Toxicology: Oxidative Stress and Dietary Antioxidants

By Victor R Preedy

Toxicology: Oxidative Stress and Dietary Antioxidants examines the nature of oxidative stress as a consequence of exposure to toxins and how antioxidant approaches can mitigate the impact of toxicant exposures. Sections covers the basic biology of oxidative stress, from molecular biology, to physiological pathology, mechanisms of action of specific toxicants, metals and other chemicals/drugs, and antioxidant approaches and therapies for toxic exposures. With contributions from an international group of experts, useful summary sections, a dictionary of terms, and applications to other areas of toxicology, this book is an informative, consolidated reference that helps bridge the interrelationship between toxicology, oxidative stress and antioxidants.

• Provides a novel collection of information linking both sides of redox biology (oxidants and antioxidants) and toxicology
• Explores the role of free radical mediated damage and toxicology
• Contains contributions from experts on toxicological science surrounding oxidative stress and on antioxidant approaches for reducing the impact of toxicant exposures

• Language: English
• Publisher: Academic Press
• Release date: Nov 17, 2020
• ISBN: 9780128190937

Book preview

libraries.

Part I

Toxicology and oxidative stress: General aspects, agricultural and industrial chemicals

Chapter 1: Agrochemicals inhibiting mitochondrial respiration: Their effects on oxidative stress

Evangelia Flampouri    Department of Biotechnology, School of Food Science, Biotechnology and Development, Agricultural University of Athens, Athens, Greece

Abstract

This chapter presents an overview of the current knowledge regarding pesticides that inhibit mitochondrial respiration and their relation to oxidative stress of mammalian organisms. Pesticides are categorized and discussed according to their mode of action and, more specifically, according to the mitochondrial respiratory protein complex they inhibit. All five complexes of the electron transport chain are targets of chemical pesticides and their impairment usually leads to electron leak and formation of free radicals. Elevated intracellular levels of reactive oxygen species, macromolecule oxidation, disturbance of the total antioxidant capability of the organism, and other oxidative stress-related effects are manifestations of toxicity induced by this group of xenobiotics.

Keywords

Electron transport chain; Mitochondrial respiration; Fungicides; Insecticides; NADH-Q oxidoreductase; Succinate-Q oxidoreductase; Q-cytochrome c oxidoreductase; Cytochrome c oxidase

List of abbreviations

ETC 

electron transport chain

CI 

complex I

Q 

ubiquinone

CII 

complex II

Cyt C cytochrome c

CIII 

complex III

ADP 

adenosine diphosphate

ROS 

reactive oxygen species

ATP 

adenosine triphosphate

CIV 

complex IV

NADH 

nicotinamide adenine dinucleotide

CV 

complex V

Introduction

The term xenobiotic refers to any foreign substance or exogenous chemical appearing in a biological system that is extrinsic to the normal metabolism of that system. The xeno in xenobiotic comes from the Greek word ξένος (xenos) meaning foreigner or stranger, while biotic comes from βίος (bios) meaning life along with the suffix -ικό (-iko) added to the Greek noun stems to form adjectives.¹

The toxicity of many xenobiotics is associated with the production of free radicals and oxidative stress.² Oxidative stress is a condition of imbalance between free radical generation and cellular antioxidant capacity. Accumulation of free radicals generated in the cells leads to dysfunction of their antioxidant systems and has been implicated in the pathophysiology of many diseases including diabetes, cancer, cardiovascular diseases, neurodegeneration, immune dysfunction, and aging.³

Among xenobiotics, pesticides are the most frequently found pollutants, because of their intensive and extensive use worldwide. A pesticide is any substance (or mixture of substances) that protects plants by preventing, destroying, repelling, and mitigating pests, weeds, or diseases. This group of xenobiotics includes insecticides, acaricides, fungicides, bactericides, herbicides, plant growth regulators, nematicides, and various other substances used to control pests.⁴

Pesticides are used in a wide range of settings throughout the world, with the most important areas being agriculture (as plant protection products) and public health (eliminating vectors of diseases, such as mosquitoes). They differ in identity, physical, and chemical properties and are usually classified based on their chemical composition, mode of action, pesticide function, and the pest organism they act upon. In this chapter, classification is based on the mode of action, focusing on pesticides that act via mitochondrial destabilization by targeting the electron transport chain (ETC). Furthermore, this chapter focuses on those pesticides that have been reported to alter the oxidative homeostasis of nontarget organisms such as mammals.

Mitochondria are subcellular organelles present in almost all types of eukaryotic cells. Their principal function is energy production, namely ATP, which is generated within several complexes or clusters of up to 42 proteins of the electron transport chain.⁵ Electron flow within four of those macromolecular transmembrane complexes leads to the transport of protons across the inner mitochondrial membrane (Fig. 1), creating a greater concentration of hydrogen ions in the intermembrane space than in the matrix. This chemiosmotic gradient, caused by the ETC, is used to drive ATP synthase to produce ATP.⁶

Fig. 1 The mitochondrial electron transport chain. Electron movement through the mitochondrial electron transport chain, with the subsequent generation of adenosine triphosphate (ATP). Respiratory complexes are marked as CI, CII, CIII, CIV, and CV.

The first complex (CI), the NADH-Q oxidoreductase, receives electrons in the form of hydride ions from NADH and passes them on to ubiquinone (Q). The second complex (CII), the succinate-Q oxidoreductase, receives electrons from succinate and after yielding fumarate and FADH2, passes them off to ubiquinone. The now reduced form of ubiquinone, ubiquinol, passes an electron off to the third complex (CIII), the Q-cytochrome c oxidoreductase (Fig. 1), which then passes it on to cytochrome c (Cyt C). Cytochrome C subsequently shuttles electrons from Q-cytochrome c oxidoreductase to the final component in the ETC, the cytochrome c oxidase (CIV), which catalyzes the reduction of O2. Ultimately, ATP synthase (CV) uses the energy created by the proton electrochemical gradient to phosphorylate ADP to ATP.

The ETC is the dominant source of mitochondrial reactive oxygen species (ROS) due to the large electron flows, even under normal mitochondrial respiration conditions.⁷, ⁸ ROS include free radicals such as superoxide anions (O2•−), hydrogen peroxide (H2O2), and hydroxyl radicals (HO•) which when in excess might detrimentally perturb cellular functions, induce apoptosis and cell death.⁹ CI and CIII have been identified as significant sites of O2•− production,¹⁰, ¹¹ while CII has also been linked with the process.¹² Xenobiotics that selectively inhibit the ETC complexes lead, in almost all cases, to an increased formation of ROS.¹³, ¹⁴ Pesticides with the above-mentioned mode of action are described below, categorized based on their targeted mitochondrial complexes.

Mitochondrial complex I inhibitors

This group of pesticides contains a large number of compounds with structural diversity, mainly insecticides and acaricides. Rotenone, an isoflavone found in the roots of several plant species, is a long-known naturally occurring high-affinity inhibitor of NADH-Q oxidoreductase, which has been used commercially as a garden insecticide since the mid-19th century.¹⁵ During the last decades, rotenone has received immense attention because of its link to Parkinson’s disease and is currently used in the research community as an experimental drug for inducing mitochondrial dysfunction and reproduce Parkinson’s disease in animal models.¹⁶ Rotenone has been extensively reviewed throughout the years in numerous publications and book chapters (for a comprehensive review see Cicchetti et al.¹⁷). Due to lack of space, rotenone will not be discussed in this chapter, which will rather focus on synthetic complex I inhibitors.

Introduced in the early 1990s, formulations of synthetic complex I inhibitors exhibit varying chemical structures and are commonly used as insecticides and acaricides. The first of these xenobiotics to be commercialized were pyrazole fenpyroximate, pyridazinone pyridaben, quinazoline fenazaquin, and pyrazolecarboxamide tebufenpyrad (Fig. 2). All these compounds have been proven highly effective against various species and life stages of insects and mites and are systematically used in crops worldwide.¹⁸

Fig. 2 Complex I inhibitors. Structures of complex I inhibitors described in the text.

