METHOD TO DETERMINE THE TOXICITY CAUSED BY EXPOSURE TO CHEMICAL AND PHYSICAL AGENTS

Abstract: It is described a method to determine the toxicity caused by exposure to chemical and physical agents consisting in exposing some biological material to said agents and in measuring with an oxygen electrode the O2 concentration change which is due to the respiratory activity of said material, after having added a convenient substratum to the solution with which the material is in contact, said method being characterised in that the biological material used is such that, at the maximum speed of its metabolism, the residual concentration of O2 is higher than zero.

Description: METHOD TO DETERMINE THE TOXICITY CAUSED BY EXPOSURE TO CHEMICAL AND PHYSICAL AGENTS Field of the invention The present invention relates to a method to determine the toxicity caused by exposure to chemical and physical agents. This method is based on the use of aerobic or facultative anaerobic cells. Prior art The toxicity of many chemical substances has been studied for many years by using living organisms as biological indicators, for instance the use of superior organisms such as guinea-pigs is well known. They often give reliable results however response times can be long, about some days (also months or years in case of chronic toxicity). If quite simpler and short living cycle of various microorganisms is used, it is possible to greatly reduce analysis times. Presently, unicellular organisms such as yeast colonies are used. These biological materials are properly joined to a suitable sensor in order to measure the toxicity of a great number of substances. This kind of join belongs to a class of analytic devices that are commonly defined as "biosensors". Various combinations between cells and sensors are known thus allowing to perform a broad range of analyses. Among known biosensors, the most common ones are those using as a transducer an electrochemical sensor: a potentiometric one, by which system changes of potential are measured, or an amperometric one, by which power intensity changes produced in the external circuit of an electrolytic cell to which a constant potential difference is applied are measured, owing to the biological process on which the biosensor operation is based upon. Among the biological materials, enzymes, antibodies, animal and vegetal tissues, both procaryote (bacteria, cyanobacteria) whole cells and eucaryote (algae, fungus, ferments, cells of upper organisms) whole cells can be used. The technical field of the present invention relates to biosensors wherein the tested biological function is the respiratory one, as this function is sensible to the action of toxic substances; cells to be used are the aerobic or facultative anaerobic ones; the biosensor is an immobilised cells one, in that it is easily moved thus allowing an easy application in situ of the measuring method. This kind of bionsensor is described in Campanella L. et al., The Science of the Total Environment 171 (1995) 227-234. The drawbacks of such biosensor are the following: it needs a fairly high number of cells/ml, from which a poor sensitivity derives, it lacks a standard from which a poor reproducibility derives; the measures depend on the substrate concentration; it lacks a purifying system in order to remove possible polluting micro-organisms present in the air. Biosensors suitable to determine the toxicity caused by exposure to physical agents are not known. Summary of the invention It is an object of the present invention a method to determine the toxicity caused by exposure to physical agents based upon the use of aerobic or facultative anaerobic cells and relevant biosensor. Another object of the present invention is a method to determine the toxicity caused the exposure to chemical agents based upon the use of aerobic or facultative anaerobic cells and relevant biosensor. Another object of the invention is a method to prepare immobilised cells to be used in the biosensor according to the invention. A further object of the invention is a kit to determine the toxicity caused by exposure to chemical and physical agents based upon the use of an aerobic or facultative anaerobic cell-based biosensor. Further objects of the present invention will be evident by the following detailed description of the present invention. Brief description of the figures Fig. 1 shows in graphic the determination of toxic effects after exposure to physical agents. Comparison between the respirometric curves of exposed cells (E) and not (B). Fig. 2 shows in graphic the determination of toxic effects after exposure to chemical agents. Respirometric curve with standard. Fig. 3A shows in graphic the results obtained after exposure (24h) of yeast cells to non-ionizing radiation (900MHz, 120V/m). Fig. 3B shows in graphic the results obtained after exposure (48h) of yeast cells to non-ionizing radiation (900MHz, 120V7m). Detailed description of the invention The principle of the method described in Campanella L. et al., The Science of the Total Environment 171 (1995) 227-234 together with the biosensor assembling, consists in measuring with an oxygen electrode (a Clark one) the change in 0 2 concentration which is due to cellular respiratory activity, when it diffuses in agar containing the immobilised cells after having added a proper substrate to the solution, commonly glucose or saccharose. A first aspect of the invention relates to the