevier Ireland Ltd. All rights reserved.
toxicokinetic effect of a toxicodynamic bio-protector). Such bilateral interdependence of toxicokinetic and toxicodynamic effects is pronounced to a varying degree in response to the action of different harmful substances but, on the whole, can be considered as a consistent pattern. The flow-chart shows also that both toxicokinetic and toxicodynamic bio-protectors can be: more or less specific with regard to a particular toxic of a particular range of toxics if bioprotection interferes with the mechanisms of toxicokinetics and toxicodynamics pertaining just to these toxics; predominantly non-specific, if their effect is realized through such integral responses at the organism level as the Selye’s general adaptation syndrome or the related but still distinct concept of “non-specifically enhanced resistance” developed by the school of late Nikolay Lazarev, an outstanding Russian toxicologist and pharmacologist. However, one and the same bio-protector may in different cases either render a largely specific effect or help the organism mainly as an agent enhancing its nonspecific defenses and thus decreasing sensitivity or increasing resistance to harmful exposures (see respective blocks and links of the same scheme). Bio-protectors acting through not fully identical mechanisms proved, in our experiments, most effective when administered not separately but in combinations (“bio-protective complexes”, or BPCs). Keeping in mind the further possible usage of bio-protectors for humans, we test in animal experiments only those substances which, on their own, are innocuous when applied for a long time in preventively effective doses. During more than 30 years, on a lot of chronic or subchronic experimental intoxications with different inorganic and organic chemicals acting separately or in different combinations (modelling actual occupational and environmental combined chemical exposures) we demonstrated bio-protective efficacy of some amino acids; vitamins (such as A, B1, E, C) and essential trace elements (selenium, iodine, copper etc) or multivitamin-multimineral preparations; fish oils rich in polyunsaturated fatty acids predominantly of the omega 3 class; pectin enterosorbents; calcium and iron supplements. In many instances, the beneficial effects and intrinsic safety of the experimentally tested BPCs were then confirmed in field investigations on volunteers as a prerequisite to a wider prophylactic usage. (For an overview of all this scientific and medico-social activity of ours see Katsnelson et al., 2014b.) As concerns the bio-protection against adverse effects of metallic nanoparticles, up to now we chose on theoretical premises and tested experimentally three BPCs protecting from nano-silver (Katsnelson et al., 2013), nano-copper oxide (Privalova
80
Letter to the Editor / Toxicology 337 (2015) 79–82
Enhancing the organism’s general defensive reactivity
Non-specific action
Toxi cokinetic Toxicokine effects Decrea Decreasing sing the sensitivity or increasing the resistance to toxic(s)
Bio-protectors Specific action
Toxicodynamic effects
Fig. 1. A mechanistic flow-chart of the anti-toxic bioprotection.
et al., 2014), and a combination of nano-nickel oxide plus nanomanganese oxide (Minigalieva et al., 2015). Different in some important details depending on specific toxicodynamic and toxicokinetic mechanisms of different metals’ toxic action, the composition of all three BPCs still have much in common and, we believe, similar BPCs should comprise most of the same components when further looking for bioprotection against other metallic nanoparticles. These components of choice are: (1) Glutamate as an effective cell membrane stabilizer acting through the intensification of ATP synthesis under exposure to the damaging action of various cytotoxic particles e.g. (Morosova et al., 1984) and, at the same time, as one of the precursors of glutathione, which is a powerful cell protector against the oxidative stress, the latter being presumably one of the key mechanisms of virtually all metallic NPs’ cytotoxicity and genotoxicity (Fröhlich, 2013). In addition to these nonspecific and almost universal bio-protective effects of glutamate, we believed that its administration might specifically increase resistance to neurotoxicity of manganese, lead and some other metallic nanoparticles due to glutamate’s major role in transmitting excitatory signals in the mammalian central nervous system and thus involvement in most aspects of normal brain functioning. Indeed, it is known, for instance, that manganese impairs the expression and function of the main glutamate transporters in astrocytes (Karki et al., 2013) and that lead interferes with glutamate release in hippocampus (White et al., 2007). We thought that additional glutamate supply to the brain might partly compensate for these adverse effects.
