УДК 57.084:[615.212+615.099]

V.V. Arkhipchuk^a, candidate of biol. sci.,
V.V. Goncharuk^a, acad. of NASU,
V.P. Chernykh^b, member-corresp. of NASU,
L.N. Maloshtan^b, doct. biol. sci.,
I. S. Gritsenko^b, doct. chim. sci.,
M.R. Vergolyas^a, T.V. Moseychuk^a

USE OF PLANT AND ANIMAL BIOASSAYS AND BIOMARKERS FOR COMPLEX ESTIMATION OF GENERAL TOXICITY, GENOTOXICITY AND CYTOTOXICITY OF DRUGS

^aInstitute of Colloid Chemistry and Water Chemistry, blvd. Vernadsky 42, 03680, Kiev, Ukraine
^bUkrainian National University of Pharmacy, Pushkinskaya 53, 61002, Kharkov, Ukraine

Abstract

A complex, integral approach based on the use of test organisms belonging to different systematic groups (plants, invertebrates, and vertebrates) as well as the nucleolar biomarker and the micronucleus test on their cells was applied to assess toxicity, cytotoxicity and genotoxicity of four pharmaceutical substances: cofalginum (metamizole sodium: caffeine-benzoate sodium=6:1), rheopyrin (phenylbutozone: aminophenazone=1:1), novalginum (propyphenazone: acetaminophen : caffeine=4:4:1), and benalginum (metamizole sodium : caffeine : thiamine hydrochloride=13:13:1) taken in IC₅₀ concentrations for mammalian cells. The studied concentrations (from 3,125 to 6,875 mg/ml) were highly toxic for animal and plant bioassays; they caused rapid mortality of both invertebrate (within 12—48 h for ceriodaphnia and 24—96 h for hydra) and vertebrate (1—24 h for crucian carps) test organisms, and practically complete inhibition of the onion root growth. At the cellular level the nucleolar biomarker demonstrated prompt changes (within 30—90 min) in morphological characteristics of nucleoli with the specific features for each drug studied. The nucleolar number was most resistant parameter, whereas the size of single nucleoli varied most significantly. Under the drug influence, the nucleolar size mainly reduced, or increased with a following decrease. Moreover, all the pharmaceutical substances caused specific effects on nucleoli in plant cells (halos, weakly-stained nucleolar cores, and destroyed nucleoli). Genotoxic effects were studied only in onion cells, where the root germination occurs. The cofalginum and benalginum concentrations stipulated the formation of cells with micronuclei and double nuclei, and the rheopyrin solution destroyed the nuclear structure. The designed complex approach may be applied for obtaining express, cost-efficient and useful supplementary data on different types of toxicity for marketed drugs as well as for drugs under development.

1. Introduction

Some international programs (Gene-Tox, International Program on Chemical Safety-IPCS, Multicenter Evaluation of In Vitro Cytotoxicity — MEIC) were devoted to the assessment of drug toxicity with the use of non-standard, alternative methods, i.e., bioassays on plants, invertebrates and non-mammalian vertebrates and their cells [1—4]. For instance, within the framework of the MEIC program, 50 reference substances, particularly drugs, were studied with the use of 68 bioassays to evaluate the relevance of toxicity tests for predicting toxicity to humans [4]. In general, human cytotoxicity was predicted well by animal cytotoxicity. Prediction of human cytotoxicity by ecotoxicological tests was only fairly good. Case studies also confirmed the effectiveness, applicability and usefulness of animal and plant bioassays for assessing different types of drug toxicity [5—8].

The replacement of long-term tests carried out with animals and analyzing carcinogenic and mutagenic properties of substances by short-term bioassays has some advantages: 1) the first tests are more expensive, require sacrificing many rodents, and yield results that are difficult to interpret [9]; 2) short-term bioassays can more exactly evaluate a risk for health of human beings, as compared to long-term tests on animals [10].

At the development of medicinal preparations, studies of various anomalies of genetic system are very important for assessing a potential risk for human health. For this purpose, the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) established a standard battery for genotoxicity testing of pharmaceuticals: a test for gene mutation in bacteria, an in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an in vitro mouse lymphoma tk assay, an in vivo test for chromosomal damage using rodent hematopoietic cells [11]. A compendium of genotoxicity information for 467 marketed drugs was issued [12].

The main point of the complex, integral approach developed in our research is as follows: different types of toxicity are studied both at the organismic and at cellular levels, and methods providing comprehensive and objective assessments of the toxic impact are applied at each of them.

