This chapter reviews the effects of vitamin C on metal-induced genotoxicity. By focusing on cutting-edge studies, including our own results in experiments with vanadium(V) and chromium(VI), the suggestion that vitamin C can be used effectively to protect against or reduce the genotoxic effects induced by metal exposure by suppressing oxidative stress is particularly explored. After explaining the chemical mechanisms involved in oxidative stress associated with heavy metals, this chapter discusses the various proposals regarding the physiological processes of vitamin C at the molecular level, its relationship with oxidative stress, levels of 8-hydroxydeoxyguanosine (8-OH-dG, 7,8-dihy-dro-8-oxodeoxyguanosine) and apoptosis, and its role in the protection and modulation of DNA damage, as well as how they fit with our own results that showed an increase in apoptosis and 8-OH-dG when vitamin C was administered in addition to the metallic compounds. The relevant gaps in our understanding of the role of vitamin C with regard to these issues are highlighted, as well as the key importance of its clinical use, and ultimately, human health.
- vitamin C
- genotoxic damage
- heavy metals
- oxidative stress
Several studies have suggested that diets rich in fresh fruits and vegetables are associated with a lower risk of cardiovascular diseases and cancer because of the high levels of antioxidants such as vitamin C and polyphenols present in these foods . The antioxidant effects of vitamin C have been observed both
A significant number of studies have focused on metal-induced toxicity and carcinogenicity by emphasizing their role in the generation of ROS. Metal-mediated formation of free radicals may cause modifications to DNA bases, lipid peroxidation, and changes in calcium and sulfhydryl homeostasis [4, 5]. However, these effects can be influenced by the action of low molecular weight antioxidants such as vitamin C, which is capable of chelating metal ions, reducing their catalytic activity, and resulting ROS formation. Since the genotoxicity of heavy metals associated with oxidative stress is based on the oxidative mechanism during reduction , vitamin C can be used effectively to protect or reduce the induced genotoxic effects by suppressing oxidative stress caused by these metallic compounds [6–8]. However, paradoxically under certain conditions (i.e., low concentration
2. Heavy metals and oxidative stress: the case of vanadium and chromium
It is well established that redox-active metals participate closely in the generation of different free radicals . Exposure to transition metal ions(n+) such as chromium (Cr) and vanadium (V) hence represent a realistic
The •OH is the most reactive of all the ROS (half-life <1 ns) and interacts with all components of the DNA molecule. The initial stage of mutagenesis, carcinogenesis, and aging involves the permanent modification of genetic material. In fact, it has been well documented that in various cancer tissues, free radical-mediated DNA damage has occurred. ROS-induced DNA damage involves single- or double-stranded DNA breaks, purine, pyrimidine, or deoxyribose modifications, and DNA cross-links [5, 13, 14].
As mentioned above, the main genotoxic mechanism of V(V) and Cr(VI) compounds has been linked to reduction and generation of •OH [15, 16]. Reduction of V(V) to V(IV) takes place outside the cell (Figure 2). In plasma, V(V) is rapidly reduced to V(IV) by nicotinamide adenine dinucleotide phosphate (NADPH) and ascorbic acid. Once reduced, it is bonded with plasma proteins that carry it into the cell, where peroxovanadyl radicals [V(IV)–OO•] and vanadyl hydroperoxide [V(IV)–HO•] are formed. The generated superoxide is further converted into H2O2 by the dismutation reaction with superoxide dismutase (SOD). V(IV) can react through the Fenton reaction with H2O2 forming a •OH (Figure 2,
The genetic damage by the production of C8-OH-adduct radical of deoxyguanosine is generated by the interaction between •OH and 2-deoxyguanosine. Therefore, there are two ways in which the protection and modulation of DNA oxidative damage could be caused. First, AscH− could react with •OH, quenching and converting it into a poorly reactive semi-hydroascorbate radical, which do not cause DNA damage (Figures 2 and 3,
Although the direct relationship between DNA damage and •OH is not completely clear, Patlolla et al.  have suggested a role for ROS in Cr(VI)-induced genotoxicity and cytotoxicity. They showed that Cr(VI) induced genomic DNA damage through the formation of 8-OH-dG. Nevertheless, Rudolf and Cérvinka  observed that Cr(VI) induced time- and concentration-dependent cytotoxicity, resulting in oxidative stress, but through suppression of antioxidant systems and by activation of p53-dependent apoptosis. Other studies have questioned the genotoxic/mutagenic effect of •OH in the context of Cr exposure, suggesting that reduction of Cr(VI) by physiological concentrations of vitamin C generates ascorbate-Cr(III)-DNA crosslinks and binary Cr(III)-DNA adducts. Therefore, Cr-DNA adducts are responsible for both the mutagenicity and genotoxicity of Cr(VI) .
