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Portable bioluminescent laboratory for ecological and medical toxicity monitoring

Name
Valentina
Surname
Kratasyuk
Scientific organization
Siberian Federal University
Academic degree
Doctor of Sciences, Professor
Position
Head of Biophysical Department, Temporary Head of Laboratory of Bioluminescent Biotechnology
Scientific discipline
Life Sciences & Medicine
Topic
Portable bioluminescent laboratory for ecological and medical toxicity monitoring
Abstract
Laboratory consists of a bioluminometer «LumiShot», a multicomponent immobilized reagent «Enzymolum» and a set of toxicity bioassays for medical, environmental and industrial application such as evaluation of water, air and soil pollution, assessment of materials toxicity, the of endotoxicosis and stress degree etc. A new educational practical courses are suggested. The laboratory allows for fast (3-5min) detection of a wide range of the chemical and biological toxic compounds. The advantages of enzymatic assays are high sensitivity, validity, simplicity of methods.
Keywords
bioluminescence, bioluminometer, practical course, immobilized reagent, portable laboratory
Summary

Laboratory consists of a bioluminometer “LumiShot”, multicomponent immobilized reagent “Enzymolum” and set of toxicity bioassays for medical, environmental, and industrial application such as evaluation of water, air and soil pollution, assessment of the toxicity of nanomaterials, medical materials and food additives, the degree of endotoxicosis, the effect of physical load on sportsmen, and in new practical courses for higher educational institutions and schools.  The multi-component reagent “Enzymolum” contains the bacterial luciferase, NADH:FMN-oxidoreductase and their substrates, co-immobilized in starch or gelatin gel.  Laboratory immediately signals chemical-biological hazards and allows us to detect a wide range of toxic substances. The advantages of enzymatic assays are their rapidity (the time of analysis does not exceed 3-5 minutes), high sensitivity and validity, simple measuring procedure, stability and safety of reagents. 

The multicomponent immobilized reagent “Enzymolum”  is the main part of laboratory [1].  In this study, we propose a new approach to design enzymatic reagent kits based on selecting the conditions for co-immobilization of various components of enzyme systems in natural gels with their subsequent drying. The method for creating multicomponent enzyme preparations is illustrated by the example of the reagents for bioluminescent analysis based on coupled enzyme system of luminous bacteria NAD(P)H:FMN- oxidoreductase and luciferase  (Red + Luc). In our study, reagent components (NAD(P)H:FMN-oxidoreductase and luciferase and their substrates NADH and myristic aldehyde) were immobilized in starch or gelatin gels. Thus obtained multicomponent immobilized reagent contains all the components required for one measurement except FMN which is added as a solution to start the reaction of light emission. The reagent represented a dried disk 6-7 mm in diameter, whose dry weight was 1.5 mg. By varying the composition of the multicomponent reagent and the microenvironment of enzymes in the reagent, it is possible to obtain a family of immobilized enzyme preparations whose characteristics vary depending on the analytical task to be solved. In the study we varied the ratio of components in the immobilized reagent; the chemical nature of the gelling agents (starch or gelatin) and their concentration; the concentration of agents stabilizing the enzyme activity and conditions of the immobilized reagent drying. We analyzed such characteristics of immobilized reagents as activity estimated by the maximum of the light intensity (Imax), time when the coupled enzyme system reached the luminescence maximum (Tmax), sensitivity to toxic compounds and stability during long-term storage. We found that a decrease in the content of enzymes or substrates in the reagent increased its sensitivity to toxic compounds. The reagent obtained by co-immobilization of the enzymes and substrates in the gelatin gel had a higher activity and stability during storage than those based on the starch gel, but it showed a lower sensitivity to toxic substances. An increase in the concentration of gelatin significantly increased the Tmax value, which, in turn, increased the duration of analysis. The best combination of high activity with stability during storage was reached by the addition of 100 μM DTT to the reagent. Multicomponent immobilized reagents (variants of “dry chemistry” reagents) can be used not only for routine assays but also as modules in biological enzymatic biosensors. Moreover, the proposed approach can be widely used for preparing reagents for not only bioluminescent but also other types of enzymatic assays. 