As a result of a series of investigations performed on organic and inorganic substances, the following set of bioassays was selected as optimal for studying various aspects of the toxic influence on organisms and their cells: representatives of plants (onion, Allium cepa), invertebrates (hydra, Hydra attenuata), and vertebrates (fishes, for example, crucian carp, Carassius auratus gibelio) [13—16]. For test organisms, including in the bioassay battery, at the cellular level the frequency of cells with micronuclei and double nuclei (as indices of genotoxicity) and quantitative characteristics of nucleoli (as an indicator of cytotoxicity) were determined. The proliferative cellular activity was evaluated by the mitotic index. Therefore, the set of methods analyzing toxic effects at the organismic level as well as changes in the structure and functional activity of the genome at the cellular level is used. The choice of these criteria is stipulated by their informative values, good reproducibility, and technical simplicity.

The purpose of the present work is to use the discussed approach for complex (integral) assessments of drug toxicity, cytotoxicity and genotoxicity on the example of four medicinal preparations widely used in Ukraine.

2. Methods and materials

2.1. Pharmacological substances
Four medicinal preparations, namely: cofalginum (tablets containing 300 mg of metamizole sodium and 50 mg of caffeine-benzoate sodium, produced by Borschagovka Pharmaceutical Company, Kiev, Ukraine), rheopyrin (125 mg of phenylbutozone and 125 mg of aminophenazone, produced by Gedeon Richter, Budapest, Hungary), novalginum (200 mg of propyphenazone, 200 mg of acetaminophen, and 50 mg of caffeine, produced by Stirol, Gorlovka, Ukraine), and benalginum (500 mg of metamizole sodium, 50 mg of caffeine, and 38,75 mg of thiamine hydrochloride, produced by Balkanpharma-Dupnitza, Bulgaria). The drugs were purchased in a pharmacy.

2.1.1. Concentrations of substances
Concentrations of the substances were chosen in accordance with results of toxicological research performed on mammalian cells. Experiments were carried out by researchers of the Ukrainian National University of Pharmacy (Kharkov). Cytotoxicity of the pharmacological substances was determined through damages in the transport function of cell membranes of red bone marrow of rats using the methylene blue stain as well as through hemolysis of erythrocytes in human blood, accounting the number of erythrocytes per 1 mm³ for each concentration of the preparation [17]. The pharmacological substances were dissolved in physiological solution, decreasing sequentially their initial concentrations 2, 4, 8, and more, times. The cytotoxic effect was estimated under a light microscope for 15, 30, 60, and 90-min exposure.

As a result, concentrations of the medicinal preparations corresponding to IC₅₀ for mammalian cells were as follows: 4,375 mg of cofalginum per 1 ml solution, 3,125 mg/ml rheopyrin and novalginum, and 6,875 mg/ml benalginum. The concentrations of the pharmacological substances were used for further studies on plant and animal (non-mammalian) organisms and cells.

2.1.2. Experimental design
The test organisms were incubated in water solutions of the drugs. Stock solutions were prepared by dissolving the substances in control water. The solution prepared with tablets was stirred up by a magnetic stirrer for 2 h, and then it was filtered through paper filters (pH of the solutions was 6,9—7,1). Required concentrations were obtained by dissolving the stock solutions with control water. Tap water, after three to five days of settling and subsequent filtration through a household carbon filter (Barrier, Russia), was used as the control and dissolvent for the drugs. Fishes, hydras and ceriodaphnias were cultivated and plant roots were germinated during the period proposed by corresponding methods; i.e., 30—90 min for determination of cytotoxic effects and 48—96 h for testing acute toxicity and genotoxicity. The pharmacological preparations were studied in several series of experiments for several months; therefore, each series was provided with its control.

The same complex, integral approach, which was approved earlier on various organic and inorganic substances, was applied for the study of medicinal preparations. In other words, drug toxicity, genotoxicity and cytotoxicity were assessed by the same scheme that was used for other chemical substances.

2.2. Drug acute toxicity
Experiments were carried out in the laboratory of biomarkers and biotesting of the Institute of Colloid Chemistry and Water Chemistry (Kiev). Various organisms representing different taxonomic groups were used to evaluate acute toxicity: invertebrates (ceriodaphnia, Ceriodaphnia affinis, and hydra, Hydra attenuata), vertebrates (crucian carp, Carassius auratus gibelio), and plants (onion, Allium cepa). Laboratory cultures of hydra and ceriodaphnia, a group of yearling crucian carps (7—9 cm long) taken from an environmental water body and then cultivated for several weeks in laboratory aquariums (100 L), and bulbs (1,5—1,7 cm in diameter) of onion purchased at the market were used as the test organisms. Some special features of cultivation of animal test organisms and storage of plants under local conditions as well as their use in biotesting have been described earlier [14].