preparation of immobilised cells that, before use, are purified in nitrogen atmosphere, preferably for about 30 min. Moreover, considering cells as a catalyst acting in heterogeneous phase and considering also that the reaction environment is open, the oxygen concentration is substantially constant in the solution body, the conclusion is that using such a substrate concentration enabling the maximum respiration reaction rate, the final value of 0 depends only on the number of cells that have been used to perform the measure. In fact, if this number is too high, as in the prior art, when substrate in excess is added, the 0 2 concentration always vanishes and does not allow data processing on comparison basis. In order to solve this drawback, according to the present invention, operations are performed in such a way to use a number of cells such that at the maximum rate of metabolism the residual concentration of 0 2 is higher than zero, preferably between 0.5 and 2.5 ppm. In order to obtain this result, for example, the culture medium can be changed in such a way that the number of cells/ml at the growing plateau (steady state) is suitable to obtain the above mentioned conditions. For example, in addition to agar, the culture medium can contain usual substances such as peptone, yeast extract and glucose, but in lower concentrations than in the prior art, particularly lower than a magnitude order. The thus prepared medium is sterilised generally in an autoclave at 120°C for 20-30 min. Cells are inoculated with a platinum loop in a test tube containing 10ml of an agar solution and modified medium as described in the above at a temperature higher than the agar solidification. After stirring, all the content is poured into a Petri capsule. All these operations are performed in substantially sterile conditions, as defined by the usual microbiological practice. The content is incubated at about 30°C for 48 hours and then the biosensor is assembled as described in Campanella L. et al., The Science of the Total Environment 171 (1995) 227-234. The thus prepared biosensor can be used in order to determine the toxicity caused by the exposure to chemical and physical agents. That is to say the biosensor is plunged in a constant temperature solution under stirring, for example distilled water or pH 7.4 phosphate buffer, or another solution chosen according to the pH and/or ionic force requirements. The electrode response can be directly expressed in ppm of 0 2 by an Orion microprocessor and recorded as a function of time. According to another aspect of the present invention, a method to determine in a reproducible and sensitive way the biological effects on aerobic or facultative anaerobic cells after their exposure to radiation is described. In this case, the biosensor is kept to stabilise in an appropriate distilled water volume (or other solution) then substrate in excess is added as described above and the new stabilised signal is waited for. The determination is performed comparing, for each measure, the obtained value of Δppm(E) 0 2 from cells exposed to radiation with the (Δppm(B) 0 2 ) one deriving from cells (blank) that have been kept for the same time and the same experimental protocol, as described hereinbelow in absence of exposure. Results can be represented in a graphic such as the one shown in fig. 1. The percentage Respiratory Inhibition Activity (IRA) can be calculated in the following way: IRA index ( p hyslc ai) - [1-(Δppm(E) 0 2 /Δppm(B) 0^ x 100 (1 ) In order to obtain a data being very closed to the real one, it is necessary to perform various comparison measures in order to reduce differences between the sample of exposed cells and the one used for blank tests. At the beginning of each test, it is necessary to wait for the system to reach the stabilisation after the substrate has been added. A further aspect of the present invention relates to an improvement of the method to determine the toxicity of chemical agents on aerobic and facultative anaerobic cells. The results can be represented in a graphic as the one shown in fig. 2. In this case it is not necessary to wait for the system stabilisation after the substrate addition, in that the operations are performed as follows. The substrate is added end the stabilisation of the system to the minimum consumption value of 0 2 is waited for, then the toxic substance is added to the solution, or the cells are exposed to a toxic gas (line (b) of the curve in figure 2, leading to the calculation of Δppm 0 2 values). The data are then normalised in order to make them independent from instrumental factors by adding a substance (a kind of internal standard) to irreversibly inhibit 100% of the cell respiratory activity (for example, formic aldehyde can be used) (line (c) of the curve shown in fig. 2, leading to the calculation of Δ'ppm 0 2 values). The percentage Inhibition of the Respiratory Activity (IRA) can be calculated in the following way: IRA index (physicair (Δppm 0 2 /Δ'ppm 0 2 ) x 100 (2) Therefore the toxicity percentage is calculated, simply and with one measure only for each test, from the ratio between the inhibition of the respiratory activity due to the toxic substance under examination and the inhibition due to the addition of the internal standard. In the field of the present invention, alternative methods for the immobilisation can be used, for example alginates, carrageenans or other kinds of immobilising