Cadmium

Cadmium (Cd) is a chemical element with the atomic number 48, in group 12 of the periodic system, also referred to the zinc group. The zinc group also contains the essential element zinc (Zn) and the toxic element mercury (Hg), with both of them Cd shares certain chemical properties. Cadmium is mostly associated with Zn, and indeed in 1817 Cd was discovered as an impurity of zinc carbonate (ICdA, 2013). Cadmium ranges in the middle of the heavy metals regarding its density (8.46 g cm^-3 at room temperature, RT) and atomic mass (112.4 u or g mol^-1). Cadmium is a rather rare element in the Earth’s crust with mean values between 0.1 and 0.5 parts per million (ppm) and holds the 64^th place of naturally occurring elements regarding the average abundance (ICdA, 2013).

Natural occurrences of Cd are linked to volcanic eruptions, burning of vegetation, and chemical weathering of rock material (Cullen and Maldonado, 2013). A much higher contribution of Cd release into the environment results from anthropogenic actions. As Cd is very often associated with Zn, it is a common by-product from Zn ores (Chizhikov, 1966;

Nriagu, 1980). With the industrial revolution, the use of Cd increased steadily (recently reviewed by Cullen and Maldonado, 2013). Cadmium is found, e.g., in plastic stabilizers used

in car tires, as a yellow pigment in paint, inks, rubbers, as a component in rechargeable Nickel-Cadmium batteries, and in tobacco smoke.

Cd is very mobile in soils and particles dissolve easily in water (unlike e.g. Fe, which is insoluble and biologically unavailable in the form of Fe³⁺). Concentrations vary from pM (10^-12 mol l^-1) in the oceans (Chung and Pai, 1996) via nM (10^-9 mol l^-1) in uncontaminated waters like Lake Constance (Petri, 2006) to µM (10^-6 mol l^-1) in stream Naraguta in Nigeria, which is heavily contaminated by tanning activities (Ahmed, 2011). The high release into the environment is a great threat because Cd is highly toxic to all organisms. Some important pollution scenarios were linked to Cd poisoning, like the “itai-itai-disease” in Japan in the early 20^th century (Kaji, 2012). The effects of Cd on humans are very well studied nowadays.

Acute or chronic toxicity can lead to multiple effects ranging from salivation, nausea, to organ (especially kidney) failure, nephrotoxicity and cancer (recently reviewed by Hartwig, 2013;

Thévenod and Lee, 2013). Besides inhalation and smoking, the main entry route of Cd into the human body is food. Plants take up Cd easily, and plant food averagely contains higher Cd concentrations than meat, eggs, or milk products (Thévenod and Lee, 2013) and allows Cd to enter the human food chain (McLaughlin et al., 1999). Therefore, the irrigation of crop plants used either directly for vegetable production, or for the production of animal feed with municipal waste water or the fertilization with sewage sludge or igneous phosphate fertilizers (Gimeno-Garcia et al., 1996; Thévenod and Lee, 2013) pose a great risk for human health (Kalavrouziotis et al., 2009).

1.2.1. Cadmium in plants

1.2.1.1. Cadmium in phytoplankton

Cd is also highly toxic for photosynthetic organisms. One exception has been found in which Cd has a metabolic function under natural conditions. The first observation was that the vertical distribution profile of Cd in the oceans resembles the one of the phytoplankton nutrient phosphate: While the concentrations are depleted at the surface water due to uptake by phytoplankton, they increase in deep water due to subsequent remineralization of sinking organic matter with a maximum in the main thermocline (Boyle et al., 1976; Bruland et al., 1978).

Indeed, a metabolic function was found in Zinc-depleted cultures of the marine diatom

low concentrations of Cd (Lane and Morel, 2000). The growth enhancement was also observed for other species of marine phytoplankton (summarized in Xu and Morel, 2013), but only when Zn was reduced and not completely removed (Xu et al., 2007). Furthermore, the growth enhancement by Cd under Zn-limited conditions was always lower than without Cd under Zn-sufficient conditions. The reason for the positive effect of Cd was identified in the form of the enzyme carbonic anhydrase (CA). The CA belongs to a large protein family and catalyses the conversion of hydrogen bicarbonate (HCO3

-) into carbon dioxide (CO2) and vice versa. In plants and algae, CO2 is fixed by the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). The conversion of HCO3

to CO2 enriches the concentration of CO2 in close proximity of RubisCO and thereby suppresses the oxygenase activity of the enzyme, and as a consequence, photorespiration. The activity of the CA is therefore important for carbon fixation.

