Today tumour diseases are the second most frequent cause of death on industrially developed countries. In the Czech Republic, breast and colorectal carcinomas are two most widespread types of tumours. In the inception and development of tumour diseases a combination of internal, genetically conditioned and external factors are present. In these factors, apart from exposure to carcinogenic substances, life style plays an important role¹. The task of epidemiology is to contribute to the detection of risk factors and, with appropriate intervention, to eliminate them by means of primary prevention, or, at least, to limit their effect in the population. Exposure to trace elements represents one of the important risk factors of the environment. Some trace elements are recognised by IARC among chemicals carcinogenic to human: arsenic, beryllium, cadmium and their compounds and chromium [VI] compounds, nickel compounds². On the other hand, certain trace elements (e.g. selenium), playing an important role in our nutrition, have been investigated as regards their effect for cancer prevention³. Epidemiological studies have consistently shown that human population having a low selenium intake and correspondingly low plasma or serum selenium levels have an increased incidence of a variety of cancers⁴. These findings are of great importance since the inhabitants of the Czech Republic have been shown to have one of the lowest selenium status in Europe^5,6.

One area of interest in present-day studies of the etiopathogenesis of tumour diseases is an attempt to establish which biologically active substances will facilitate the determination not only of the earliest possible diagnosis but more particularly the prognosis of further development of the tumour disease. Nowadays there are several method used based on histological and histochemical examinations, determining and assessing the dynamics of tumour marker levels in biological fluids and determining of specific cellular proteins and enzymes in the cytosol of tumour cells. Moreover, it has been observed that certain trace element concentrations in biological fluids and cancerous tissues of patients are altered as secondary effects of tumour growth. Knowledge about trace element metabolism and its disturbances in tumor tissue or in the organism carrying a malignancy, might help to elucidate the relation of the elements to cancer and to decide whether these elements could be used as additional biochemical markers for the diagnosis or prognosis of tumour development.

It is known that many trace elements participate in biological processes in the organism, which include the activation or inhibition of enzymatic reactions, competition between elements and metalloproteins for binding positions, and modifications in the permeability of cellular membranes. It can therefore be presumed that these elements might influence carcinogenic processes. There are few studies concerning the determination of trace element levels in blood (urine, hair) of breast or colorectal cancer patients. Most of them deal with zinc, copper and selenium levels because of the role of these elements in the antioxidant defence system of the organism. The results are contradictory and not consistent.

Some authors^7-10 found the mean serum copper level and the mean Cu/Zn ratio in patients with breast cancer significantly higher than the control group, and the mean serum zinc level in patients significantly lower than the controls. Garofalo et al.¹¹ and Piccinini et al.¹² reported that neither the serum Cu nor Zn levels are of value in discriminating between the patients with benign and malign breast cancer and the healthy controls, and in determining the severity of the disease. On the contrary, Cavallo et al.¹³, in a two-centred study performed in Northern Italy and Southern France observed serum zinc levels to be significantly higher in patients in both centres than in controls, whereas serum copper levels were found to be significantly high in controls in Italy and in patients in France. As for selenium, several authors reported decreased selenium level in patients with cancers breast and colorectal cancers^14-16. However, this drop could be interpreted as either the consequence or even the cause of the disease. It could be due to the selenium needs of tumour, to the metabolic consequences of the tumour or even to a low dietary intake of Se.

Multielemental techniques such as neutron activation (NAA) and inductively coupled plasma mass spectroscopy (ICP-MS), make possible the simultaneous determination of ultra-trace quantities of elements in human tissues and body fluids. By using such techniques, it is possible to determine whether the simultaneous monitoring of the less abundant trace metals has diagnostic or prognostic significance. Such systematic studies which include a broad spectrum of trace elements with large patient populations are very scarce. They concern trace element levels in cancerous and normal breast tissue^17-19. Again, data reported by different authors are not consistent although they agree as to the significant enhancement of certain elements in the cancerous vs adjacent normal tissue. According to the authors, the elevations of concentrations of elements are probably due to the increased cellular activity in malignant tissue and active enzymatic systems leading to increased amounts of trace elements.