(2) The other two glutathione precursors: glycine and cysteine (the latter in a highly active and metabolically well available form of N-acetylcysteine), taking into consideration both the above-mentioned general important role played by the oxidative stress as a mechanism of metallic nanoparticles toxicity and some experimental data demonstrating that glutathione deficiency potentiates metals toxicity – e.g. manganese-induced damage to the rat striatum and brainstem (Desole et al., 1994). (3) Other agents of the organism’s anti-oxidant system (vitamins A, E, and E, and selenium). (4) omega-3 polyunsaturated fatty acids whose intracellular derivatives are eicosanoids that activate DNA replication and thus play an important part in its repair. (5) Iodine, taking into consideration well-known disturbances of the thyroid function caused by lead, manganese and some other intoxication (6) Pectin enterosorbent as an agent that hinders the reabsorption of toxic metals excreted into the intestines with bile (which, again, is of special importance for manganese as it is excreted predominantly by this route). In all the studies we found that, as expected, the toxicity and even genotoxicity of metallic nanoparticles could be really attenuated against the background of adequately composed BPCs. It should be understood that we do not claim to be the first to show a possibility of inhibiting some metallic nanoparticles’ toxicity with this or that agent aimed at a certain mechanism of this toxicity. However, this possibility had been demonstrated by others, as a rule, in experiments in vitro and was used as an
Table 1 Some morphometric indices for tubular epithelium damage in the kidneys of rats after repeated intraperitoneal injections of some metallic oxides nanoparticles with or without background oral administration of a BPC (X s.e.). Groups of
Brush border loss (% lengthwise)
Epithelial desquamation
Rats given NiO nanoparticles + Mn3O4 nanoparticles Water (control) Nanoparticles Nanoparticles BPC
5.44 0.9 12.33 2.3a 7.08 1.7
0.00 0 2.43 1.0a 0.00 0b
CuO nanoparticles Water (control) Nanoparticles Nanoparticles BPC
5.39 0.42 8.36 0.76a 5.98 0.46b
0.33 0.13 1.16 0.38a 0.98 0.35
a b
(% Lengthwise)
Statistically significant difference from the control group. From the group given nanoparticles without the BPC (p < 0.05 by Student’s t-test).
Letter to the Editor / Toxicology 337 (2015) 79–82
evidence for the importance of the said mechanism rather than a foundation of a holistic bio-protective system. Meantime, it is just such a prevention-oriented system (a “biological prophylaxis” in our terminology) that is the goal of our mechanistically substantiated approach, and we believe we were the first indeed who began to investigate effects of bio-protectors against metallic nanoparticles on the whole mammalian organism in chronic or subchronic animal experiments. A high beneficial efficacy of this bioprotection was assessed in those experiments with a lot of indices, but here we propose not to repeat original publications referred to above but only to illustrate this efficacy with several examples. Thus, all the above-mentioned nano-metals proved markedly nephrotoxic exerting, in particular, a significant damage to epithelial cells of proximal convoluted renal tubules. On the PAS-stained histological preparations of kidneys In rats exposed intraperitoneally during 6–7 weeks to those nanoparticles we saw marked degenerative and necrotic changes of these cells up to their disappearance with partial destruction of the brush border, while in rats exposed to the same nanoparticles against the background BPC administration a marked alleviation of the tubular damage was manifest. In Table 1 we give respective morphometric results obtained in the experiment with nickel oxide and manganese oxide nanoparticles (Minigalieva et al., 2015) and similar results of the experiment with copper oxide nanoparticles (Privalova et al., 2014). Another adverse effect proved specific for the toxicity of copper oxide and of manganese oxide nanoparticles was a marked damage to some specialized structures of the brain (especially, to the striatum and the hippocampus), and this damage also was significantly attenuated by the BPC. Examples are given by Fig. 2 (Privalova et al., 2014) and Table 2 (Minigalieva et al., 2015).
81
Fig. 2. Number of cells without a nucleolus per 100 Golgi cells in nucleus caudatus of rats exposed (A) to water (Control); (B) to water suspension of copper oxide nanoparticles; (C) to the same against the background of bioprotective complex (BPC) administrations, (D) to the BPC only (Average values with 95% CI). Differences are statistically significant between (B) and (A); (C) and (B) (p < 0.05 by Student’s ttest).