3. Protective effects of vitamin C against genotoxic damage from vanadium(V) and chromium(VI)
For humans, vitamin C is an essential micronutrient that plays multiple biological roles. It must be obtained from the ingestion of particular foods, mainly fresh fruits and vegetables, since our body is incapable of synthesizing it. The consequences of the intake of very high doses of vitamin C (>2 g/day) remain a subject of intense debate. However, it has been observed that supplementation of vitamin C reduces the incidence of stomach, lung and colorectal cancer; likewise, low serum levels of vitamin C in high-risk populations may contribute to increased risk of gastric metaplasia or chronic gastritis, which are both precancerous lesions [5, 25]. Nevertheless, analyses of the effects of vitamin C are rather complicated because diet and vitamin supplementation determine the levels of vitamin C in plasma.
Cameron and Pauling highlighted the beneficial properties of vitamin C in the 1970s. They suggested that high doses of vitamin C (>10 g/day) cure and prevent cancer by promoting collagen synthesis . However, researchers now suggest that vitamin C prevents cancer by neutralizing ROS before they can damage DNA and initiate tumor growth. Furthermore, it has been proposed that vitamin C may also act as a pro-oxidant, helping the body’s own ROS destroy early-stage tumors [27, 28]. Currently, the recommended dietary allowance (RDA) in many countries ranges from 40–90 mg/day, although the results of various studies suggesting that the protective vitamin C concentrated in plasma for the minimum risk of free radical diseases corresponds to an intake of 124.2 mg/day (in the range of 92–181 mg) [10, 29].
Vitamin C possesses double bonds with an associated electron deficiency, making it highly reactive to free radicals from molecular oxygen. It donates two electrons from C-2 and C-3 double-bonded carbons, resulting in the formation of tricarbonyl ascorbate radical (AscH•), which is present in the nonprotonated form, a semidehydroascorbate radical (Asc•−). The resulting ascorbate free radicals reduce to a neutral ascorbate molecule (Figures 2 and 3,
In a previous study, we observed that the frequency of micronuclei in polychromatic erythrocytes (MN-PCE) increased with the administration of 40 mg/kg of V2O5 through ip , consistent with other studies testing soluble vanadium compounds (Na3VO4, SVO5, and NH4VO3) [34–36]. However, the
Despite the important studies on the cytotoxic and anticarcinogenic effects of antioxidants in tumor model systems, it is clear that the molecular mechanisms underlying the benefits of antioxidants in cancer prevention are not yet well understood. Some ascorbyl forms of stearate inhibited cell proliferation by interfering with the cell cycle, reversing the phenotype and inducing apoptosis in human brain tumor glioblastoma (T98G) cells. Therefore, it has been proposed that the chemopreventive properties of antioxidants are related to their ability to target specific cellular signaling pathways that regulate cellular proliferation and apoptosis . This proposal is consistent with our results since the frequencies of apoptotic cells (particularly, late apoptotic cells) indeed increased significantly with the administration of vitamin C, and their administration prior to treatment of V2O5 increased them even further . Additionally, other studies have reported that the apoptosis-inducing activity of antioxidants might be synergistically enhanced by a combined treatment with chemopreventive  or genotoxic agents . Therefore, it is plausible that enhanced induction of apoptosis following a combined treatment may positively contribute to the elimination of the cells with V2O5-induced DNA damage (MN-PCE).