agents can be defined. It is also possible to work with cells in liquid phase, provided that their number is checked in order to verify the existence of the previously described conditions required for the measure. The liquid phase is advantageously any liquid being compatible with the cell vitality such as distilled water, pH or ionic force buffer, physiological solution. The measures according to the methods of the present invention can be performed by a kit comprising a biosensor such as the one prepared for example with immobilised cells on agar according to the present invention and an amperometric electrode for the measure of 0 , for example a Clark electrode. Other possible kit components are the common laboratory instruments such as paddles, beckers, wash bottles, agitators, magnetic stirrer, pipettes. The methods described in the present invention allow to obtain a meaningful increase in sensitivity, reproducibility and analysis quickness, particularly in case of chemical pollutants determination. Moreover, the results are characterised by a very high sensitivity. The answering times of the analyses performed with the method according to present invention can be comprised in the order of 1-2hours or less. Advantageously, all the chemical substances and rays of any kind, interfering with the cell vital cycle can be determined with the method of the invention. Chemical polluters that can be determined can be the following ones: metals, pesticides, antiseptics, surfactants, phenol and its derivatives. Physical polluters can be: electromagnetic rays, rays produced by radioelements, X rays. The present invention is now described with reference to the following examples and enclosed figures that are to be considered illustrative and not restrictive of the scope of the invention. Examples Reagests and equipment Cells belonging to a wild diploid group of Saccharomyces cerevisiae have been used. The following reactive substances have been used: Yeast extract for microbiology, universal peptone for microbiology, D(+)-Glucose for microbiology, all products produced by Merck, saccharose (Carlo Erba). The following equipment has been used: Glass measure cell, capacity 50 ml. Orion 0 2 Electrode, electrode Model 97-08-99 Orion Microprocessor ion alyzer/901 potentiometric Amel Model 868 recorder Amel Model 291/LF magnetic stirrer Certo Clav 11/1600 (1600W- 220V) Autoclave Purification and immobilisation of cells Before being used, cells are purified in nitrogen atmosphere for 30 min. Then a culture medium containing peptone at a concentration of 0.17% p/v, ferment extract 0.17% p/v, D(+)-Glucose 0.17% p/v and agar 1.0% p/v is prepared. The thus prepared medium is sterilised in autoclave at 120°C for 30 min. Cells are then inoculated by a platinum loop in a test tube containing 10ml of the above mentioned solution at a temperature of about 40°C. After stirring, the content is poured into a Petri capsule. The content is kept to incubate at about 30°C for 48 hours. Toxicity tests From the culture medium contained in the Petri capsule a diskette is obtained and the biosensor is assembled as described in Campanella L. et al., The Science of the Total Environment 171 (1995) 227-234. Tests are performed plunging the biosensor tip in distilled water at room temperature under stirring. The electrode response is directly expressed in ppm of 0 2 and recorded as a funtion of time. Tests on physical agents To the distilled water in which the biosensor is kept to stabilise, saccharose is added up to a concentration of 0.5 M and the new stabilisation of the signal is waited for. The radiation source was a GTEM chamber operating at 900 MHz and 120V/m, corresponding to the frequency source used by the 1 st generation mobile phones. The measurement is performed comparing, for each measure, the obtained value of Δppm(E) 0 2 from cells exposed to radiation with the Δppm(B) 0 2 one deriving from cells (blank) that are kept for the same period and the same experimental protocol in the absence of exposure. The obtained results for exposure times of 24 and 48 hours are summarised in fig. 3. The inhibition % of the activity is correlated to the damage suffered by the cells that, as clearly shown in the graphs, is always present in all the performed measures (no test was rejected) and it seems to be proportional to the exposure time. Tests on chemical agents The procedure of the previous test has been performed without waiting for the system stabilisation after the glucose addition. Therefore, glucose is added up to a concentration of 0.5M, the system stabilisation to the minimum consumption value of 0 2 is waited for and the toxic substance is added to the solution (SDS). The signal stabilisation is waited for and then formic aldehyde 1 % p/v is added. Data are summarised in table 1 Table 1

