## 4.2.3. Oxidative stress - II.

Induction by chemical exposure and possible effects

Author: Frank van Belleghem

Reviewers: Raymond Niesink, Kees van Gestel, Éva Hideg

Learning objectives:

You should be able to

- explain how xenobiotic compounds can lead to an increased production of.
- explain what oxidative stress does with
  - proteins,
  - lipids,
  - DNA and
  - gene regulation.

Keywords: prooxidant-antioxidant balance, bioactivation, oxidative damage,

How xenobiotic compounds induce generation of ROS

The formation of reactive oxygen species (ROS; see section on Oxidative stress I) may involve endogenous substances and chemical-physiological processes as well as xenobiotics. Experimental evidence has shown that oxidative stress can be considered as one of the key mechanisms contributing to the cellular damage of many toxicants. Oxidative stress has been defined as “a disturbance in the prooxidant-antioxidant balance in favour of the former”, leading to potential damage. It is the point at which the production of ROS exceeds the capacity of antioxidants to prevent damage (Klaassen et al., 2013).

Xenobiotics involved in the formation of the superoxide anion radical are mainly substances that can be taken up in so reactive oxygen species -called redox cycles. These include quinones and hydroquinones in particular. In the case of quinones the redox cycle starts with a one-electron reduction step, just as in the case of benzoquinone (Figure 1). The resulting benzosemiquinone subsequently passes the electron received on to molecular oxygen. The reduction of quinones is catalyzed by the NADPH-dependent cytochrome P-450 reductase.

Figure 1. The bioactivation of benzoquinone by the cytochrome P450 system under the generation of ROS. Figure adapted from Niesink et al. (1996) by Steven Droge.

Obviously, hydroquinones can enter a redox cycle via an oxidative step. This step may be catalyzed by enzymes, for example prostaglandin synthase.

Other types of xenobiotic that can be taken up in a redox cycle, are the bipyridyl derivatives. A well-known example is the herbicide paraquat, which causes injury to lung tissue in humans and animals. Figure 2 schematically shows its bioactivation. Other compounds that are taken up in a redox cycle are nitroaromatics, azo compounds, aromatic hydroxylamines and certain metal (particularly Cu and Zn) chelates.

Figure 2. The bioactivation (via electron donation) of paraquat by the cytochrome P450 system under the generation of ROS. Figure adapted from Niesink et al. (1996) by Steven Droge.

Xenobiotics can enhance ROS production if they are able to enter mitochondria, microsomes, or chloroplasts and interact with the electron transport chains, thus blocking the normal electron flow. As a consequence, and especially if the compounds are electron acceptors, they divert the normal electron flow and increase the production of ROS. A typical example is the cytostatic drug doxorubicin, a well-known chemotherapeutic agent, which is used in treatment of a wide variety of cancers. Doxorubicin has a high affinity for cardiolipin, an important compound of the inner mitochondrial membrane and therefore accumulates at that subcellular location.

Xenobiotics can cause oxidative damage indirectly by interfering with the antioxidative mechanisms. For instance it has been suggested that as a non-Fenton metal, cadmium (Cd) is unable to directly induce ROS. However, indirectly, Cd induces oxidative stress by a displacement of redox-active metals, depletion of redox scavengers (glutathione) and inhibition of antioxidant enzymes (protein bound sulfhydryl groups) (Cuypers et al., 2010;Thévenod et al., 2009).

The mechanisms of oxidative stress

As mentioned before, oxidative stress has been defined as “a disturbance in the prooxidant-antioxidant balance in favour of the former”. ROS can damage proteins, lipids and DNA via direct oxidation, or through redox sensors that transduce signals, which in turn can activate cell-damaging processes like apoptosis.

Oxidative protein damage

Xenobiotic-induced generation of ROS can damage proteins through the oxidation of side chains of amino acids residues, the formation of protein-protein cross-links and fragmentation of proteins due to peptide backbone oxidation. The sulfur-containing amino acids cysteine and methionine are particularly susceptible for oxidation. An example of side chain oxidation is the direct interaction of the superoxide anion radical with sulfhydryl (thiol) groups, thereby forming thiyl radicals as intermediates:

As a consequence, glutathione, composed of three amino acids (cysteine, glycine, and glutamate) and an important cellular reducing agent, can be damaged in this way. This means that if the oxidation cannot be compensated or repaired, oxidative stress can lead to depletion of reducing equivalents, which may have detrimental effects on the cell.