As expected, based on its target site, fenpyroximate inhibition of NADH-Q oxidoreductase has been reported to cause mitochondrial formation of O2•− in rat neuronal cells,¹⁹ elevated intracellular ROS production in human lymphocytes,²⁰ and impaired oxygen utilization at the tissue level via oxidative damage in acute human intoxication.²¹ Similarly, pyridaben has been shown to initiate oxidative damage in dopaminergic neuronal cells,²² increase ROS levels and reduce antioxidant markers in mice,²³ and elevate oxidative stress biomarkers in human neuroblastoma cells.²⁴ Fenazaquin was also found to increase H2O2 production in mitochondrial fractions of dopaminergic rat and human neuroblastoma cell lines²⁵ and cause mitochondrial formation of O2•− in rat neuronal cells.¹⁹ Intracellular ROS generation in tebufenpyrad-treated rat dopaminergic neural cells has been observed,²² along with ROS-related genotoxicity in human lymphocytes.²⁰ Other less studied compounds of this class include pyrimidifen and tolfenpyrad, with the latter been reported to induce oxidative damage in dopaminergic neural cells²² and human lymphocytes.²⁰

Considering that complex I inhibitors exert their toxic action through ROS formation, treatment with antioxidants or ROS scavengers is expected to ameliorate their effects. Indeed, many research studies have reported that oxidative damages caused by these pesticides could be partially attenuated with alpha-tocopherol,²⁴ Trolox, or N-acetyl-l-cysteine.¹⁹

Mitochondrial complex II inhibitors

Complex II is the only membrane-bound component of the Krebs cycle and in addition functions as a member of the electron transport chain in the mitochondria (Fig. 1).²⁶ It is not a significant contributor to the production of ROS; however, research shows than when complex I and complex III are inhibited and succinate concentration is low, complex II can generate superoxide or H2O2.²⁷ Compounds inhibiting mitochondrial complex II are not expected to have a direct effect on the electron flux to the mitochondrial respiratory chain, but to disturb the tricarboxylic acid cycle²⁸ instead.

Succinate-Q oxidoreductase inhibitors of this pesticide group mainly exhibit fungicidal action, with some recently developed substances acting as acaricides. Structurally, many of the compounds share a central amide moiety essential for hydrogen-bond interactions in the Q-binding site of complex II. The most common fungicides of this group are the pyridine carboxamides boscalid and sedaxane, and the pyrazole carboxamides bixafen, fluxapyroxad, isopyrazam, and penthiopyrad (Fig. 3). Additionally, some modern complex II acaricides are the beta-ketonitrile derivatives cyenopyrafen and cyflumetofen, and the carboxanilide pyflubumide.²⁹ Xenobiotics of this group have not been clearly described in respect to their oxidative damage potential. Only bixafen and cyflumetofen have been reported to increase ROS formation in human lymphocyte²⁰ and neurobast³⁰ cells, respectively, while boscalid is reported to have no effect on ROS levels of primary cultures of cortical neurons.³¹

Fig. 3 Complex II inhibitors. Structures of complex II inhibitors described in the text.

Mitochondrial complex III inhibitors

Xenobiotics belonging to this group mainly exhibit fungicidal and insecticidal action. They act through binding to the ubiquinol oxidation site of cytochrome b (complex III) and thereby interfere with electron transfer between cytochrome b and cytochrome c,³² which halts reduced nicotinamide adenine dinucleotide (NADH) oxidation and adenosine triphosphate (ATP) synthesis.³³

Depending on their binding location at the cytochrome bc1 complex, they can be categorized into Quinone inside Inhibitors (QiI), which act on the quinol inner binding site and Quinone outside Inhibitors (QoI), which act on the quinol outer binding site.

Commercial QoI fungicides are among the best-selling agricultural fungicides worldwide, and are primarily used as plant protectants against fungal and oomycete pathogens. Strobilurins represent the largest part of the QoI group, with their synthesis based on secondary metabolites of the wood-rotting fungus Strobirullus tenacellus. The most common synthetic strobilurins are azoxystrobin, kresoxim-methyl, trifloxystrobin, pyraclostrobin, fluoxastrobin, and picoxystrobin (Fig. 4). Although designed to target fungi, strobilurins have been shown to exert their action on nontarget organisms such as mammalian cells. Azoxystrobin was found to dose dependently increase mitochondrial superoxide anion generation in rat cardiomyocytes,³⁴ while kresoxim-methyl exposure to primate renal cells and rat neural cells is reported to induce a marked release of H2O2³⁵ and elevated mitochondrial superoxide generation,³⁶ respectively. Other QoIs include famoxadone and fenamidone, with the latter reported to cause mitochondrial superoxide production and microtubule destabilization in neural cells.³⁷ Similar to previously discussed ETC inhibitors, oxidative effects of complex III pesticides have been minimized using ROS scavengers such as MitoTEMPO.³⁸

Fig. 4 Complex III inhibitors. Structures of complex III inhibitors described in the text.

Mitochondrial complex IV inhibitors

Complex IV is the terminal enzyme of the electron transport chain, which catalyzes the final step of the electron transfer from the reduced cytochrome C to oxygen (Fig. 1). Chemicals belonging to this class can be categorized into phosphides and cyanides (Fig. 5). Phosphides include metal phosphides such as aluminum phosphide, calcium phosphide, phosphine, and zinc phosphide. Metal phosphides are highly toxic fumigants rodenicides, insecticides, and fungicides, which are still commonly used in developing countries. Their toxic effects are due to the deadly phosphine gas (PH3), which inhibits cytochrome C oxidase and induces the formation of highly reactive hydroxyl radicals.³⁹, ⁴⁰ Phosphine-induced oxidative damage has been reported in mammalian cells,⁴¹ rats,⁴² and poisoned human patients.⁴³ Cyanide is a potent poison that induces histotoxic anoxia by inhibiting cytochrome C oxidase and impairing cellular oxygen utilization. As a result of mitochondrial electron transport inhibition by cyanide, excess ROS are generated at complexes I and III, producing intense oxidative stress that contributes to cellular dysfunction.⁴⁴

Fig. 5 Complex IV inhibitors. Structures of complex IV inhibitors described in the text.

Mitochondrial complex V inhibitors

Although ATP synthase is not involved in the transport of electrons, it is considered part of the respiratory chain (Fig. 1). Within the process of oxidative phosphorylation, complex V discharges the electrochemical potential gradient created across the inner mitochondrial membrane, catalyzes the phosphorylation of ADP, and releases ATP into the cell. Very few pesticides have been reported as inhibitors of complex V, with no reports on oxidative damage in nontarget organisms. This class includes mostly acaricides such as diafenthiuron, tetradifon, chlorfenson, chloropropylate, bromopropylate, flubenzimine, propargite, and organotin miticides such as azocyclotin, cyhexatin, and fenbutatin oxide (Fig. 6).

Fig. 6 Complex V inhibitors. Structures of complex V inhibitors described in the text.

Applications to other areas of toxicology

In this chapter, pesticides acting on the respiratory complexes of the mitochondrial electron transport chain were reviewed with respect to their effects on oxidative stress on mammalian organisms. It was shown that many of this diverse group of chemicals exert their mode of action on nontarget organisms and induce reactive species formation and oxidative stress. This is supported by studies showing that exposure to this type of pesticides leads to free radical generation, induction of macromolecule oxidation, and disturbance of the total antioxidant capability in mammalian cell lines, animals, and humans. Among the cell lines that showed altered oxidative status, few were of malignant origin. Of the agents with anticancer activity currently investigated worldwide, the ones that act on the mitochondria have massive potential as efficient anticancer drugs.⁴⁵ Furthermore, in some recent studies complex III inhibitors/strobilurin analogs exhibited potent effects against cancer cells.⁴⁶ This suggests that designing and synthesizing new substances based on those pesticides could provide a starting point for new classes of anticancer drug development.

