HEAVY METAL TOXICITY IN HUMAN LUNG FIBROBLASTS AND INHIBITION OF HUMAN TOPOISOMERASE-I AS A POTENTIAL MECHANISM
Although there is a major decline of heavy metal usage in most Western countries, the industrial use of the metals is not prohibited world-wide and their effects on human health are still obvious. Heavy metal resuspension, through the environmental dust and soil accumulation, make them a dangerous enemy for human health, as the exposure to them cannot be avoided. The genotoxic effects of most of the metals are mostly well-established; the most common ones have identified to be the generation of ROS and the inhibition of DNA repair pathways. However, a big variety of co-interacting mechanisms makes it particularly difficult to identify the specific action of heavy metals inside the body. The enzyme Topoisomerase-I acts on the DNA topological stability and previous works have shown inhibitory effects on the enzyme, can affect DNA sustainability. In this project, the toxicity of cadmium, lead, copper and zinc on human lung fibroblasts at physiological levels has been studied. Also, the inhibition of the enzyme Topoisomerase-I, is suggested as a potential mechanism and the purification of the protein was attempted for future work. The present study gives additional evidence of the genotoxic effects of the heavy metals and their inhibitory ability on Topoisomerase-I through cleavage and relaxation assays; and suggests further research in order for the leading pathways of heavy- metal induced cancer to be comprehended.
Introduction to heavy metals
The term "heavy metal" is often used to describe a specific group of metals; however, it has been associated with various metal properties throughout the years. The very first and most common use of the term "heavy metal" was to describe metals with high environmental contamination risk and potential toxicity. In addition, it has been used to describe metals based on their density (Jarup, 2003). Later on, authors started to associate this term with the mass or even the atomic weight of the metals. Although it was a much more accurate way to describe the metals and closer to the periodic table, there is no description of it in the International Union of Pure and Applied Chemistry (IUPAC) Compendium of Chemical Terminology (Duffus, 2002). Due to the imprecision of the term, it should be noted that in this work the term is used to describe metals which combine toxicity and high specific density.
In this thesis, metals such as cadmium, lead, zinc and copper have been utilised and their toxic effects are investigated. These heavy metals have atomic masses greater than non-trace metals like potassium, calcium, and magnesium. Their ions are noted for their toxicity and they are also chemical elements with a specific gravity at least five times greater than the specific gravity of water, which is the defining value that is currently most.
Heavy metals are found naturally in the Earth's crust and are released to the environment via natural phenomena, such as rock erosion and volcanic explosion. However, the industrial development added anthropogenic factors to metal emissions, such as mining and excavation of metals. Furthermore, the excessive industrial exploitation of metals has contributed to water and soil pollution due to industrial waste; consequently, the exposure, of livestock and finally humans, to metals became inevitable. The heedless usage of metals, without assessing their toxicity first, led to numerous diseases. A clear example is that of leaded gasoline, which was used for years, polluting the environment irreversibly, causing exposed individuals to suffer from bone diseases and neurological impairment. The most important fact about heavy metal pollution is that metals do not degrade over the years- as organic waste does- whereas they
resuspend via the air dust and accumulate within the soil (Beyersmann and Hartwig, 2008).
The health impact of long-term heavy metal exposure is well known. The cancerogenic effects of heavy metals have been demonstrated in an extensive bibliography, including meta-analysis and animal studies, with the findings being certified by the International Agency for Research on Cancer (IARC) and World Health Organisation (WHO) committees’ agreements. Among metals, cadmium, beryllium, arsenic, chromium and nickel have been classified as human carcinogens and several others were classified as possible carcinogens (IARC, 1993; WHO/IFCS/FSC/WG, 2008). Metal toxicity can arise from the oxidation state, the charge and the ionic radii of the metal ions. Regarding metal compounds, key factors are their coordination number and geometry. These properties, when found to be comparable to those of other essential elements, could replace such elements and lead to the malfunctioning of the related enzymes and proteins, as it happens at cadmium which can replace calcium affecting the bone structure (reviewed in Beyersmann and Hartwig, 2008).
Although most heavy metals are toxic, there are others that are essential trace elements, such as copper, zinc, nickel and calcium. However, when their levels exceed a certain range, toxic or deficiency effects could appear as well. Overdose or deficiency could occur either by a high exposure or malnutrition respectively, or by mutation in genes of their carrier’s enzymes. For example, mutations in genes related to copper transport enzymes can cause the very well- known Menkes and Wilson diseases, causing copper toxicity or deficiency respectively. However, the low physiological levels of those metals, in combination with the lack of early biomarkers of exposure set the early diagnosis as a difficult task.