Fortunately, antioxidant defence mechanisms limit the oxidative stress and the cell has repair mechanisms to reverse the damage. For example, heat shock proteins (hsp) are able to renature damaged proteins and oxidatively damaged proteins are degraded by the proteasome.

Oxidative lipid damage
Increased concentrations of reactive oxygen radicals can cause membrane damage due to lipid peroxidation (oxidation of polyunsaturated lipids). This damage may result in altered membrane fluidity, enzyme activity and membrane permeability and transport characteristics. An important feature characterizing lipid peroxidation is the fact that the initial radical-induced damage at a certain site in a membrane lipid is readily amplified and propagated in a chain-reaction-like fashion, thus dispersing the damage across the cellular membrane. Moreover, the products arising from lipid peroxidation (e.g. alkoxy radicals or toxic aldehydes) may be equally reactive as the original ROS themselves and damage cells by additional mechanisms. The chain reaction of lipid peroxidation consists of three steps:

1. Abstraction of a hydrogen atom from a polyunsaturated fatty acid chain by reactive oxygen radicals (radical formation, initiation).
2. Reaction of the resulting fatty acid radical with molecular oxygen (oxygenation or, more specifically, peroxidation, propagation)
3. These events may be followed by a detoxification process, in which the reaction chain is stopped. This process, which may proceed in several steps, is sometimes referred to as termination.

Figure 3 summarizes the various stages in lipid peroxidation.

Figure 3. The different steps in the lipid peroxidation chain reaction. LH = polyunsaturated fatty acid, OH● = hydroxyl radical, L● = lipid radical; LOO●/LO●2= lipid peroxyl radical; LOOH = lipid peroxide. Figure adapted from Niesink et al. (1996) by Steven Droge.

In step II, the peroxidation of biomembranes generates a variety of reactive electrophiles such as epoxides (LOO•) and aldehydes, including malondialdehyde (MDA). MDA is a highly reactive aldehyde which exhibits reactivity toward nucleophiles and can form MDA–MDA dimers. Both MDA and the MDA–MDA dimers are mutagenic and indicative of oxidative damage of lipids from a variety of toxicants.

A classic example of xenobiotic bioactivation to a free radical that initiates lipid peroxidation is the cytochrome P450-dependent conversion of carbon tetrachloride (CCl4) to generate the trichloromethyl radical (•CCl3) and then the trichloromethyl peroxylradical CCl3OO•. Also the cytotoxicity of free iron is attributed to its function as an electron donor for the Fenton reaction (see section on Oxidative stress I) for instance via the generation of superoxide anion radicals by paraquat redox cycling) leading to the formation of the highly reactive hydroxyl radical, a known initiator of lipid peroxidation.

Oxidative DNA damage
ROS can also oxidize DNA bases and sugars, produce single- or double-stranded DNA breaks, purine, pyrimidine, or deoxyribose modifications and DNA crosslinks. A common modification to DNA is the hydroxylation of DNA bases leading to the formation of oxidized DNA adducts. Although these adducts have been identified in all four DNA bases, guanine is the most susceptible to oxidative damage because it has the lowest oxidation potential of all of the DNA bases. The oxidation of guanine and by hydroxyl radicals leads to the formation 8-hydroxyguanosine (8-OH-dG) (Figure 4).

Figure 4. The hydroxylation of guanine. Drawn by Steven Droge.

Oxidation of guanine has a detrimental effect on base paring, because instead of hydrogen bonding with cytosine as guanine normally does, it can form a base pair with adenine. As a result, during DNA replication, DNA polymerase may mistakenly insert an adenosine opposite to an 8-oxo-2'-deoxyguanosine (8-oxo-dG), resulting in a stable change in DNA sequence, a process known as mutagenesis (Figure 5).

Figure 5. Base paring with 8-oxo-2'-deoxyguanosine (8-oxo-dG). Drawn by Steven Droge.