The toxicity of drug solutions was tested under laboratory conditions (20±20C, a 16-h day light cycle), following the methods proposed by Fiskesjo for the onion 96-h mean root elongation test; and Trottier et al. (1997) for the hydra 48-h sublethal and lethal effects test [18, 19]. The ceriodaphnia mortality was observed in experimental samples after 24 h and 48 h exposure. The fish lethality was determined after 96 h of their cultivation in the studied solutions. Fin edge cells of crucian carps were used for the cytological analysis.

2.3. Drug cytotoxicity
The drug cytotoxicity was analyzed by quantitative parameters of nucleoli (the nucleolar biomarker), which objectively characterize the cell biosynthetic activity, and by changes in the mitotic index reflecting the proliferative activity of cells.

2.3.1. Nucleoli and nucleolar biomarker
Results of many studies conducted by various techniques have proved convincingly that morphological characteristics of nucleoli are associated with the most important molecular-genetic processes and are objective indicators of cell metabolism features. Such a relation is easily explicable, because the nucleolus is the site of transcription of ribosomal DNA, processing of the rDNA transcripts, and the formation of pre-ribosomal components [20—22]. In addition to ribosome biosynthesis, the nucleolus of eukaryotic cells also participates in many other aspects of gene expression, according to the so-called plurifunctional nucleolus concept [23, 24]. For example, there are abundant experimental data on nucleolar quantitative parameters as a useful indicator for diagnosing and prognosing different types of malignant tumors [25—29]. Nucleoli are also considered as a key factor controlling aging mechanisms [30, 31].

In earlier studies, the following parameters, which objectively and comprehensively reflected nucleolar activity of cells with a small number of nucleoli (the typical nucleolar constitution for plant and animal cells), were selected for the analysis: the average number of nucleoli per cell; the volume of a single nucleolus; and the percentage of cells with heteromorphic (differing in size) paired nucleoli [32—34]. Moreover, the idea of using the nucleolar biomarker for express assessment of water cytotoxicity was proposed [13, 15, 32]. One of advantages of the proposed biomarker is related to rapid assessment (during the first hours of exposure) of the adverse influence on functions of the cell genome. This feature is important for analyzing drug effects.

2.3.2. Slide preparation
Cytological analyses were carried out on onion root tips, fish fins, and adult hydra, fixed in ethanol : acetic acid (3:1), 30 and 90 min after exposure. Small pieces of the caudal fin were used as cytological samples, which made it possible to determine periodically the nucleolar indices of the same fish without any damage to its organs and physiological functions [35].

A mixture of cells of organisms under analysis was used to prepare cytological slides. One sample (2 slides) required 12—20 whole hydras and 10—14 plant root tips. Cytological slides (air-dried for animals and squashed for plants) were stained with 50% AgNO₃ solution, following the technique of Howell and Black (1980), and Dev and Tantravahi (1982) [36, 37]. Two slides for every sample were prepared, coded, and then independently analyzed by two assistants.

2.3.3. Microscopy
For each sample, nucleoli were counted in 1400—1800 cells using an Amplival light microscope (Carl Zeiss, Germany) equipped with 100x objective lens and 16x eyepieces. The size of spherical and intact nucleoli was measured in 200 cells at the same magnification with the use of a micrometer eyepiece. Paired nucleoli were visually classified into homomorphic (i.e., of nearly equal sizes) and heteromorphic (i.e., of different sizes) at the maximum magnification (1600x) according to the procedure described by Arkhipchuk and Palamarchuk [34].

2.4. Drug genotoxicity
The drug genotoxicity was estimated by increasing the frequency of cells with micronuclei formed as a result of chromosome aberrations, and the frequency of cells with double nuclei reflecting damages of cell divisions. Development of micronuclei is one type of mitosis pathologies; its formation is caused by chromosome lagging during metaphases and anaphases, or by the chromosome fragmentation. Cells with double nuclei result from damages during the cell wall formation, when two daughter cells arise from the single mother cell.

In our experiments, values of the discussed indices were calculated per 1000 cells, i.e. in pro mil. For each sample the micronuclei and double nuclei frequency was determined for 4—5 thousand cells of onion root tips or fish fin edges. The material was fixed after 4—5 days of incubation of crucian carps and onion bulbs in water solutions of the pharmacological substances studied. Smear cytological slides of animal cells and squashed slides of plant cells were prepared. Animal slides were stained with azure-eosin by Romanovsky (stock solution), and those with plant cells, by aceto-orcein (2% solution). Slides were analyzed under light microscopes at 1600x magnification.

