214
8.4.2 Oxidative Stress
Oxidative stress is an important parameter for evaluating NMs toxicity (CaballeroDiaz and Cases 2016; Czerska et al. 2015). Oxidative stress is defined as a biochemical imbalance that leads to the production of free radicals (pro-oxidants) and
the molecules responsible for their removal (antioxidant defense mechanisms)
(Czerska et al. 2015).
Reactive oxygen (ROS) and nitrogen (RNS) species are considered the prooxidant markers of oxidative stress, and for their detection several molecular probes
have been developed (Gunsolus and Haynes 2016; Jing et al. 2015; Czerska et al.
2015). The non-polar 2′,7′-dichlorohydrofluorescein diacetate (DCFH-DA) is one
of the mainly used probes (Aranda et al. 2013). Once internalized, DCFH-DA is
converted into the polar derivative DCFH by cellular esterases and subsequently
oxidized to a highly fluorescent 2′,7′-dichlorofluorescein (DCF) by radical species.
As both ROS and RNS are responsible for its formation (Crow 1997), DCF fluorescence is an indicator of oxidative stress without identifying any specific radical
species (Marchesi et al. 1999). This molecular probe can be monitored by fluorimetry, flow cytometry, or by fluorescence microscopy, making it highly flexible for
the observation of the formation of radical species (Love et al. 2012; Stone
et al. 2009).
Considering also the antioxidant defense mechanisms, the monitorization of the
regulation of biological oxidant molecules can also provide insight on the oxidative
stress imbalance. For instance, glutathione (GSH) is an important antioxidant in
plants, animals, fungi, and some bacteria and archaea, playing a role in preventing
damages caused by reactive oxygen species. It exists in both reduced (GSH) and
oxidized (GSSG) states, and in healthy conditions more than 90% of the total glutathione presents in its reduced form. Thus, an increased GSSG-to-GSH ratio is considered indicative of oxidative stress (Doktorovova et al. 2014b, 2016). Several in
vitro assays based on colorimetric, fluorescent, or luminescent molecular probes
can be used to estimate GSSG-to-GSH ratio (Meloni and Nicolay 2003). Moreover,
the activation and upregulation of the antioxidant enzyme superoxide dismutase
(SOD) represents another cellular defense against oxidative stress. SOD activation
can be assessed measuring superoxide-dependent conversion of substrates such as
dihydroethidium (DHE) or nitroblue tetrazolium (NTB) into the red fluorescent
DHE or to blue formazan, respectively (Love et al. 2012; Stone et al. 2009). SOD
upregulation can be quantified by immunoblotting techniques (Love et al. 2012).
While molecular probes such as DCFH-DA directly measure radical species formation, oxidative stress generation can be indirectly investigated by analyzing
oxidative- dependent damages to biological molecules as proteins, lipids, sugars,
and nucleic acids. For instance, lipid peroxidation and protein carbonylation protein
oxidation reactions promoted by ROS and their products can be considered as oxidative stress biomarkers. Depending on the biomolecular reaction, specific mass
spectrometry and chromatographic techniques have been adopted, as well as
M. C. Teixeira et al.
8.4.2 Oxidative Stress
Oxidative stress is an important parameter for evaluating NMs toxicity (CaballeroDiaz and Cases 2016; Czerska et al. 2015). Oxidative stress is defined as a biochemical imbalance that leads to the production of free radicals (pro-oxidants) and
the molecules responsible for their removal (antioxidant defense mechanisms)
(Czerska et al. 2015).
Reactive oxygen (ROS) and nitrogen (RNS) species are considered the prooxidant markers of oxidative stress, and for their detection several molecular probes
have been developed (Gunsolus and Haynes 2016; Jing et al. 2015; Czerska et al.
2015). The non-polar 2′,7′-dichlorohydrofluorescein diacetate (DCFH-DA) is one
of the mainly used probes (Aranda et al. 2013). Once internalized, DCFH-DA is
converted into the polar derivative DCFH by cellular esterases and subsequently
oxidized to a highly fluorescent 2′,7′-dichlorofluorescein (DCF) by radical species.
As both ROS and RNS are responsible for its formation (Crow 1997), DCF fluorescence is an indicator of oxidative stress without identifying any specific radical
species (Marchesi et al. 1999). This molecular probe can be monitored by fluorimetry, flow cytometry, or by fluorescence microscopy, making it highly flexible for
the observation of the formation of radical species (Love et al. 2012; Stone
et al. 2009).
Considering also the antioxidant defense mechanisms, the monitorization of the
regulation of biological oxidant molecules can also provide insight on the oxidative
stress imbalance. For instance, glutathione (GSH) is an important antioxidant in
plants, animals, fungi, and some bacteria and archaea, playing a role in preventing
damages caused by reactive oxygen species. It exists in both reduced (GSH) and
oxidized (GSSG) states, and in healthy conditions more than 90% of the total glutathione presents in its reduced form. Thus, an increased GSSG-to-GSH ratio is considered indicative of oxidative stress (Doktorovova et al. 2014b, 2016). Several in
vitro assays based on colorimetric, fluorescent, or luminescent molecular probes
can be used to estimate GSSG-to-GSH ratio (Meloni and Nicolay 2003). Moreover,
the activation and upregulation of the antioxidant enzyme superoxide dismutase
(SOD) represents another cellular defense against oxidative stress. SOD activation
can be assessed measuring superoxide-dependent conversion of substrates such as
dihydroethidium (DHE) or nitroblue tetrazolium (NTB) into the red fluorescent
DHE or to blue formazan, respectively (Love et al. 2012; Stone et al. 2009). SOD
upregulation can be quantified by immunoblotting techniques (Love et al. 2012).
While molecular probes such as DCFH-DA directly measure radical species formation, oxidative stress generation can be indirectly investigated by analyzing
oxidative- dependent damages to biological molecules as proteins, lipids, sugars,
and nucleic acids. For instance, lipid peroxidation and protein carbonylation protein
oxidation reactions promoted by ROS and their products can be considered as oxidative stress biomarkers. Depending on the biomolecular reaction, specific mass
spectrometry and chromatographic techniques have been adopted, as well as
M. C. Teixeira et al.