Fortunately, there is an extensive repair mechanism that keeps mutations to a relatively low level. Nevertheless, persistent DNA damage can result in replication errors, transcription induction or inhibition, induction of signal transduction pathways and genomic instability, events that are possibly involved in carcinogenesis (Figure 6). It has to be mentioned that mitochondrial DNA, is more susceptible to oxidative base damage compared to nuclear DNA due to its proximity to the electron transport chain (a source of ROS), and the fact that mitochondrial DNA is not protected by histones and has a limited DNA repair system.

Figure 6. Oxidative damage by ROS leading to mutations and eventually to tumour formation. Figure adapted from Boelsterli (2002) by Evelin Karsten-Meessen.

One group of xenobiotics that have clearly been associated with eliciting oxidative DNA damage and cancer are redox-active metals, including Fe(III), Cu(II), Ag(I), Cr(III), Cr(VI), which may entail, as seen before, the production of hydroxyl radicals. Other (non-redox-active) metals that can induce ROS-formation themselves or participate in the reactions leading to endogenously generated ROS are Pb(II), Cd(II), Zn(II), and the metalloid As(III) and As(V). Compounds like polycyclic aromatic hydrocarbons (PAHs), likely the largest family of pollutants with genotoxic effects, require activation by endogenous metabolism to become reactive and capable of modifying DNA. This activation is brought about by the so-called Phase I biotransformation (see Section on Xenobiotic metabolism and defence).

Genetic detoxifying enzymes, like cytochrome P-450A1, are able to hydrophylate hydrophobic substrates. Whereas this reaction normally facilitates the excretion of the modified substance, some polycyclic aromatic hydrocarbons (PAHs), like benzo[a]pyrene generate semi stable epoxides that can ultimately react with DNA forming mutagenic adducts (see Section on Xenobiotic metabolism and defence). The main regulator of phase I metabolism in vertebrates, the Aryl hydrocarbon receptor (AhR), is a crucial player in this process. Some PAHs, dioxins, and some PCBs (the so-called coplanar congeners; see section on Complex mixtures) bind and activate AhR and increase the activity of phase I enzymes, including cytochrome P-450A1 (CYP1A1), by several fold. This increased oxidative metabolism enhances the toxic effects of the substances leading to increased DNA damage and inflammation (Figure 7).

Figure 7. Environmental pollutants such as Dioxines, PCBs, PAHs (such as benzo[a]pyrene) bind to AhR and induce ROS production, DNA damage, and inflammatory cytokine production. Drawn by Frank van Belleghem.

Oxidative effects on cell growth regulation

ROS production and oxidative stress can act both on cell proliferation and apoptosis. It has been demonstrated that low levels of ROS influence signal transduction pathways and alter gene expression.

Figure 8. Role of ROS in altered gene expression. Figure adapted from Klaassen (2013) by Evelin Karsten-Meessen.

Many xenobiotics, by increasing cellular levels of oxidants, alter gene expression through activation of signaling pathways including cAMP-mediated cascades, calcium-calmodulin pathways, transcription factors such as AP-1 and NF-κB, as well as signaling through mitogen activated protein (MAP) kinases (Figure 8). Activation of these signaling cascades ultimately leads to altered gene expression or a number of genes including those affecting proliferation, differentiation, and apoptosis.

References

Boelsterli, U.A. (2002). Mechanistic toxicology: the molecular basis of how chemicals disrupt biological targets. CRC Press.

Cuypers, A., Plusquin, M., Remans, T., Jozefczak, M., Keunen, E., Gielen, H., ... , Nawrot, T. (2010). Cadmium stress: an oxidative challenge. Biometals 23, 927-940.

Furue, M., Takahara, M., Nakahara, T., Uchi, H. (2014). Role of AhR/ARNT system in skin homeostasis. Archives of Dermatological Research 306, 769-779.

Klaassen, C.D. (2013). Casarett & Doull's Toxicology: The Basic Science of Poisons, Eighth Edition, McGraw-Hill Professional.

Niesink, R.J.M., De Vries, J. & Hollinger, M. A. (1996). Toxicology: Principles and Applications. CRC Press.

Thévenod, F. (2009). Cadmium and cellular signaling cascades: to be or not to be? Toxicology and Applied Pharmacology 238, 221-239.