2.5. Statistical analysis
Average values and standard deviations were calculated for the data obtained. The data were then checked for statistical significance using the STATISTICA software (StatSoft Inc., USA, 1995) for Microsoft Windows.

3. Results

3.1. Drug acute toxicity
Results on acute toxicity of the four pharmacological substances are presented in table 1. the concentrations studied (from 3,125 to 6,875 mg/ml) were highly toxic for all test organisms used. the most definite reaction was observed for a representative of vertebrates, fish. for instance, in three solutions (benalginum, novalginum, and rheopyrin) 100% mortality of crucian carps was registered during 1—2 h. the same drug concentrations caused the complete lethality in ceriodaphnia after 12-h exposure. another representative of invertebrates, hydra, remained alive for a longer time, from 1 to 4 days. all the medicinal preparations were very severe to plants, i.e. they were highly phytotoxic, because the onion root growth was inhibited almost completely.

3.2. Drug cytotoxicity (plant)
All the pharmaceutical substances studied produced specific effects on nucleoli in onion cells. For example, the cofalginum solution caused the appearance of nucleoli with halos in 20-30% of plant cells; after the exposure with benalginum and rheopyrin solutions, the weakly-stained nucleolar core was observed for about 50 and 70% of onion cells respectively; and after the novalginum influence, most (60—70%) nucleoli were destroyed.

The results of studying the drug cytotoxicity with using the nucleolar biomarker are presented in table 2. the share of cells with heteromorphic-paired nucleoli (pnhet) decreased after exposure with the drugs, with the only exception: the percentage of these cells having non-destroyed paired nucleoli increased after the novalginum impact. the process developed most significantly in cofalginum and rheopyrin solutions; in 90 min the values reduced by 35,8% and 24,6%, respectively.

The influence of the pharmaceutical substances studied stipulated the growth in a number of nucleoli in plant cells (consequences of the 90-min cofalginum exposure was the only exception). The most prominent enlargement was revealed in the rheopyrin solution (Table 2).

The nucleolar size in onion cells reacted in different ways to the exposure of various medicinal preparations. For instance, similarly to its action on the nucleolar number, the cofalginum solution caused some decrease in the size of nucleoli by 16,2—50,7% during the whole period of observations (Table 2). The novalginum concentration initially decreased the parameter (by 10,4%, P<0,001), then increased it (by 7,5%, P<0,01) in cells with non-destroyed nucleoli. After the impact of rheopyrin and benalginum solutions, the volume of single nucleoli increased.

3.3. Drug cytotoxicity (invertebrates)
Results of the drug influence on hydra cells are presented in table 3. the share of cells with pnhet increased in two solutions with cofalginum (p<0,01) and novalginum (p<0,05) and decreased essentially (p<0,001) in the benalginum concentration, in 30 min, and in the rheopyrin one, in 90 min.

Similarly with the data obtained on plant cells, the cofalginum solution decreased reliably the number of nucleoli in hydra cells after 90-min exposure, whereas other three solutions increased the studied parameter during the same period (P<0,001). Moreover, some similarity with plant cells was determined for changes in the size of nucleoli of hydra cells. For instance, the cofalginum impact significantly reduced values of the parameter (Table 3). The benalginum solution initially increased the nucleolar volume in the comparison with the control, and, after 90 min, decreased it. For rheopyrin and novalginum solutions, the following tendencies were revealed: decreasing the nucleolar size by 7,7% (P<0,05) in 30 min, and its growing by 17,7% (P<0,001) in 90 min for the first drug; and reduction of the parameter during the whole period by 15,7—53,2% (P<0,001), for the second substance.

3.4. Drug cytotoxicity (vertebrates)
Results of the medicinal preparations' influence on carp cells based on the nucleolar biomarker are presented in table 4. three drug solutions (rheopyrin, novalginum, and benalginum) reliably lowered (p<0,001) the percentage of cells with pnhet in fish after 90-min exposure. only the cofalginum concentration increased the value. three solutions (cofalginum, rheopyrin, and benalginum) initially reduced the nucleolar number, but then enlarged it. most dramatic variations in the number of nucleoli were observed for cofalginum.