Diatoms express two different kinds of CAs, depending on the metal availability in the oceans: Under conditions where Zn is not limiting, diatoms possess the δ-form of the CA (Lane and Morel, 2000). This form contains either a Zn or a Cobalt (Co) ion in its active center. Under Zn limitation, however, a protein is expressed that contains either Cd or Zn (ζ-CA). Although the catalytic activity of the ζ-CA is higher when Zn is bound instead of Cd, the substitution allows the diatoms to cope with Zn limitation, yield fast growth rates and therefore are able to compete with other algae in the low metal environment of the oceans (Lane and Morel 2000; Xu et al. 2008). However, the cdca gene coding for the ζ-CA was found in some (like Thalassiosira pseudonana), but not all other species (like Emiliania huxleyi or Tetraselmis maculata, Lee and Morel, 1995; Xu et al., 2007) that showed a growth enhancement when supplemented with low Cd concentrations. In those species, Cd must have another positive role for other, yet unknown reasons.

Whether Cd has a positive effect in land plants is still to be determined. However, there is a strong hint that also in the hyperaccumulator Noccaea (formerly Thlaspi) caerulescens Cd is used in the active center of the CA. When Cd was added to the medium, growth was enhanced and the enzyme was shown to be more active (Liu et al., 2008) compared to non-supplemented media.

1.2.1.2. Hyperaccumulators

As plants are immobile and cannot avoid unfavorable conditions, like high concentrations of heavy metals, they have to adjust and adapt to them. Some plants actively accumulate large amounts of heavy metals and can therefore grow on polluted soils. Those

“hyperaccumulators” do not take the metals up for metabolic reasons, but store them in tissues and organs (mostly cell wall and vacuole) where they interfere least with the metabolic processes (Küpper and Leitenmaier, 2013). Besides occupying an ecological niche, the hyperaccumulators also gain advantage over related non-accumulators as they are protected from herbivores or pathogens. This “defense hypothesis” was verified for many plants, metals and pathogens. A summary of the available data including experimental approaches and other challenges when studying the elemental defense hypothesis was released by Boyd (2007).

1.2.2. Cd toxicity in plants

Beside the two mentioned scenarios of positive effects, there is a vast number of studies emphasizing the toxicity of Cd (recently reviewed by Andresen and Küpper, 2013). Some of the main threats that also concern the following specialized chapters will be summarized below.

1.2.2.1. Damage to the photosynthetic apparatus

For phototrophic organisms the inhibition of photosynthesis is certainly a main threat. Cd can inhibit photosynthesis by direct interaction with the photosynthesis-related molecules. The substitution of Mg²⁺ in chlorophyll (Chl) by different divalent metal ions was shown for different plant and algae species (Kowalewska et al., 1992, 1987; Kowalewska and Hoffmann, 1989; Küpper et al., 1996, 1998, 2002, 2006). Those heavy-metal substituted Chls ([hms]-Chls) are unsuitable for photosynthesis for many reasons. The singlet excited state of [Cd]-Chl is very unstable and the return to the ground state is achieved by thermal relaxation rather than fluorescence or electron transfer (= photosynthesis) (Watanabe and Kobayashi, 1988; Watanabe et al., 1985). The capacity of electron release from the excited state is highest for [Mg]-Chl compared to any other [hms]-Chl (Watanabe et al., 1985). And many [hms]-Chls cannot bind axial ligands (Boucher and Katz, 1967), making the structure of the

Depending on the light conditions, the substitution of Mg²⁺ by other heavy metals takes place in different molecules: Under low light conditions, it will be in the LHC molecules forming the antenna system (shade reaction), while under high light conditions the replacement will be directly in the reaction centers (RC; sun reaction) damaging the PS II core (Küpper et al., 2002). Although these reactions are well studied for e.g. Cu, in vivo proof is lacking for [Cd]-Chl, which is hard to detect, as its absorption spectrum resembles that of [Mg]-Chl and cannot be distinguished by UV-Vis spectroscopy. However, [Cd]-Chl is rather unstable (unlike [Cu]-Chl) and easily degraded (Küpper et al., 1996). Only a small fraction (3-10%) of [Mg]-Chl will be substituted by Cd under environmentally relevant conditions (see below).