The presented project aims to help to elucidate the role of trace elements status of patients with breast and colorectal carcinomas with respect to their environment and life style. We will investigate trace element levels in blood and cancerous tissues of the patients together with the assessment of life style risk factors (using an epidemiological questionnaire) and the determination of cytosolic and serial examination of tumour markers. In our knowledge, such complex approach has not been undertaken so far and it will consist in the co-operation of experts from different fields - epidemiology, oncology, immunology, analytical chemistry. The project will be the pilot study for further research on the significance of trace elements in carcinogenesis.

During the preliminary study, the sampling protocol will be established, trace elements selected and this study will also lead to the optimisation of the ICP-MS technique. Aside from essential trace elements such as zinc, copper and selenium, we aim to determine elements such as Rb, Sr, Pb, Tl, ..., whose function in the carcinogenesis has not been taken into account so far.

The project is considered to 3 years. The study group will consist of 100 patients with breast cancer and 100 patients with colorectal cancer from Oncological centre of Faculty Hospital Plzeň. In 1st and 2nd year there will be the examination of patients with first diagnosis of tumour. Each patient will undergo:

• determination of selected trace elements in serum and cancerous tissues by ICP-MS technique (comparison with 200 randomly selected age-matched healthy controls, all from the west Bohemia region) — responsible: Department of Analytical Chemistry of Charles University Praha
• completing of the Epidemiological questionnaire — the data processing will be performed by Institute of hygiene and Epidemiology of 1st Medical faculty Praha)
• tumour markers assessment and determination of other serial parameters — responsible: Laboratory of immunoanalysis, Faculty Hospital Plzeň

During the project, patients will be followed in 3-month intervals and there will be the assessment of the same tumour markers and trace elements as during the first examination. Trace elements levels will be correlated with tumour marker levels and with the evolution of the disease and treatment.

References:

3. Investigation of metallothioneins using modern electrochemical methods

(differential pulse voltammetry, cyclic voltammetry, adsorptive stripping voltammetry and high performance liquid chromatography with electrochemical detection)

Metallothioneins (MT) are low molecular mass proteins (6-7 kDa), characterised by high cysteine content and ability to bind metals , most important being Zn, Cd, Cu, Hg. Formation of metal - thiolate bonds possessing specific spectroscopic features and arranged in metal- thiolate clusters is important characteristic of metallothioneins, which is studied extensively and summarised in several books and monographs^1-5. Besides most frequently studied mammalian MT, metallothioneins isolated from mussels, fungi and plants have been reported. At the end of 1997, more than 170 amino acid sequences of metallothionein from 50 species have become known and analysed⁵.

Metallothioneins are considered to be important in homeostatic control, metabolism and detoxification of several essential or toxic trace metals. Also, they act as antioxidants and radical scavengers, protecting DNA damage. Several studies also suggest that metallothionein can protect DNA damage caused by metals and other chemicals e.g. chromium, nickel, alkylating agents^4-6.. Cd and Zn containing metallothioneins are studied extensively on mercury electrodes^7-15 and also on carbon electrodes¹⁶.

The aim of the research in this field is:

1. Use of electrochemical methods for speciation of cations bound to cysteine moiety
2. estimation of apparent stability constants of complexes in different media (pH, presence of other ions etc).
3. Combination of electroanalytical methods with separation techniques, e.g. HPLC, capillary electrophoresis.
4. The investigation of the interactions of MT in systems producing radicals or containing other species inducing damage of DNA.
5. Environmental monitoring - MT proposed as a tool for trace metal monitoring - marine environment, different types of algae.
6. Development of new types of electrodes and electrochemical software suitable for electrochemical investigation of MT

References:

4. Development of new analytical methods for trace elements