Claims: CLAIMS 1. Method to determine toxicity caused by exposure to chemical and physical agents consisting in exposing some biological material to said agents and in measuring with an oxygen electrode the change of 0 2 concentration due to the respiratory activity of said material after addition of a substrate to the solution with which it is in contact, said method being characterised in that the biological material to be used is made to grow in a culture medium so that at the maximum metabolism rate of said material the residual concentration of 0 2 is higher than zero in the solution. 2. Method according to claim 1 wherein the residual concentration of 0 2 is comprised between 0.5 and 2.5 ppm. 3. Method according to claim 1 wherein the biological material is constituted by aerobic or facultative anaerobic cells. 4. Method according to claim 3 wherein the cells are yeast cells. 5. Method according to claim 3 wherein the cells, before use, are purified in nitrogen atmosphere. 6. Method according to claim 1 wherein the substrate is selected between glucose and saccharose. 7. Method according to claim 1 wherein the culture medium comprises peptone, yeast extracts and glucose in concentrations that, at the maximum metabolism rate of said material, the residual concentration of 0 2 is higher than zero. 8. Method according to claim 1 wherein the biological material to be used is inoculated in a test tube containing an immobilising agent solution and culture medium at a temperature that is higher than the solidification one of the immobilising agent, the composition is stirred and poured in a Petri capsule, it is kept to incubate at about 30°C for 48 hours and then the thus immobilised material is assembled with the 0 2 electrode to obtain a biosensor. 9. Method according to claim 8 wherein the immobilising agent is selected between agar and carrageenans. 10. Method according to claims 1-7 wherein the biological material is kept in liquid phase and in this phase it is joined with the oxygen electrode in order to obtain a biosensor. 1 1. Method according to claims 8-10 wherein the biosensor to determine the toxicity is plunged in a constant temperature solution under stirring and the electrode response is directly expressed in ppm of 0 2 . 12. Method to determine toxicity caused by exposure to physical agents comprising the following steps: combine some biological material with a sensor for the 0 2 determination in order to obtain a biosensor, keep the biosensor to stabilise in a solution compatible with the vitality of the biological material, add substrate in excess so that the residual concentration of 0 2 is higher than zero, wait for a new signal stabilisation, expose the biological material to a physical agent, compare, for each performed measure, the obtained value of Δppm(E) 0 2 from the biological material exposed with the (Δppm(B) 0 2 ) one deriving form the non exposed biological material (blank), that is kept for the same time in the same conditions, measure the percentage Respiratory Activity Inhibition (RIA) with the following formula: RIA index (physica l ) - [1-(Δppm(E) 0 2 /Δppm(B) O∑] x 100 (1 ) 13. Method according to claim 12 wherein the biological material is constituted by aerobic or facultative anaerobic cells. 14. Method according to claim 12-13 wherein physical agents are: electromagnetic rays, rays produced by radioelements, X rays. 15. Method to determine toxicity caused by exposure to chemical agents comprising the following steps: combine some biological material with a sensor for the 0 2 determination in order to obtain a biosensor, plunge the biosensor into a solution compatible with the biological material vitality, survey the initial consumption of oxygen in the solution body, add substrate in excess so that the residual concentration of 0 2 is higher than zero, wait for the system stabilisation to the minimum consumption value of 0 2 , add the chemical agent to the solution, or expose the biological material to said agent, calculate the corresponding value of Δppm 0 2 , add a substance to irreversibly inhibit 100% of the biological material respiratory activity, calculate the corresponding Δ'ppm 0 2 value, calculate the percentage Respiratory Inhibition Activity value (RIA) with the following formula : RIA index (C hemicai) = (Δppm 0 2 /Δ'ppm 0 2 ) x 100 (2). 16. Method according to claim 15 wherein the biological material is constituted by aerobic or facultative anaerobic cells. 17. Method according to claims 15-16 wherein chemical agents are: metals, pesticides, antiseptics, surfactants, phenol and its derivatives. 18. Method according to claims 12-17 wherein the residual concentration value of oxygen is comprised between 0.5 and 2.5 ppm. 19. Method according to claims 12-18 wherein the liquid phase is chosen between distilled water, pH or ionic force buffer physiological solution. 20. Biosensor to determine toxicity caused by exposure to chemical and physical agents characterised in that the biological material, before its use, is made to grow in a culture medium that at the maximum metabolism rate of said material, the residual concentration of 0 2 is higher than zero. 21. Biosensor according to claim 20 wherein the value of the oxygen residual concentration is comprised between 0.5 and 2.5 ppm. 22. Biosensor wherein the biological material is obtained according to claims 8-9. 23. Biosensor wherein the biological material is obtained according to claims 10- 11. 24. Kit to determine toxicity caused by exposure to chemical and physical agents comprising a biosensor according to claims 20-23, an amperometric electrode to measure 0 2 , paddles, beckers, wash bottles, agitators, magnetic stirrers, pipettes.

Inventors: CAMPANELLA LUIGI, DRAGONE ROBERTO, FAVERO GABRIELE, CAMPANELLA, LUIGI, DRAGONE, ROBERTO, FAVERO, GABRIELE
Applicants: CAMPANELLA LUIGI, DRAGONE ROBERTO, FAVERO GABRIELE, UNIV ROMA, CAMPANELLA, LUIGI, DRAGONE, ROBERTO, FAVERO, GABRIELE, UNIVERSITA' DEGLI STUDI DI ROMA "LA SAPIENZA"
Application year: 2000
Main classification: C12Q1
IPC codes: C12Q1, G01N33