As an illustration of the efficacy of bioprotection against less specific toxic effects of metallic nanoparticles we give some results of the same experiment with nickel plus manganese oxides nanoparticles (Table 3). This Table demonstrates as well that a BPC which has significantly attenuated the adverse shifts caused by nanoparticles has itself no effect on the respective indices. This is quite typical for all our experiments. It should be stressed also that a significant attenuation of toxic effects was not obligatorily associated with a decrease in target organs’ load with a toxic metal (including that in the form of nanoparticles), thus demonstrating an important role of predominantly toxicodynamic mechanisms of bio-protection. Nevertheless, a beneficial toxicokinetic effect of BPCs was also observed in some cases, as illustrated by Table 4 (Privalova et al., 2014). The last but not the least result we would like to stress is that, while all the nanoparticles studied by us up to now were more or
Table 2 Some morphometric indices for the state of rat’s brain after repeated intraperitoneal injections of NiO and Mn3O4 nanoparticles with or without background oral administration of a BPC (X s.e.). Golgi neurons (%%)
Rats injected with water (control)
Rats injected with nanoparticles
Rats injected with nanoparticles and administered a BPC
Nucleus caudatus Without a nucleolus With a distinct centrally located nucleolus
30,50 2,77 25,12 1,16
60,30 2,26a 12,35 0,95b
37,15 2,89b 23,28 1,09b
Hippocampus (CA 1) Without a nucleolus With a distinct centrally located nucleolus
30,50 2,30 46.4 2.92
70,40 3,75a 11.0 1,13a
41,30 2.14a,b 30.5 1.96a,b
a b
Statistically significant difference from the control group. From the group given nanoparticles without the BPC (p < 0.05 by Student’s t-test).
Table 3 Some functional indices for the condition of rat after repeated intraperitoneal injections of NiO and Mn3O4 nanoparticles and/or oral administration of a BPC (x s.e.). Index
Groups given: Water (control)
Nanoparticles
Nanoparticles and the BPC
BPC
Leukocytes, 103/ ml Bilirubin in blood serum, mmol/L Albumin in blood serum, g/L Diuresis, ml Urine relative density Creatinine in urine, mmol/L d–ALA in urine, mmol/day
4.3 0.4 2.02 0.4 46.6 0.8 32.7 1.8 1.017 0.001 1.09 0.1 0.23 0.07
6.1 0.5a 1.15 0.1a 38.6 0.8a 17.9 2.9a 1.023 0.001a 1.8 0.2a 0.54 0.13
5.7 0.6b 1.5 0.1b 41.8 1.1b 30.2 2.7b 1.019 0.001b 1.2 0.1b 0.22 0.02b
4.3 0.4 1.7 0.1 47.3 1.2 31.2 4.5 1.019 0.001 1.2 0.1 0.25 0.08
a b
Statistically significant difference from the control group. From the group given NiO-NPs + Mn3O4-NPs (without the BPC) (p < 0.05 by Student’s t-test with Bonferroni correction).
82
Letter to the Editor / Toxicology 337 (2015) 79–82
Table 4 Copper content of some organs (mcg/g of dry-frozen tissue) in rats after repeated intraperitoneal injections of copper oxide nanoparticles and/or oral administration of a BPC (x s.e.) Group of rats given
Kidneys
Liver
Spleen
Brain
Water (control) Nanoparticles Nanoparticles and BPC BPC
42.4 2.9 62.5 7.1a 59.4 10.0 50.4 5.6
12.2 2.4 28.8 6.3a 22.1 3.5a 10.6 0.3
22.5 2.1 24.2 1.5 18.0 2.5b 25.3 2.2
18.9 0.7 21.5 1.7 18.8 1.4 20.8 1.5
a b
Statistically significant difference from the control group. from the group given nanoparticles without the BPC (p < 0.05 by Student’s t-test).
Table 5 Coefficients of the genomic DNA fragmentation in rats exposed to subchronic administration of silver nanoparticles with or without BPC based on the results of RAPD test (x s.e). Group of given:
Water (controls) Nano-particles Nanopar-ticles and BPC a b
Tissues Liver
Bone marrow
Spleen
Kidney
Nuclea-ted cells of blood
0.40 0.001 0.46 0.002a 0.41 0.011b
0.39 0.003 0.46 0.032a 0.37 0.003a,b
0.38 0.002 0.46 0.001a 0.42 0.003a,b
0.39 0.003 0.42 0.008a 0.40 0.006a,b
0.38 0.001 0.41 0.012a 0.39 0.007
Statistically significant difference from the control group. From the group given nanoparticles without the BPC (p < 0.05 by Student’s t-test).