This review describes the principle and applications of bioluminescent enzymatic toxicity assays. The new approach to develop the bioluminescent enzymatic biosensors, toxicity bioassays and reagents has been described. To solve the problem of how to detect, identify, and measure the contents of the numerous chemical compounds in environmental monitoring, food product monitoring, and medical diagnostics, the bioluminescent enzymatic toxicity assays were, wherein the bacterial coupled enzyme system NAD(P)H:FMN-oxidoreductase-luciferase substitutes for living organisms. Historically, the application of bacterial luminescence in toxicology began with the usage of luminous bacteria for ecological monitoring and they are still widely used. These methods made it possible to determine environmental pollution by comparing the light emission intensity of luminous bacteria in the control and in the analyzed samples. As opposed to other test objects such as paramecia, algae, crustaceans, and so on, the bioluminescent assay was faster (the time of analysis didn’t exceed 30 min). However, as with other living organisms, the essential disadvantage of this method was low accuracy of measurement caused by the ‘‘petulance’’ of living luminous bacteria: failure to maintain the stable state of bacterial culture during measurements and storage. The bacteria reacted to the appearance of toxic substances either by decreasing or by increasing the luminous intensity, which often led to ambiguous interpretation of results. That’s why only qualified staff could work with bacteria. Because of these shortcomings an assay based on luminous bacteria didn’t show very good results in ecological laboratories. To overcome those difficulties it was suggested to use enzymes of luminous bacteria in soluble and immobilized forms. Since 1990, a new trend in bioluminescent toxicity analysis named bioluminescent enzymatic toxicity assay has been developed [2]. At present, it is actively used in ecology, medicine, agriculture, and other areas [3, 4]. 

The main principle of the bioluminescent toxicity enzymatic assays is inhibition of couple enzyme system: Red + Luc by the toxic components of analyzed samples. The principles of bioluminescent enzymatic toxicity assay were successfully used for the analysis of aquatic environments as well as air and soil pollutions. With bioluminescent enzymatic toxicity assay there is possibility to solve a problem of complex evaluation of environmental toxicity. It is well-known that to estimate environmental toxicity it is necessary to use the battery of bioassays. Usually they represent different levels of life organizing such us cells, organs, organisms and ecosystems.  Due to the coupling with bacterial luciferase, it is possible to design new enzymatic assays in toxicology and combine them into a set to provide the toxicity control at the enzymatic level. The set includes enzymes of different classes, or key enzymes of metabolic processes in living organisms. The bacterial luciferase may be the terminal enzyme in coupling chains for more than 100 enzymes including such as lactate dehydrogenase, trypsin, glucose-6-phosphate dehydrogenase, and others, making it possible to measure the enzyme activities according to the light emission intensity. To develop the set of bioluminescent enzymatic toxicity assays different enzyme interaction mechanisms were suggested. As an example  to estimate toxicity of water samples two enzymes were chosen: alcohol dehydrogenase (ADH) and trypsin, because they belong to different classes (oxidoreductases and hydrolases), and secondly, because they interact differently with bacterial luciferase, providing different sensitivity to the toxic substances. The effect of toxic substances on the activities of the triple enzyme system with ADH and trypsin were measured using the bioluminescence decay constant. The set of bioluminescent enzymatic toxicity assays was used for monitoring natural and laboratory aquatic ecosystems and for studying the seasonal dynamics of zooplankton non consumptive mortality.