Summary points

•This chapter focuses on pesticides that inhibit mitochondrial respiration and their relation to oxidative stress of mammalian organisms.

•Pesticide inhibitors of the protein complexes involved in the respiratory chain are able to induce free radical generation, macromolecule oxidation, and disturbance of the total antioxidant capability on nontarget organisms.

•Of the five protein complexes, I and III seem to be to two principal sites for chemically induced superoxide generation.

•Inhibition of complex V has not yet been reported to be involved in ROS formation, while only a few reports exist for complex II.

•Pesticides that target mitochondrial respiration have exhibited anticancer activity.

References

1 Soucek P. Xenobiotics. In: Encyclopedia of Cancer. Berlin, Heidelberg: Springer-Verlag; 2009 3215-32–18.

2 Pagano G. Redox-modulated xenobiotic action and ROS formation: a mirror or a window?. Hum Exp Toxicol. 2002;21:77–81.

3 Liguori I., Russo G., Curcio F., et al. Oxidative stress, aging, and diseases. Clin Interv Aging. 2018;13:757–772.

4 Food and Agriculture Organization of the United Nations. International code of conduct on the distribution and use of pesticides (revised version) : adopted by the hundred and twenty-third session of the FAO Council in November 2002. Food and Agriculture Organization of the United Nations; 2003. http://www.fao.org/3/y4544e/y4544e00.htm.

5 Friedman J.R., Nunnari J. Mitochondrial form and function. Nature. 2014;505:335–343.

6 Nicholls D.G., Ferguson S.J. Respiratory chains. In: Bioenergetics. Academic Press; 2007:89–XIII.

7 Chance B., Sies H., Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev. 1979;59:527–605.

8 Turrens J.F., Boveris A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem J. 1980;191:421–427.

9 Ghosh N., Das A., Chaffee S., Roy S., Sen C.K. Reactive oxygen species, oxidative damage and cell death. In: Immunity and inflammation in health and disease. Academic Press; 2017:45–55.

10 Nishikawa T., Edelstein D., Du X.L., et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature. 2000;404:787–790.

11 Orrenius S., Gogvadze V., Zhivotovsky B. Mitochondrial oxidative stress: implications for cell death. Annu Rev Pharmacol Toxicol. 2007;47:143–183.

12 Ralph S.J., Moreno-Sánchez R., Neuzil J., Rodríguez-Enríquez S. Inhibitors of succinate: quinone reductase/complex II regulate production of mitochondrial reactive oxygen species and protect normal cells from ischemic damage but induce specific cancer cell death. Pharm Res. 2011;28:2695–2730.

13 Li N., Ragheb K., Lawler G., et al. Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J Biol Chem. 2003;278:8516–8525.

14 Meyer J.N., Leung M.C.K., Rooney J.P., et al. Mitochondria as a target of environmental toxicants. Toxicol Sci. 2013;134:1–17.

15 Fernando P., Yan X., Lockwood J., et al. Toxicological evaluation of a rotenone derivative in rodents for clinical myocardial perfusion imaging. Cardiovasc Toxicol. 2014;14:170–182.

16 Uversky V.N. Neurotoxicant-induced animal models of Parkinson’s disease: understanding the role of rotenone, maneb and paraquat in neurodegeneration. Cell Tissue Res. 2004;318:225–241.

17 Cicchetti F., Drouin-Ouellet J., Gross R.E. Environmental toxins and Parkinson’s disease: what have we learned from pesticide-induced animal models?. Trends Pharmacol Sci. 2009;30:475–483.

18 Van Leeuwen T., Vontas J., Tsagkarakou A., Dermauw W., Tirry L. Acaricide resistance mechanisms in the two-spotted spider mite Tetranychus urticae and other important Acari: a review. Insect Biochem Mol Biol. 2010;40:563–572.

19 Marella M., Byoung B.S., Matsuno-Yagi A., Yagi T. Mechanism of cell death caused by complex I defects in a rat dopaminergic cell line. J Biol Chem. 2007;282:24146–24156.

20 Graillot V., Tomasetig F., Cravedi J.P., Audebert M. Evidence of the in vitro genotoxicity of methyl-pyrazole pesticides in human cells. Mutat Res Genet Toxicol Environ Mutagen. 2012;748:8–16.

21 Lee H.Y., Lee B.K., Jeung K.W., Lee G.S., Jung Y.H., Jeong I.S. A case of near-fatal fenpyroximate intoxication: the role of percutaneous cardiopulmonary support and therapeutic hypothermia. Clin Toxicol. 2012;50:858–861.

22 Charli A., Jin H., Anantharam V., Kanthasamy A., Kanthasamy A.G. Alterations in mitochondrial dynamics induced by tebufenpyrad and pyridaben in a dopaminergic neuronal cell culture model. Neurotoxicology. 2016;53:302–313.

23 Ghodrat E.M., Kazem P., Shapour H., Parichehr Y., Golamreza N. The effects of pyridaben pesticide on gonadotropic, gonadal hormonal alternations, oxidative and nitrosative stresses in Balb/C mice strain. Comp Clin Pathol. 2014;23:297–303.

24 Sherer T.B., Richardson J.R., Testa C.M., et al. Mechanism of toxicity of pesticides acting at complex I: relevance to environmental etiologies of Parkinson’s disease. J Neurochem. 2007;100:1469–1479.

25 Seo B.B., Marella M., Yagi T., Matsuno-Yagi A. The single subunit NADH dehydrogenase reduces generation of reactive oxygen species from complex I. FEBS Lett. 2006;580:6105–6108.

26 Cecchini G. Function and structure of complex II of the respiratory chain. Annu Rev Biochem. 2003;72:77–109.

27 Quinlan C.L., Orr A.L., Perevoshchikova I.V., Treberg J.R., Ackrell B.A., Brand M.D. Mitochondrial complex II can generate reactive oxygen species at high rates in both the forward and reverse reactions. J Biol Chem. 2012;287:27255–27264.

28 Nicholls D.G. Mitochondrial function and dysfunction in the cell: its relevance to aging and aging-related disease. Int J Biochem Cell Biol. 2002;34:1372–1381.

29 Stammler G., Wolf A., Glaettli A., Klappach K. Respiration inhibitors: complex II. In: Fungicide resistance in plant pathogens. Tokyo: Springer Japan; 2015:105–117.

30 Zhao H., Chen Y., Li N., Yang X., Li S., Liu W. Cytotoxicity of cyflumetofen on SH-SY5Y cells and possible mechanism. Chin J Pharmacol Toxicol. 2017;31:318–324.

31 Regueiro J., Olguín N., Simal-Gándara J., Suñol C. Toxicity evaluation of new agricultural fungicides in primary cultured cortical neurons. Environ Res. 2015;140:37–44.

32 Gisi U., Sierotzki H., Cook A., McCaffery A. Mechanisms influencing the evolution of resistance to Qo inhibitor fungicides. In: Pest management science. John Wiley & Sons, Ltd; 2002:859–867.

33 Balba H. Review of strobilurin fungicide chemicals. J Environ Sci Health B. 2007;42:441–451.

34 Rodrigues E.T., Pardal M., Laizé V., Cancela M.L., Oliveira P.J., Serafim T.L. Cardiomyocyte H9c2 cells present a valuable alternative to fish lethal testing for azoxystrobin. Environ Pollut. 2015;206:619–626.

35 Flampouri E., Mavrikou S., Mouzaki-Paxinou A.C., Kintzios S. Alterations of cellular redox homeostasis in cultured fibroblast-like renal cells upon exposure to low doses of cytochrome bc1 complex inhibitor kresoxim-methyl. Biochem Pharmacol. 2016;113:97–109.