And although only these few Chl substitutions can be tremendous for the plant, especially under high light conditions, their detection becomes a challenge. However, inhibition of photosynthesis due to Cd treatment was clearly observed as reduced electron flow through PS II, reduced variable fluorescence, or enhanced non-photochemical fluorescence quenching (Küpper et al., 2007a; Pietrini et al., 2010) suggesting the occurrence of the substitution.

1.2.2.2. Induction of reactive oxygen species

Oxidative stress due to heavy metal treatment is a well studied phenomenon in plants and algae (reviewed by Pinto et al., 2003; Shaw et al., 2004). Reactive oxygen species (ROS) are a normal by-product of metabolic processes (respiration and photosynthesis; Asada 2006;

Asada and Takahashi, 1987) and also occur in specialized organelles like peroxisomes (Corpas et al., 2001; del Río et al., 2002). Low concentrations of e.g. nitric oxide (NO) and hydrogen peroxide (H2O2) serve as signaling molecules in the cell (reviewed by Mittler et al., 2011; Van Breusegem et al., 2008). Plants and other phototrophic organisms possess several antioxidant enzymes and scavenging molecules to dispose off the harmful ROS. While under unstressed conditions, the formation and neutralization of ROS is balanced, under stress conditions, the imbalance is in favor of the accumulation of ROS, leading to the oxidative stress. The two most reactive species are the hydroxyl radical (HO^•) and singlet oxygen (¹O₂).

They are considered to be specifically dangerous to all living cells as they react fast and non-specific with all kinds of biomolecules, including DNA, proteins, enzymes, and lipids (Shaw et al., 2004). The peroxidation of unsaturated fatty acids of plasma- and organelle membranes can lead to their disruption and thereby leakage of the organelle and cell contents. One further danger of lipid peroxidation is the production of mutagenic aldehydes (Halliwell and Gutteridge, 2007).

As Cd is redox-inert and does not react directly with molecular oxygen, the most likely origin of ROS under Cd stress is the dysfunction of photosynthesis and respiration. Increased concentrations of superoxide radicals (O2

•-) and hydrogen peroxide (H2O2) were observed in Cd-treated pea plants by specific dyes (Romero-Puertas et al., 2004; chapter 2.1. of this thesis). Both, the enhancement of ROS production and the reduction of enzymatic activity of the antioxidant system were connected to Cd toxicity. An upregulation of gene expression of those enzymes is clearly a response to enhanced oxidative stress. The reduction in activity, e.g. of the superoxide dismutase (SOD) may be the result of the substitution of the Zn ion in its active center by Cd (see below), thereby inactivating the enzyme (Sandalio et al., 2001) which will lead to higher amounts of ROS.

1.2.2.3. Cadmium-induced genotoxicity

Cadmium can induce genotoxicity by directly interacting with the nucleotides (Müller, 2010;

Sigel et al., 2013), by inhibiting DNA-repairing enzymes, and via the induction of ROS leading to lipid peroxidation, membrane leakage, and the production of mutagenic aldehydes (Lin et al., 2007). In Vicia species, the root tip micronucleus assay is well established (Ma et al., 1995). Micronuclei (MN) originate during cell division by the exclusion of whole chromosomes or by chromatin fragmentation (Savage, 2000). In studies with Allium cepa, Allium sativum, and Vicia faba, treated with different Cd concentrations from 75 nM up to 200 µM, the MN formation was significantly higher in the roots treated with Cd compared to the control (Manier et al., 2009; Seth et al., 2008; Ünyayar et al., 2006). Cd-induced DNA damage was also shown as increasing length of tail DNA in the so called Comet Assay. In this assay, nuclei of Cd-treated cells were isolated, lysed and damaged DNA was determined by the amount that left the nucleus when gel electrophoresis was applied. The higher the damage, the longer will be the tail of the comet (Cvjetko et al., 2010; Koppen and Verschaeve, 1996).

Cadmium-induced damage to plants can have several reasons and can reveal itself in many ways. Most of the proposed mechanisms are summarized in the following graph.

Figure 1: Scheme of damage pathways and interactions of Cd toxicity which were proposed in the literature. (Picture taken from Andresen and Küpper, 2013).