Examples of application the set for toxicity analysis of pesticides and sanitary assessment of natural polymers polyhydroxyalkanoates are given too. Bioluminescent enzymatic assays are used  in other sectors, such as agriculture and food industry. The first example is evaluation of wheat grain infection with Fusarium. Mycotoxins of fungi of the genus Fusarium in feeding causes poisoning and even death of animals. International standards for grain quality and medical and biological requirements for food quality require that grain contamination with Fusarium should be controlled at the stages of crop harvesting, purchase, and processing. To develop rapid analysis of wheat grain infection with Fusarium the effects of their mycotoxins on the coupled enzyme system were studied at first and the strong inhibition of enzymatic activity was observed. The sensitivity of the coupled enzyme system Red + Luc to mycotoxins decreased in the following order: zearalenone, deoxynivalenol, toxin T-2, and diacetoxiscripenol. Further, in study it has been showed that the efficiency of Red + Luc activity inhibition by wheat extracts depended on the severity of grain infection with Fusarium. Moreover, the inhibition was caused not only by mycotoxins but also by other metabolites of Fusarium, which were accumulated in infected grain. The inhibition of bioluminescence depended on the geographical origin and growth conditions of the grain. These differences were able to minimize due to the method of sample preparation. Another example is assessment of food additives safety. The sodium benzoate (Е 211), potassium sorbate (Е 202) and sorbic acid (Е 200) were tested. The effects of nanomaterials such as Ag, Cu, Сu2О which have the prospect of introduction to the food technology also were identified. The loss of light emission intensity of the coupled enzyme system Red + Luc in the presence of food additives was estimated. Also the toxic effects of additives on the bioluminescence of the three triple enzyme systems Red + Luc + trypsin, Red + Luc + ADH and Red + Luc + LDH (lactate dehydrogenase) were analyzed. Results were compared to the well-known tests based on survival and chemotaxis ciliates Paramecium caudatum, germinating of shoots and roots of cress "Cudriavyy", survival of Daphnia magna, changes in the level of chlorophyll fluorescence of algae Scenedesmus spp. and foaming by the yeast Saccharomyces cerevisiae. The effects of the food additives on organisms were evaluated using parameters EC50 or LD50. The coupled enzyme system Red + Luc and triple enzyme system with LDH showed a great sensitivity to the analyzed food preservatives. Values of EC50 were equal 0.03, 0.14, 0.008 and 0.66, 0.13, 0.07 mM for sodium benzoate, potassium sorbate and sorbic acid, respectively. The values of EC50 estimated by enzymatic tests were over two times less than that for the biological tests mentioned above. The maximum decrease in the relative activity of trypsin and ADH assays did not exceed 45 % in the studied range of preservatives concentration. It was shown that both copper and copper oxide (I) nanoparticles had a strong inhibitory effect on Red + Luc system. Values of EC50 were equal 4 μM and 1.5 μM for copper nanoparticles and Cu2O, respectively. Value of EC50 for silver nanoparticles was 0.18 mM. The bioluminescent enzymatic toxicity assay indicated the negative effect of food additives in the much lower concentrations than its actual maximum content in food products. There is a problem which is extremely topical both for agriculture and food industry. It is pesticides. Pesticides vary in their toxicity mechanism and character, e.g. they can be carcinogenic or mutagenic, or they can affect the respiratory, endocrine, immune or nervous systems. There are two different types of pesticides: organophosphates and pyrethroids. Organophosphorous substances are complex esters of phosphoric acid and their toxic effect is accounted for by their ability to inhibit acetyl cholinesterase, the key enzyme in synaptic transmission in nerves. Pyrethroid insecticides, synthetic analogues of natural pyrethrins, act through intestinal contact, thereby affecting the nervous and the immune systems. In our study the set of bioluminescent enzymatic toxicity assays was applied to analyze toxicity of organophosphorous and pyrethroid pesticides. The sensitivities of the bioluminescence assays were close to those determined by other biological assays or even higher. The triple enzyme systems with ADH and trypsin have been shown to be more sensitive to organophosphorous compounds (0.13–11 mg·L-1). Sensitivities of the triple enzyme systems to pyrethroid pesticides were similar to those of in vivo assay based on luminous bacteria (0.9–5 mg·L-1). 

The bioluminescent enzymatic toxicity assay is also very promising for medical research, for example, for evaluating the gravity of endotoxicosis during treatment in surgery and therapy. It is based on the fact that the effect of donor’s blood serum on enzymatic activity differed markedly from that of the patients’: blood serum of a patient inhibits bioluminescence less than that of donor. Two modifications of the assay using luciferase and coupled enzyme system Red + Luc have been developed. Comparative analysis of application efficiency for luciferase index LI and other laboratory parameters to assess the severity of patients with peritonitis have been made. Bioluminescent enzymatic toxicity assay allows to estimate the severity of a patient’s condition as satisfactory, of middle seriousness, severe and the critical one. It can be used also for disease course prediction, estimation of the efficiency of the used detoxification methods, and control of drainage procedure with semipermeable membranes. Most important is applying LI in prognostic plan as far as the long positive LI dynamics could indicate the need for change of treatment plan. It was reported that the assay can be used as a reliable criterion to monitor the course of disease for therapeutic patients with bronchitis, ulcerous disease or chronic colecystitis. The most important advantages of the proposed approach are the very short time interval between sample collection and results, high sensitivity, low traumatism, and simplicity. 

A very interesting and promising trend in the development of bioluminescent enzymatic toxicity assay is the creation of rapid analysis for the assessment of human organism reaction to physical and mental stress. Analysis is made by comparing the light emission intensity of the coupled enzyme system Red + Luc in the presence of a person’s saliva taken before and after a certain stress load. The main advantage of the assay is noninvasiveness, because human saliva is analyzed, which reflects the functional state of a person just as blood does.

 

1. Esimbekova E.N., Lonshakova-Mukina V.I., Bezrukikh A.E., Kratasyuk V.A. Design of multicomponent reagents for enzymatic assays. // Doklady Biochemistry and Biophysics, 2015, Vol. 461, P. 472-475.  

2. Kratasyuk, V.A. Principle of luciferase biotesting. In: Proceeding of the first international school “Biological luminescence”, Wroclaw, Poland, 20-23 June 1989. World Scientific Publishing Co.: Singapore, 1990; pp. 550-558.

3. Esimbekova, E.; Kratasyuk, V.; Shimomura, O. Application of enzyme bioluminescence in ecology. Adv. Biochem. Eng. Biotechnol., 2014, 144, 67-109.

4.Kratasyuk V., Esimbekova E. Applications of luminous bacteria enzymes in toxicology //Combinatorial Chemistry & High Throughput Screening, 2015. V. 18, Issue 10. Р. 952-959.