36 Flampouri E., Theodosi-Palimeri D., Kintzios S. Strobilurin fungicide kresoxim-methyl effects on a cancerous neural cell line: oxidant/antioxidant responses and in vitro migration. Toxicol Mech Methods. 2018;28:709–716.

37 Pearson B.L., Simon J.M., McCoy E.S., Salazar G., Fragola G., Zylka M.J. Identification of chemicals that mimic transcriptional changes associated with autism, brain aging and neurodegeneration. Nat Commun. 2016;7:11173.

38 Jang Y., Kim J.-E., Jeong S.-H., Paik M.-K., Kim J.S., Cho M.-H. Trifloxystrobin-induced mitophagy through mitochondrial damage in human skin keratinocytes. J Toxicol Sci. 2016;41:731–737.

39 Chefurka W., Kashi K.P., Bond E.J. The effect of phosphine on electron transport in mitochondria. Pestic Biochem Physiol. 1976;6:65–84.

40 Bolter C.J., Chefurka W. Extramitochondrial release of hydrogen peroxide from insect and mouse liver mitochondria using the respiratory inhibitors phosphine, myxothiazol, and antimycin and spectral analysis of inhibited cytochromes. Arch Biochem Biophys. 1990;278:65–72.

41 Hsu C.H., Quistad G.B., Casida J.E. Phosphine-induced oxidative stress in Hepa 1c1c7 cells. Toxicol Sci. 1998;46:204–210.

42 Hsu C.H., Han B.C., Liu M.Y., Yeh C.Y., Casida J.E. Phosphine-induced oxidative damage in rats: attenuation by melatonin. Free Radic Biol Med. 2000;28:636–642.

43 Kariman H., Heydari K., Fakhri M., et al. Aluminium phosphide poisoning and oxidative stress: serum biomarker assessment. J Med Toxicol. 2012;8:281–284.

44 Sun P., Rane S., Gunasekar P., Borowitz J., Isom G. Modulation of the NMDA receptor by cyanide: enhancement of receptor-mediated responses. J Pharmacol Exp Ther. 1998;280:1341–1348.

45 Neuzil J., Dyason J.C., Freeman R., et al. Mitocans as anti-cancer agents targeting mitochondria: lessons from studies with vitamin e analogues, inhibitors of complex II. J Bioenerg Biomembr. 2007;39:65–72.

46 Lee S., Kwon O.S., Lee C.-S., Won M., Ban H.S., Ra C.S. Synthesis and biological evaluation of kresoxim-methyl analogues as novel inhibitors of hypoxia-inducible factor (HIF)-1 accumulation in cancer cells. Bioorg Med Chem Lett. 2017;27:3026–3029.

Chapter 2: Nanoparticle toxicity and reactive species: An overview

Marcin Kruszewskia,b; Agnieszka Grzelakc    a Centre for Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa, Poland

b Department of Molecular Biology and Translational Research, Institute of Rural Health, Lublin, Poland

c Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland

Abstract

This chapter reviews the role of oxidative stress in nanoparticle toxicity. There is accumulating evidence that oxidative stress plays a crucial role in induction of cell death after nanoparticle exposure. Production of reactive oxygen species increases in a dose- and time-dependent manner shortly after exposure to nanoparticles. This correlates with induction of damage to biological macromolecules, such as DNA, proteins and lipids, and cell death. A likely source of oxidative stress in the malfunction of mitochondria, however involvement of nonmitochondrial enzymatic systems, such as NADPH oxidases, was also suggested. Despite the damage to macromolecules and associated harmful effects, oxidative stress triggers cellular signaling pathways that might lead to cell death or survival, depending on the cellular context. Regardless of the nanoparticle and the mechanism of reactive species production, the primary reactive species produced is superoxide which is further dismutated to hydrogen peroxide. Hydrogen peroxide, in turn, in the presence of trace amounts of transition metals undergoes Fenton reaction and gives rise to the formation of highly reactive hydroxyl radicals.

Keywords

Oxidative stress; Antioxidant defense; Transition metal ions; Cell signaling

List of abbreviations

Akt 

serine-threonine protein kinase B

AP-1 

activator protein 1

CAT 

catalase

DSB 

double strand breaks

EGF 

epidermal growth factor

GPX 

glutathione peroxidase

HIF 

1 hypoxia-inducible factor 1

IKK 

IκB kinase

KEAP1 

Kelch-like ECH-associated protein 1

MAPK 

mitogen activated protein kinases

MN 

micronuclei

MWCO 

molecular weight cut-off

NF-κB 

nuclear factor κB

NOX 

NADPH oxidase

NP 

nanoparticle

Nrf2 

nuclear factor eryhroid 2-related factor 2

PI3K 

phosphoinositide 3-kinase

PM10 

particulate matter less than 10 μm

PM2.5 

particulate matter less than 2.5 μm

PVP 

polyvinylpyrrolidone

ROS 

reactive oxygen species

SOD 

superoxide dismutase

Introduction

In recent years, a large number of engineered nanoparticles (NPs) have been developed with promising technical benefits for consumers, medical appliances, and the industry. Despite the potential benefits that nanotechnology may bring to society, nanomaterials may trigger undesirable hazardous interactions with biological systems with potential to generate harmful effects. Presently, there is increasing concern about the detrimental health effects due to NP exposure. NPs have been reported to induce oxidative stress, DNA damage, inflammation, and many other adverse effects, which are known to be crucial for the development of lifestyle diseases.

Oxidative stress is defined as an imbalance between the generation of free radicals and their neutralization by cellular antioxidative defense mechanisms that causes disturbance of the cellular redox equilibrium. This imbalance of cellular redox equilibrium may have different manifestations depending on the magnitude of oxidative stress, from triggering signal transduction by low concentrations of ROS, through the induction of oxidative damage to cellular components and organelles, to induction of cell death by immense ROS production. At the organismal level, oxidative stress usually appears as changes in tissue and organ functioning, often leading to inflammation and tissue necrosis. Oxidative stress might be induced by different physical, chemical, and biological factors (Table 1).

Table 1

In boldface letters factors that might be associated with intentional or unintentional exposure to nanomaterials.

Being highly reactive, ROS are capable to interact with cellular components, either causing changes in their functionality or damaging them irreversibly. While changes in the function of redox sensitive proteins might be helpful, e.g., switching on cellular antioxidant defense mechanisms, damage to cellular components, if not repaired, might lead to detrimental processes, such as carcinogenesis or cell death.

In this review, we summarize the current knowledge about oxidative stress induced by exposure to NPs and its associated harmful effects on molecular, cellular, and organismal level.

Nanoparticle-generated oxidative stress

Role of mitochondria in oxidative stress induction

Exposure to NPs markedly increases ROS formation, likely due to the interference with the mitochondria and/or nonmitochondrial ROS-producing enzymes. It was also shown that NPs of various size and shape accumulate in the mitochondria and might induce mitochondrial damage and malfunction of the respiratory chain, resulting in ROS generation.¹, ² Although the mechanism of NP-induced mitochondrial toxicity is not yet clarified, it has been reported that the presence of NPs inside the mitochondria hinders the mitochondrial electron chain.³ NPs has been shown to induce mitochondrial membrane depolarization; however, whether this is an effect of direct interaction with mitochondrial membranes or interaction of NPs with respiratory chain proteins needs further clarification. The last cannot be excluded as respiratory chain complexes have different susceptibility to AgNPs.⁴

In normal cells, mitochondria are responsible for majority of energy production, thus damage to the mitochondria may result in insufficient energy supply and hamper ATP-dependent cellular processes. Aa an alternative source of energy, glycolysis is less efficient. Moreover, it was shown that cells that rely on glycolysis for their energy production, such as cancer cells, are more susceptible to NPs due to the less efficient antioxidant defense system.⁵

Nonmitochondrial induction of oxidative stress

Oxidative stress can be generated by different factors, physical, chemical, and biological. Ionizing and UVA radiation are the most common free radicals inducing physical factors that affect cells. Visible light in the presence of photosensitizers also produces free radicals and the system is used in medicine. Among the chemical factors autooxidation of catecholamines and thiol groups, redox cycling of quinones and thiols, and redox reactions of different xenobiotics are of utmost importance from the point of view of ROS production.