Metal genotoxicity/ carcinogenicity
Metal toxicity and carcinogenicity depend on very complex mechanisms, which are still not entirely clear for most metals. The toxicity of a metal depends on the metal compound bioavailability, i.e. the mechanisms regulating the metal absorbance at the cell membrane, the intracellular distribution and the binding to cellular macromolecules. On the other hand, carcinogenicity can depend on the combination of the production of Reactive Oxygen Species (ROS), DNA repair modulation and disturbances of signal transduction pathways (reviewed in Beyersmann and Hartwig, 2008). The involved mechanisms are so inter- connected and complex that it is difficult to single out one mechanism. For example, there are metals like cadmium, which are not directly redox active, but act indirectly, by inhibiting antioxidant enzymes, such as the superoxide dismutase, catalase and glutathione peroxidase. All these pathways will be further discussed separately in each metal section, summarising the existing literature.
The environmental emission of cadmium on the Earth’s crust is mainly due to sedimentary rocks and in water it occurs due to the weathering and erosion of rocks, which end up in rivers and the sea. Other major natural sources of cadmium are volcanic activity and forest fires (WHO/IPCS, 1992; Cook, 1995; Nriagu, 1989). Anthropogenic sources of cadmium emission include nickel- cadmium batteries, pigments, ceramics, paints, stabilized Polyvinyl Chloride (PVC) products and electronic components (Bertin and Averbeck, 2006). Sources of cadmium emission also include, non-ferrous metal production, stationary fossil fuel combustion and waste incineration, steel and cement production and phosphate fertilizers (WHO, 2007).
Cadmium is known for its toxicity and its health effects on humans, which can lead to organ damage and cancer. Cadmium accumulation in the kidneys makes it nephrotoxic, while several bone damages have been recorded as well
(reviewed in IARC, 1993). Cadmium can also be blamed for having significant cellular effects, as it affects cell proliferation, the cell cycle, cell signaling, DNA replication and repair, differentiation and apoptosis. ROS are also implicated in cadmium toxicity (Bertin and Averbeck, 2006).
It has been shown that the human body absorbs cadmium via inhalation, ingestion and dermal exposure. The major source of cadmium exposure which consists the 90% of the total intake among the non-smoking population in non- polluted areas is through diet (WHO/IPCS, 1992; WHO, 2007). It is estimated that approximately 80% of the ingested cadmium comes from cereals and vegetables, while in drinking water usually is between 0.01 and 1μg/l of the total ingestion (WHO, 2007). However, the very low cadmium concentration in drinking water makes it an insignificant contribution to total body cadmium levels (Olsson et al., 2002).
Although inhaled cadmium makes up a smaller part of the total cadmium body burden than the ingested one, pulmonary cadmium absorption is higher, ranging from 10% to 50% (WHO/IPCS, 1992), while gastrointestinal absorption accounts for only a small percentage (Järup et al., 1998). As tobacco contains cadmium at high levels, it is important to highlight that smokers have about 4-5 times higher cadmium blood levels (nearly 1.5μg/l) and two times higher kidney cortex cadmium concentration (20-30μg/g per weight) than non-smokers (WHO, 2007).
Cadmium kinetics in human body
Following absorption, cadmium accumulates primarily in the kidneys and has a biological half-life ranging between 10 to 35 years. Long-term cadmium exposure could be revealed by urinary cadmium concentrations as the kidney releases it gradually, whereas recent exposure could be revealed by blood cadmium levels (WHO/IFCS/FSC/WG, 2008).
The metabolism and mobilisation of cadmium in humans and animals mainly depends on enzyme's metallothionein ability to bind to metals, as its name declares. Metallothioneins (MT) are a family of cysteine-rich proteins which have the capacity to bind to heavy metals through the thiol group of their
cysteine residues. In the human body, MT are mainly synthesised in the liver and the kidneys, and tend to accumulate there. MT production is induced by cadmium presence in the body, creating a defense mechanism against cadmium toxicity in tissues and organs, because it has the ability to bind with metals (reviewed in Astrid et al., 2009).
After cadmium exposure, either at the gastrointestinal or the respiratory system, a part of it is absorbed by the body and binds to MT forming a cadmium- Metallothionein (Cd-MT) complex, which can travel within the bloodstream. When this complex reaches the kidneys, it is filtered at the glomerulus and may be re-absorbed from the filtrate in the proximal tubules (Foulkes, 1982). Inside the tubules, the Cd-MT complex is degraded and cadmium is released. As a result, the free cadmium accumulates in kidney tubules and damages the tubular cells (Dorian et al., 1992).
Health problems following cadmium exposure
Several health effects have been monitored after cadmium exposure. The kidney is the main organ where cadmium accumulates causing renal malfunction (WHO/IPCS, 1992). Cadmium effects on calcium metabolism makes bones a tissue sensitive to cadmium exposure, as several bone diseases have been noted after exposure to it. It should also be noted, that cadmium is carcinogenic for several organs, with lungs being the most well established ones (Figure 1) (WHO/IPCS, 1992). In 1993, the IARC classified cadmium as a human carcinogen (Group 1) (IARC, 1993), after strong evidence was found in animal models and human epidemiological studies. For example, lung adenocarcinomas were developed in rats after cadmium inhalation (Glaser et al., 1990; Takenaka et al., 1983)..