less genotoxic, all the three tested BPCs significantly attenuated this most worrying effect. Table 5 demonstrates it by the example of silver nanoparticles (Katsnelson et al., 2013). To conclude, we maintain that the notoriously high toxicity of metallic nanoparticles make it necessary not only to try and keep respective dangerous exposures as low as possible but also to look for ways of increasing the organism’s resistance to them. As we believe, we have demonstrated that against the background of adequately composed combinations of some bioactive agents used in innocuous doses, the integral and specific toxicity of metallic nanoparticles and even their genotoxicity could be markedly attenuated. Therefore we strongly recommend to further develop this vector of nanotoxicological research. Our previous positive experience in organizing first a selective and then a large-scale “biological prophylaxis” of adverse health effects of other toxicants makes us expect that it would be no less practicable and effective in the field of nanotechnologies as well. References Desole, M.S., Miele, M., Esposito, G., Migheli, R., Fresu, L., De, N., atale, G., Miele, E., 1994. Dopaminergic system activity and cellular defense mechanisms in the striatum and striatal synaptosomes of the rat subchronically exposed to manganese. Arch. Toxicol. 68, 566–570. Fröhlich, E., 2013. Cellular targets and mechanisms in the cytotoxic action of nonbiodegradable engineered nanoparticles. J. Curr. Drug Metab. 14, 976–988. Karki, P., Lee, E., Aschner, M., 2013. Manganese Neurotoxicity: a Focus on Glutamate Transporters. Ann. Occup. Environ. Med. 25, 4. doi:http://dx.doi.org/10.1186/ 2052-4374-25-4. Katsnelson, B.A., Privalova, L.I., Gurvich, V.B., Makeyev, O.H., Shur, V., Ya Beikin YaB. Sutunkova, M.P., Kireyeva, E.P., Minigalieva, I.A., Loginova, N.V., 2013. Comparative in vivo assessment of some adverse bio-effects of equidimensional gold and silver nanoparticles and the attenuation of nanosilver’s effects with a complex of innocuous bioprotectors. Int. J. Mol. Sci. 14, 2449–2483. Katsnelson, B.A., Privalova, L.I., Gurvich, V.B., Kuzmin, S.V., Kireyeva, E.P., Minigalieva, I.A., Sutunkova, M.P., Loginova, N.V., Malykh, O.L., Yarushin, S.V., Soloboyeva, J.I., Kochneva, N.I., 2014a. Enhancing population’s resistance to toxic exposures as an auxilliary tool of decreasing environmental and occupational health risks (a self-overview). J. Environ. Protect. 5, 1435–1449. Katsnelson, B.A., Privalova, L.I., Sutunkova, M.P., Gurvich, V.B., Loginova, N.V., Minigalieva, I.A., Kireyeva, E.P., Shur, V.Y., a, Shishkina, E.V., 2014b. Some inferences frm in vivo experiments with metal and metal oxide nanoparticles: the pulmonary phagocytosis response, subchronic systemic toxicity and genotoxicity, regulatory proposals, searching for bioprot ectors (a selfoverview). Int. J. Nanomed. 10, 3013–3029.
Minigalieva, I.A., Katsnelson, B.A., Privalova, L.I., 2015. Attenuation of combined nickel (II) oxide and manganese (II,III) oxide nanoparticles’ adverse effects with a complex of bioprotectors. Int. J. Mol. Sci. 16, 22555–22583. Morosova, K.I., Katsnelson, B.A., Rotenberg, Y., u, Belobragina, S., GV, 1984. A further experimental study of the antisilicotic effect of glutamate. Br. J. Ind. Med. 41 (4), 518–525. Privalova, L.I., Katsnelson, B.A., Loginova, N.V., Gurvich, V.B., Shur, V.Y., a, Valamina, I. E., Makeyev, O.H., Sutunkova, M.P., Minigalieva, I.A., 2014. Subchronic toxicity of copper oxide nanoparticles and its attenuation with the help of a combination of bioprotectors. Int. J. Mol. Sci. 15, 12379–12406. White, L.D., Cory-Slechta, D.A., Gilbert, M.E., Tiffany-Castiglioni, E., Zawia, N.H., Virgolini, M., Rossi-George, A., Lasley, S.M., Qian, Y.C., 2007. New and evolving concepts of the neurotoxicology of lead. Toxicol. Appl. Pharmacol. 225, 1–27.
Boris A. Katsnelson* Larisa I. Privalova Marina P. Sutunkova Ilzira A. Minigalieva Vladimir B. Gurvich Vladimir Y. Shur Oleg H. Makeyev Irina E. Valamina Ekaterina V. Grigoryeva The Ekaterinburg Medical Research Center for Prophylaxis and Health Protection in Industrial Workers, Ekaterinburg, Russia The Institute of Natural Sciences, the Ural Federal University, Ekaterinburg, Russia The Central Research Laboratory, the Ural State Medical University, Ekaterinburg, Russia The Ekaterinburg Medical Research Center for Prophylaxis and Health Protection in Industrial Workers, Ekaterinburg, Russia * Corresponding author. E-mail address: [email protected] (B. Katsnelson). Received 8 September 2015 Accepted 8 September 2015 Available online 10 September 2015