Although, ROS are generated primarily in the mitochondria during oxidative phosphorylation, nonmitochondrial cellular sources of ROS are also recognized (Table 2). Mammalian peroxisomes are key players in various metabolic pathways, including fatty acid β-oxidation, glyoxylate metabolism, amino acid catabolism, polyamine oxidation, and pentose phosphate pathway. Many of the enzymes involved in these processes produce H2O2 as a byproduct of reactions. Whether peroxisome-produced H2O2 may affect the cellular function remains unknown, as catalase present in peroxisomes likely prevents its release to the cytoplasm (for a review see Ref. 6). Peroxisomes also contain xanthine oxidase that produces O2•−; however, SOD present in peroxisomes likely convert the radical to H2O2. In the endoplasmic reticulum, ROS are produced by cytochromes (b5 and p450), diamine oxidase (EC 1.4.3.22), and thiol oxidase (EC 1.8.3.2), all producing H2O2.

Table 2

Membrane-associated NADPH oxidases (NOXs) are the main source of ROS in cytosol. NOXs are also able to produce ROS extracellularly (Fig. 1A). The phagocyte NOX produces large amounts of O2•− that serve a host defense role. Nonphagocytic cells, like fibroblasts, B lymphocytes, endothelial cells, and platelets express, express also other isoforms of NOX that produce O2•−, however to a lesser extent. Dual oxidase, another NOX isoform expressed in thyroid glands and airway epithelial cells, produces extracellularly H2O2, likely as a part of the host defense system (Fig. 1B). NP-dependent increase in the production of O2•− by NOX, accompanied by intracellular production of the other ROS has been recently reported after treatment with AgNPs⁷ and ultrafine particles.⁸ Additional source of ROS in organisms is through oxidation of hemoproteins (hemoglobin, myoglobin), autooxidation of adrenaline, noradrenaline, dioxyphenylanine, and biosynthesis of prostaglandins.

Fig. 1 ROS production and regulation of NADPH oxidase (A) and dual oxidase (B). In red —catalytic subunit, in gray —regulatory subunits, in yellow —activating factor. P , phosphate group.

Oxidative stress-associated nanoparticle toxicity

Nanoparticle-induced toxicity

Although the generation of ROS and associated oxidative stress seems to be a main cause of NP toxicity, a direct interaction with macromolecules cannot be excluded. NPs with charged surfaces can interact with charged macromolecules, such as DNA or proteins. Direct interaction of NPs with proteins and formation of protein corona on the surface of NPs is a well-known phenomenon. In addition, it was reported that in response to SiO2NPs treatment aberrant clusters of topoisomerase I were formed in the nucleoplasm that impairs DNA processing.⁹ It was also shown that AuNPs may interact with monomeric and/or oligomeric α-synuclein protein during the process of nucleation, thus affecting the formation of microfibrils that contribute to the formation of intracellular Lewy body in Parkinson’s disease.¹⁰ AuNPs have also been shown to impair fibrillation of β-amyloid protein that is implicated in the development of Alzheimer’s disease.¹¹ When in the nucleus or during interphase NPs can also directly interact with DNA, as it has been shown that NPs can dissociate double-stranded DNA.¹²

Toxicity of nanomaterials correlate with several universal parameters, such as the size or diameter of NPs, concentration of NPs, the method of synthesis of NPs, coating, time and ways of exposure, and the biological model used to evaluate toxicity (for a review regarding AgNP toxicity see Ref. 13). The primary mechanism of NP toxicity seems to be the generation of oxidative stress as the addition of antioxidants attenuates NP toxicity.¹⁴ Intense oxidative stress induced by NPs lead to damage to organelles and biological macromolecules, such as malfunction of the mitochondria, oxidation of proteins, lipids and DNA, decrease in antioxidant concentration, and antioxidant enzyme activity (Fig. 2). Kusaczuk et al.¹⁵ reported that in glioblastoma SiNP-induced oxidative stress resulted in mitochondrial damage, deregulated expression of genes encoding the antioxidant enzymes SOD, and CAT, ATP depletion, elevated expression of BAX, PUMA, and NOXA and simultaneous downregulation of BCL2/BCL2L1, followed by activation of caspase 9 and apoptosis. SiNP-mediated apoptosis was also demonstrated in other cellular models. Ahamed¹⁶ reported that SiNPs treatment resulted in excessive oxidative stress, which led to the upregulation of CASP9 and CASP3 genes and initiation of mitochondria-mediated apoptosis in A431 and A549 cells. In line, upregulation of BAX and CASP3 genes together with downregulation of the antiapoptotic BCL2 gene was observed in HepG2 cells.¹⁷ Besides induction of apoptosis, several reports described SiNPs-mediated induction of necrosis or both necrosis and apoptosis. The mode of cell death after NPs treatment depends on the cellular context, as exposure of human umbilical vein endothelial cells to SiNPs resulted in enhanced necrosis, while similar exposure of alveolar macrophages resulted in 80% apoptosis and 20% necrosis.¹⁸

Fig. 2 Mechanism of NP-induced toxicity associated with oxidative stress induction.

The role of ions in nanoparticle toxicity

Release of ions from NPs is an important issue repeatedly raised in many publications. The ion release from NPs depends on many factors, including redox properties of the surrounding environment, NPs shape, and composition. However, their actual role in NPs toxicity depends also on the experimental setup. For example, dissolution of PVP-coated AgNPs (100 μg/cm³) in RPMI-1640 medium was studied by Loza et al.¹⁹, who calculated a maximum possible concentration of Ag+ in the conditioned medium to be 0.327 μg/cm³. The RPMI 1640 medium, however, contains 6 mg/cm³ of NaCl. The AgCl has a particularly low solubility (1.92 × 10− 4 g/100 g H2O), which leads to its precipitation from the culture medium. Thus, even if Ag+ would be released from AgNPs, the 120-fold excess of Cl− ions and precipitation of one of the reaction products from the reaction environment will favor the formation of AgCl, thus all dissolved Ag+ will precipitate in the form of AgCl. Indeed, almost no silver was recovered from the cell culture medium in which AgNPs (100 μg/cm³, 20 nm) were kept for up to 192 h, if the medium was ultrafiltrated through the Amicon Ultra-15 tube 3000 MWCO.²⁰ This, however, must be distinguished from the situation inside the cell. Inside the cell, AgNPs that had been transported to the mitochondria could have undergone oxidative dissolution, while compartmentalization of cells might lead to a situation when Ag+ are present and participate in the detrimental effects of AgNPs. Investigations by He et al.²¹ revealed that in the environment containing hydrogen oxide and superoxide, such as this in the mitochondria, AgNPs underwent cyclic reaction of oxidative dissolution and reduction to the solid state. As pointed by De Matteis et al.,²² the toxicity of AgNPs is mainly dependent on the intracellular release, but not the silver ions liberated in the culture medium. Yet another story is the fate of NPs in the gastric tract. Swallowed NPs likely dissolve in the gastric fluid and penetrate intestines in the ionic form. Whether they are reduced back to nanoparticles or exist as ions is still a matter of debate.²³

Nanoparticle-induced damage to organelles and cell components

NPs easily pass cell membranes and enter organelles. Microphotographs from electron microscope clearly proved the presence of NPs in all major organelles, such as the mitochondria, nucleus, endoplasmic reticulum, Golgi apparatus, vacuoles, plastids and different vesicles, including endosomes, lysosomes. Kinetics of NPs uptake depends on their shape, size, surface charge, coating, and opsonization.