On the other hand, some compounds including vanadium-oxide(V) have been proposed for clinical use as therapeutic drugs for cancer because the intracellular cascade mechanisms may be involved in causing apoptotic cell death. For many decades, vanadium was considered a low-toxicity essential trace element with anticarcinogenic properties . However, important events have taken place since then. In 2006, the International Association for Research on Cancer (IARC) classified vanadium pentoxide (V2O5) as a Group 2B substance (possibly carcinogenic to humans) based on results in experimental animals . In 2009, the American Council of Government and Industrial Hygienists (ACGIH) placed V2O5 in category A3 (confirmed animal carcinogen with unknown relevance to humans) .
The low levels of ROS promoting mRNA formation and encoding proteins known to be regulated by vanadium can induce the activation of transcription factors. In contrast, high levels of ROS are cytotoxic to the cells and trigger apoptotic mechanisms. Therefore, it has been proposed that the cytotoxic effects of vanadium compounds should be used to generate ROS and reactive nitrogen species to combat cancer cell lines [48, 49]. Of all the proposed mechanisms of V(V) toxicity, the induction of oxidative stress is of particular importance for biological systems [50, 51]. As explained above, antioxidants can deactivate highly reactive molecules such as ROS that are generated during various biochemical processes in the cells . As a consequence, substances with antioxidant properties emerge as putative preventatives and co-adjuvants in the treatment of chronic degenerative diseases related to oxidative stress and DNA damage . Additionally, the promising low costs of vanadium-based drugs make it particularly attractive, and the ability to overcome the adverse effects of vanadium compounds during therapeutic action is an urgent and crucial issue for its future use in medicine . Our findings strongly suggest that vitamin C can be used effectively in therapy either alone (antioxidant) or in combination with other agents such as V2O5 to reduce their genotoxicity .
With regard to Cr(VI) compounds, they have been of particular interest and broadly studied because of their importance in different industrial applications including chrome plating, metallurgy, pigment manufacturing, leather tanning, and wood preservation and, most relevant to this chapter, because they are associated with the induction of cancer [52, 53]. Cr usually exists in various oxidation states, primarily Cr(III) and Cr(VI). The former is an essential micronutrient that plays a key role in protein, sugar, and fat metabolism. The latter is particularly effective in inducing genotoxicity by producing several types of DNA lesions and gene mutations. Some of the major factors that may play a significant role in determining cellular genotoxicity are Cr(VI)-induced DNA-DNA interstrand crosslinks, oxidative DNA damage, and mutations in the tumor suppressor gene p53 [19, 54]. It has been observed that Cr(VI) induces DNA damage through changes in the 8-OH-dG levels in DNA in rats. Furthermore, both endogenic (enzyme system) and exogenic (antioxidant consumption) antioxidant systems might counteract ROS and free radicals. In a recent study, we observed that administration of Cr(VI) increased MN-PCE (genotoxic damage), nonviable cells (cytotoxic damage), and glutathione (GSH) levels (a molecule that intervenes in its reduction to Cr(V), (Figure 3,
Vitamin C is a potent antioxidant found mainly in fresh fruits and vegetables. It can be readily absorbed and concentrated in tissues and biofluids at a physiologically relevant level, presenting effects in both the aqueous and membrane domains. Furthermore, it plays an essential role in the organism since it scavenges free radicals, chelates redox metals, and regenerates other antioxidants within the “antioxidant network.” All these characteristics make the study of the effects of the
The authors wish to thank Estefani Y. Hernández-Cruz for his excellent technical assistance. Financial support was obtained from DGAPA-UNAM PAPIIT-IN219216.
|AscH2, AscH−, Asc2−||Forms of ascorbic acid (vitamin C)|
|AscH•||Tricarbonyl ascorbate radical|
|MN-PCE||Micronucleated polychromatic erythrocytes|
|NADPH||Nicotinamide adenine dinucleotide phosphate|
|NAD(P)+||Oxidated form of nicotinamide adenine dinucleotide phosphate|
|ROS||Reactive oxygen species|
- The authors declare that they do not have any competing interests.