Nanoparticles present in cellular organelles might have detrimental effects on their integrity and functionality. Mitochondria seem to be a main target organelle with regard to NPs toxicity. Once inside the mitochondrion NPs affect its functionality changing the mitochondrial potential, lowering ATP production, and enhancing ROS production. ROS produced by the failing mitochondria might damage biological macromolecules, such as DNA, proteins, and lipids. In vitro studies revealed induction of DNA strand breaks and base oxidation by many NPs, including silver, cobalt, nickel, copper, zinc oxide, titanium dioxide NPs, quantum dots, carbon black, and single- and multiwall carbon nanotubes. As a consequence, mutations and structural and numeric aberrations of chromosomes (breaks, translocations, and aneuploidy) appear that might lead to cell death.²⁴ Formation of oxidative damage to DNA well correlates with a number of NPs up taken by the cell and production of ROS. While the formation of DNA breaks and oxidized bases seem to be a general phenomenon in NPs-exposed cells, induction of double-strand breaks (DSB) and micronuclei (MN) seems to be dependent on the type of NPs and cellular model and experimental setup. Whereas many NPs were reported to induce micronuclei both in vitro and in vivo, TiO2NPs were poor inducer of MN (reviewed in Ref. 25). In line, we were not able to prove induction of DSB nor MN in three human cell lines treated with AgNPs.²⁰

Treatment with NPs results also in protein oxidation observed usually as oxidation of protein thiol groups and/or formation of protein carbonyls. In some cases, modifications of aminoacids by reactive hypochlorite and peroxynitrite were also observed. Protein modifications result usually in conformational changes that in turn impairs protein functions.

NPs-induced oxidative stress may lead also to lipid peroxidation. Indeed, the formation of 4-hydroxynonenal, malondialdehyde, isoprostanes, or conjugated dienes was reported in many models, both in vitro and in vivo. Intense lipid peroxidation may result in the impairment of mitochondria and ER functionality, and in the worst scenario may lead to loss of membrane integrity and cell death.²⁶

Endocytosed NPs entrapped in endosomes are eventually degraded when endosomes fuse with lysosomes. However, the presence of NPs in endosome/lysosome may alter their function, as it was shown that ZnONPs induce a loss of lysosome membrane integrity in BV2 cells.²⁵ NPs can interact also with other cellular structures, such as the endoplasmic reticulum and Golgi apparatus.

Silica NPs accumulate in the endoplasmic reticulum and trigger autophagy,²⁷ while accumulation of AuNPs leads to reduction of endoplasmic reticulum stress.²⁸ There is some evidence that NPs can accumulate in the Golgi apparatus; however, no further information is available as to how they affect the activity of the Golgi apparatus.²⁹, ³⁰

Role of transition metals in nanoparticle toxicity

Damaged mitochondria and the resulting leakage of ROS is a commonly accepted mechanism of NP toxicity. Damage to the mitochondria results in the generation of superoxide anion radical (O2•−). The O2•− is a relatively inert chemical moiety and is rather unlikely to damage cell components or induce cell death. The species reacts slowly with DNA and even slower with proteins, peptides, and lipids. This makes a discrepancy between the production of O2•− by the failing mitochondria as the main mechanism of NP-induced toxicity and observed the detrimental effects of NPs on the cellular and the organismal level.

On the other hand, O2•− is effectively scavenged by nitric oxide to form very reactive peroxynitrite or dismutated to H2O2. Since the concentration of NO in the mitochondria is relatively low, the prevailing process is dismutation of O2•− by mitochondrial superoxide dismutase and production of H2O2 (Fig. 3). This is in agreement with a number of works on isolated mitochondria, in which production of H2O2 was used as a measurement of mitochondrial failure, clearly indicating that H2O2 is a final ROS released by collapsing mitochondria. Indeed, a due analysis of ROS generated in HepG2 cells treated with AgNPs using ROS-specific mitochondrial and cytoplasmic fluorescent probes revealed the generation of H2O2 instead of O2•− inside the failing mitochondria and in the cytoplasm. Consequently, no increase in O2•− level was observed in the cytosol nor in the mitochondria of AgNP-treated cells. Altogether, these results suggest that O2•− generated during NP-induced mitochondrial collapse is rapidly dismutated to H2O2.³¹ Generation of H2O2 due to mitochondrial dysfunction was also confirmed in the mitochondria isolated from rats treated to TiO2NP.³² It is also in good agreement with steady-state concentrations of O2•− (0.2–0.3 nM) and H2O2 (10–100 nM) in the mitochondria.³³

Fig. 3 Putative mechanism of induction of oxidative stress in AgNP-treated cells. [1] In the mitochondrion: Superoxide anion radical (O 2 •− ) generated by leaking mitochondria electron transport chain is either dismutated to hydrogen peroxide H 2 O 2 by superoxide dismutase 2 (SOD2, in matrix) or superoxide dismutase 1 (SOD1, in the intermembrane space), or protonated to form hydroperoxyl radical (HO 2 • ). Unlike O 2 •− , HO 2 • , and H 2 O 2 easily cross cell membranes, and so can penetrate between mitochondrial compartments or to the cytoplasm. H 2 O 2 formed in the mitochondrion can migrate to the cytoplasm or undergo iron-catalyzed Fenton reaction to form highly reactive hydroperoxyl radical ( • OH). [2] In the cytoplasm: HO 2 • can be deprotonated to form O 2 •− , that is further dismutated to H 2 O 2 by SOD1. O 2 •− is also generated by NADPH oxidase (NOX). H 2 O 2 formed in the cytoplasm can migrate to the mitochondrion or undergo iron-catalyzed Fenton reaction to form very reactive • OH. QI-QIV—electron transport chain. mtCIP, cytCIP—mitochondrial or cytosol chelatable iron pool. DFO—iron chelator, deferoxamine. (Reprinted with permission from Grzelak A, Wojewodzka M, Meczynska-Wielgosz S, Zuberek M, Wojciechowska D, Kruszewski M. Crucial role of chelatable iron in silver nanoparticles induced DNA damage and cytotoxicity. Redox Biol 2018; 15: 435–40.)

Nonetheless, prevalent production of H2O2 over O2•− by collapse of the mitochondria of NP-treated cells does not explain the observed nanoparticle genotoxicity and cytotoxicity. H2O2 is not very reactive, and therefore rather unlikely to damage DNA or cause cell death. However, H2O2 is reduced by trace amounts of transition metal ions (such as iron) to generate highly reactive hydroxyl radical (•OH) in the so-called Fenton reaction. The hydroxyl radical is commonly accepted as the main source of oxidative damage to the cell.³⁴ Transition metal-dependent generation of oxygen-derived free radicals is known to induce oxidation of proteins, lipids, and lipoproteins; nucleic acids, carbohydrates, and other cellular components. Iron salts induced DNA damage and decreased cell survival in vitro,³⁵ while animals suffering from iron overload is characterized by elevated levels of DNA damage and lipid peroxidation.³⁶ A positive correlation between the concentration of Fenton reaction available iron and the level of 8-oxo-7,8-dihydro-2-deoxyguanosine in the DNA was reported in human lymphocytes, indicating a direct link between the unshielded iron and DNA damage.³⁷ On the other hand, iron depletion by means of iron-specific chelators diminishes the deleterious effects of H2O2.

Antioxidants

Imbalance in the production and removal of free radicals might have fatal consequences for the cell. Cells have developed mechanisms enabling fast adaptation to temporary oxidative stress. The most commonly observed mechanism is an increase of activity of antioxidant enzymes and low molecular weight antioxidants, e.g., glutathione. Some NPs, e.g., AgNPs or AuNPs, have strong affinity to thiol groups, thus, their toxicity is attenuated by the presence of glutathione, but on the other hand, these NPs may directly bind to thiol-containing antioxidant enzymes, such as diminishing their activity and antioxidant potential of the whole cell. Indeed treatment of neural cells with AgNPs caused a decrease of intracellular thiols that led to a decrease in cell proliferation and apoptosis, whereas addition of thiol antioxidant (N-acetyl-cysteine) resulted in increased resistance to NPs action.¹⁴

Removal of excess of free radicals is a complex process involving different antioxidant systems (Table 3). In addition to enzymatic antioxidant systems several proteins decrease oxidative stress by elimination of transition metal ions, the catalysts of •OH production. These include transferrin, ferritin, ceruloplasmin, haptoglobin, hemopexin, and metallothionein. Finally, the enzymatic and nonenzymatic proteinous antioxidant systems are supported by a battery of low molecular weight antioxidants, such as glutathione, ascorbate, uric acid, cysteine, creatinine, carnosine, bilirubin, and many others. Many other compounds present in our diet have also antioxidative properties that might affect cellular response to NPs.

Table 3

Nanoparticle-induced nitrosative stress

Many NPs induce expression and activity of nitric oxide synthase.³⁸ Reaction of nitric oxide with superoxide leads to the formation of peroxynitrite. Peroxynitrite is a very reactive chemical moiety that might damage cellular components and affect cell functions. However, formation of peroxynitrite seems to depend on the composition of NPs, as despite the induction of nitric oxide synthase, exposure to AgNPs did not lead to excess production of peroxynitrite in HepG2 cells, because the formed peroxynitrite was decomposed by the NPs.³⁹ Whether peroxynitrite is formed after exposure to other NPs and produced peroxynitrite-induced damage to cells needs further clarification.

Nanoparticle-induced signal transduction in response to oxidative stress

Increasing oxidative stress and decreasing capability of the cell to detoxicate ROS leads to activation of signaling pathways responsible for keeping cellular homeostasis. Activation of these pathways leads to an increase of activity of antioxidant enzymes, e.g., catalase, superoxide dismutase, or glutathione peroxidase, and nonenzymatic low-molecular weight antioxidants, such as glutathione. On the other hand, overactivation of these pathways leads to apoptosis and/or autophagy.

In physiological conditions, ROS are crucial for proper function of cellular signaling pathways directly affecting the activity of signal messengers. Many transcription factors (including NF-κB, AP-1, HIF-1, and p53) has redox sensitive thiol groups in DNA-binding domains. Oxidation of these groups as a result of increasing oxidative stress seems to be a key mechanism affecting functionality and activity of these transcription factors, and indirectly nanoparticle toxicity.⁴⁰

The key factors responding to the changes of cellular redox state are NF-κB and Nrf2/KEAP1 transcription factors. The first described was activation of NF-κB by H2O2 through the canonic pathway dependent on IKK.⁴¹ In line, activation of NF-κB by NP-induced ROS was described in many cellular and animal models.⁴²–⁴⁴

Another well-described signaling pathway responding to ROS overproduction is the NRF2/KEAP1 pathway. It is likely a crucial pathway in cellular response to oxidative stress, as it regulates expression of the majority of proteins involved in antioxidant defense.⁴⁵ It was shown that palladium NPs activate NRF2/KEAP1 pathway in human keratinocytes.⁴⁶ NRF2/KEAP1 activation was also crucial for induction of expression of heme oxygenase-1 gene in endothelial cells treated with gold NPs.⁴⁷ Metallic NPs activate also transcription factor AP-1 ⁴⁸ that is responsible for cell proliferation and differentiation, apoptosis and carcinogenesis.⁴⁹

NPs-induced oxidative stress induces also signaling pathways independent of the oxidation of thiol groups oxidation, such as the mitogen-activated protein kinases (MAPK). MAPK activation is necessary for many basic cell functions, such as cell cycle progression, gene expression and cell death; however, its overactivation may have detrimental effects, such as carcinogenesis.⁵⁰ Mechanism of MAPK activation depends on NPs and types of cells. AgNPs stimulate MAPK directly activating EGF receptor or activating stress-activated protein kinase pathway.⁵¹ AgNPs-induced ROS activate also JNK signaling pathway inducing apoptosis.⁵² Whereas CeO2NPs activate p38 MAPK pathway.⁵³

In addition, NPs specifically modify activity of other signaling pathways, e.g., carbon NPs activate phosphoinositide 3-kinase (PI3K) pathway and serine-threonine protein kinase B (Akt) directly activating EGF receptor and integrin β1.⁵⁴ The EGFR pathway was also activated by PM2.5 and diesel exhaust particles in isolated human bronchial cells that led to activation of ERK pathway and chronic inflammation.

Summary

Despite the benefit that nanotechnology brings to society, in recent decades there is increasing concern on the detrimental effects that development on nanotechnology might bring to humans and the environment. It has been shown that toxicity of nanomaterials correlates with several universal parameters, such as the NP size or diameter, NP concentration, the method of NP synthesis, coating, time and ways of exposure, and the biological model used to evaluate toxicity (for a review regarding AgNP toxicity, see Ref. 13). NPs that enter the cell induce oxidative stress and activate ROS-dependent cellular signaling pathways. ROS generation is likely associated with mitochondrial damage and malfunction, whereas moderate oxidative stress is balanced by antioxidant defense systems; massive ROS generation leads to collapse of energy production and triggers apoptosis.

Applications to other areas of toxicology

Oxidative stress understood as an imbalance between the generation of free radicals and their neutralization is a common phenomenon associated with normal cell functioning. Occurrence of oxidative stress is associated with exposure to many xenobiotics commonly present in our everyday life, such as crop prevention products, e.g., paraquat, diquat, organophosphates, organochlorines, or pyrethroids; many drugs, e.g., bleomycin, anthracyclines, menadione, nonsteroidal antiinflammatory drugs, antibiotics, etc.; or exposure to transition metal ions, like iron, copper, and chromium. Oxidative stress is also induced by exposure to environmental pollutants, such as PM10 or PM2.5 particles, polycyclic aromatic hydrocarbons, diesel exhaust, etc. In general, since cellular mechanisms of antioxidant defense have limited efficacy, every chemical or physical factor producing free radicals can elicit oxidative stress, if in excess amount. Occurrence of the oxidative stress is also associated with many pathological processes, such as infections, ischemia-reperfusion, inflammation, diabetes, neurodegenerative disorders, and asthma.

General mechanisms of free radical formation, their action, and mechanisms of their neutralization are common despite the origin and source of radicals. Hence, mechanisms and processes described in this chapter are relevant to many other situations, both physiological and pathological.

Summary points

•This chapter focuses on the role of oxidative stress in NP toxicity.

•Oxidative stress plays a crucial role in induction of cell death after NP exposure.

•Production of reactive oxygen species in nanoparticle-treated cells correlates with induction of damage to DNA, proteins, and lipids.

•The primary source of oxidative stress is malfunction of mitochondria. Nonmitochondrial enzymatic complexes, such as NADPH oxidases, are also involved.

•Nanoparticle-induced oxidative stress triggers cellular signaling pathways that lead to cell death or survival, depending on the cellular context.

•The primary reactive species produced is superoxide that is further dismutated to hydrogen peroxide. Hydrogen peroxide, in turn, in the presence of trace amounts of transition metals gives rise to the formation of highly reactive hydroxyl radicals.

References

1 AshaRani P.V., Hande M.P., Valiyaveettil S. Anti-proliferative activity of silver nanoparticles. BMC Cell Biol. 2009;10:65.

2 Bressan E., Ferroni L., Gardin C., et al. Silver nanoparticles and mitochondrial interaction. Int J Dent. 2013;2013:312747.

3 Maurer L.L., Meyer J.N. A systematic review of evidence for silver nanoparticle-induced mitochondrial toxicity. Environ Sci Nano. 2016;3(2):311–322.

4 Costa C.S., Ronconi J.V., Daufenbach J.F., et al. In vitro effects of silver nanoparticles on the mitochondrial respiratory chain. Mol Cell Biochem. 2010;342(1–2):51–56.

5 Zuberek M., Wojciechowska D., Krzyzanowski D., Meczynska-Wielgosz S., Kruszewski M., Grzelak A. Glucose availability determines silver nanoparticles toxicity in HepG2. J Nanobiotechnol. 2015;13:72.

6 Fransen M., Nordgren M., Wang B., Apanasets O. Role of peroxisomes in ROS/RNS-metabolism: implications for human disease. Biochim Biophys Acta. 2012;1822(9):1363–1373.

7 Rinna A., Magdolenova Z., Hudecova A., Kruszewski M., Refsnes M., Dusinska M. Effect of silver nanoparticles on mitogen-activated protein kinases activation: role of reactive oxygen species and implication in DNA damage. Mutagenesis. 2015;30(1):59–66.

8 Mo Y., Wan R., Chien S., Tollerud D.J., Zhang Q. Activation of endothelial cells after exposure to ambient ultrafine particles: the role of NADPH oxidase. Toxicol Appl Pharmacol. 2009;236(2):183–193.

9 Chen M., von Mikecz A. Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO2 nanoparticles. Exp Cell Res. 2005;305(1):51–62.

10 Alvarez Y.D., Fauerbach J.A., Pellegrotti J.V., Jovin T.M., Jares-Erijman E.A., Stefani F.D. Influence of gold nanoparticles on the kinetics of alpha-synuclein aggregation. Nano Lett. 2013;13(12):6156–6163.

11 Mirsadeghi S., Dinarvand R., Ghahremani M.H., et al. Protein corona composition of gold nanoparticles/nanorods affects amyloid beta fibrillation process. Nanoscale. 2015;7(11):5004–5013.

12 Yang J., Pong B.K., Lee J.Y., Too H.P. Dissociation of double-stranded DNA by small metal nanoparticles. J Inorg Biochem. 2007;101(5):824–830.

13 Akter M., Sikder M.T., Rahman M.M., et al. A systematic review on silver nanoparticles-induced cytotoxicity: physicochemical properties and perspectives. J Adv Res. 2018;9:1–16.

14 Haase A., Rott S., Mantion A., et al. Effects of silver nanoparticles on primary mixed neural cell cultures: uptake, oxidative stress and acute calcium responses. Toxicol Sci. 2012;126(2):457–468.

15 Kusaczuk M., Kretowski R., Naumowicz M., Stypulkowska A., Cechowska-Pasko M. Silica nanoparticle-induced oxidative stress and mitochondrial damage is followed by activation of intrinsic apoptosis pathway in glioblastoma cells. Int J Nanomedicine. 2018;13:2279–2294.

16 Ahamed M. Silica nanoparticles-induced cytotoxicity, oxidative stress and apoptosis in cultured A431 and A549 cells. Hum Exp Toxicol. 2013;32(2):186–195.

17 Ahmad J., Ahamed M., Akhtar M.J., et al. Apoptosis induction by silica nanoparticles mediated through reactive oxygen species in human liver cell line HepG2. Toxicol Appl Pharmacol. 2012;259(2):160–168.

18 Joshi G.N., Knecht D.A. Silica phagocytosis causes apoptosis and necrosis by different temporal and molecular pathways in alveolar macrophages. Apoptosis. 2013;18(3):271–285.

19 Loza K., Diendorf J., Sengstock C., et al. The dissolution and biological effects of silver nanoparticles in biological media. J Mater Chem B. 2014;2(12):1634–1643.

20 Kruszewski M., Gradzka I., Bartlomiejczyk T., et al. Oxidative DNA damage corresponds to the long term survival of human cells treated with silver nanoparticles. Toxicol Lett. 2013;219(2):151–159.

21 He D., Garg S., Waite T.D. H2O2-mediated oxidation of zero-valent silver and resultant interactions among silver nanoparticles, silver ions, and reactive oxygen species. Langmuir. 2012;28(27):10266–10275.

22 De Matteis V., Malvindi M.A., Galeone A., et al. Negligible particle-specific toxicity mechanism of silver nanoparticles: the role of Ag + ion release in the cytosol. Nanomedicine. 2015;11(3):731–739.

23 Walczak A.P., Fokkink R., Peters R., et al. Behaviour of silver nanoparticles and silver ions in an in vitro human gastrointestinal digestion model. Nanotoxicology. 2013;7(7):1198–1210.

24 Magdolenova Z., Collins A., Kumar A., Dhawan A., Stone V., Dusinska M. Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles. Nanotoxicology. 2014;8(3):233–278.

25 Swedish Chemicals Agency. Nanomaterials and genotoxicity—a literature review. Report 13/16 Stockholm: Swedish Chemicals Agency; 2016.

26 Zuberek M., Grzelak A. Nanoparticles-caused oxidative imbalance. Adv Exp Med Biol. 2018;1048:85–98.

27 Wei F., Wang Y., Luo Z., Li Y., Duan Y. New findings of silica nanoparticles induced ER autophagy in human colon cancer cell. Sci Rep. 2017;7:42591.

28 Gunduz N., Ceylan H., Guler M.O., Tekinay A.B. Intracellular accumulation of gold nanoparticles leads to inhibition of macropinocytosis to reduce the endoplasmic reticulum stress. Sci Rep. 2017;7:40493.

29 Cao Z., Peng F., Hu Z., et al. In vitro cellular behaviors and toxicity assays of small-sized fluorescent silicon nanoparticles. Nanoscale. 2017;9(22):7602–7611.

30 Silva E., Barreiros L., Segundo M.A., Costa Lima S.A., Reis S. Cellular interactions of a lipid-based nanocarrier model with human keratinocytes: unravelling transport mechanisms. Acta Biomater. 2017;53:439–449.

31 Grzelak A., Wojewodzka M., Meczynska-Wielgosz S., Zuberek M., Wojciechowska D., Kruszewski M. Crucial role of chelatable iron in silver nanoparticles induced DNA damage and cytotoxicity. Redox Biol. 2018;15:435–440.

32 Stapleton P.A., Nichols C.E., Yi J., et al. Microvascular and mitochondrial dysfunction in the female F1 generation after gestational TiO2 nanoparticle exposure. Nanotoxicology. 2015;9(8):941–951.

33 Cadenas E., Boveris A. Mitochondrial free radical production, antioxidant defenses and cell signaling. In: Grune T., ed. Oxidants and antioxidant defense systems. Berlin, Heidelberg: Springer-Verlag; 2005:219–234.

34 Liochev S.I., Fridovich I. The relative importance of HO and ONOO− in mediating the toxicity of O. Free Radic Biol Med. 1999;26(5–6):777–778.

35 Abalea V., Cillard J., Dubos M.P., Anger J.P., Cillard P., Morel I. Iron-induced oxidative DNA damage and its repair in primary rat hepatocyte culture. Carcinogenesis. 1998;19(6):1053–1059.

36 Galleano M., Puntarulo S. Hepatic chemiluminescence and lipid peroxidation in mild iron overload. Toxicology. 1992;76(1):27–38.

37 Gackowski D., Kruszewski M., Bartlomiejczyk T., Jawien A., Ciecierski M., Olinski R. The level of 8-oxo-7,8-dihydro-2′-deoxyguanosine is positively correlated with the size of the labile iron pool in human lymphocytes. J Biol Inorg Chem. 2002;7(4):548–550.

38 Barcinska E., Wierzbicka J., Zauszkiewicz-Pawlak A., Jacewicz D., Dabrowska A., Inkielewicz-Stepniak I. Role of oxidative and nitro-oxidative damage in silver nanoparticles cytotoxic effect against human pancreatic ductal adenocarcinoma cells. Oxid Med Cell Longev.