As to the size of single nucleoli, two pharmacological substances (cofalginum and novalginum) reduced it significantly during the whole exposure period; whereas two other preparations (rheopyrin and benalginum) initially increased the volume (by 47,3% and 84,2% respectively), and then decreased it down to the control level (Table 4).

3.5. Drug genotoxicity (onion and fish)
Onion roots did not grow under the studied concentration of novalginum during 5 days of experiments. Therefore, the mitotic index was zero, and it was impossible to estimate genotoxic effects by the micronucleus test. For other drug solutions the analysis was practicable because of the root availability. The proliferative activity of onion cells decreased more than 3 times (from 38,2‰ to 11,4‰) under the influence of cofalginum. Although frequencies of cells with micronuclei and double nuclei in the cofalginum solution did not differ essentially from those in the control, the difference calculated relative to the number of dividing cells (ratios mN/MI and 2N/MI) was significant (table 5). after 96-h benalginum influence, the cell division in onion roots was completely inhibited; though many double nuclei and micronuclei varied by their sizes were detected. as a result of the rheopyrin impact, all nuclei in plant cells were destroyed or deformed.

Because of rapid carp mortality (within 1—24 h of exposure), no assessment of genotoxic effects of the medicinal preparations for fish cells was carried out.

Thus, the analysis of toxicity of four pharmacological substances taken in IC₅₀ concentrations for mammalian cells revealed that these substances were highly toxic for animal and plant bioassays; they caused rapid mortality of both invertebrate (within 12—96 h) and vertebrate (1—24 h) test organisms, and practically complete inhibition of the plant root growth. At the cellular level the nucleolar biomarker demonstrated prompt changes (within 30—90 min) in morphological characteristics of nucleoli, with specific features for each drug studied. The nucleolar number was most stable parameter, whereas the size of single nucleoli varied most significantly. Under the drug influence, the nucleolar size mainly reduced, or increased with a following decrease. Moreover, all the pharmaceutical substances studied caused specific effects on nucleoli in plant cells, namely: halos (cofalginum impact), weakly-stained nucleolar cores (benalginum and rheopyrin solutions), and destroyed nucleoli (novalginum influence). Genotoxic effects were studied only in onion cells, where the root germination took place. The cofalginum and benalginum concentrations stipulated the formation of cells with micronuclei and double nuclei, and the rheopyrin solution destroyed the nuclear structure.

4. Discussion

In the present work, water solutions of four pharmacological substances studied in concentrations that are toxic for mammalian cells (at the level of IC₅₀) demonstrated evident toxic, cytotoxic and genotoxic effects for plant, invertebrate and vertebrate organisms and their cells. Toxic consequences of the drug influence were studied by standard bioassays on animal and plant test organisms. The specific cytotoxic impact of the medicinal preparations on cells of the same test organisms was assessed by the nucleolar biomarker, and genotoxic effects were estimated by the micronucleus test.

The prompt reaction of nucleoli to external impacts through changes in their morphological parameters has been revealed earlier [32]. In these experiments the drug solutions during the first hours of their influence changed significantly the size of nucleoli in both animal and plant cells; i.e. they affected first and foremost the transcriptional activity of ribosomal genes. Sharp fluctuations of the nucleolar activity during short-time periods indicate some abnormalities in the function of nucleoli in cell, because similar variations are not observed under the optimum (control) conditions. Decreasing the nucleolar size reflects the inhibition of biosynthetic activity of nucleoli, whereas increasing the parameter may indicate an adaptive reaction of ribosomal genes via intensification of their activity at a harmful influence.

The studied drugs revealed different effects in cells of the test organisms studied. This is evidence that the same pharmacological substance, first, undergoes dissimilar biotransformations in plant and animal organisms and in their cells, and, second, there is some specificity of reactions of different species and their cells on toxic influence. This phenomenon was discussed earlier [13, 14]. Consequently, it is necessary, on the one hand, to evaluate toxic effects on test organisms belonging to different taxonomic groups, and, on the other hand, to use both organismic and cellular levels.

The analysis of different types of drug toxicity using cells of plants (onion) and animals (fish) is important and topical, because it is known that plant bioassays, especially with Allium, could be used as an alternative to in vivo and in vitro tests on mammals, particularly on human beings [38]; bioassays with fish, for example, the micronucleus test, estimate objectively the risk of the studied water samples for human health [39].

Our study confirmed the expediency of using biotesting methods for assessing drug toxicity, cytotoxicity and genotoxicity. The obtained results proved high effectiveness of the proposed complex approach. It could be applied for a preliminary screening of newly developed drugs and their components, as well as for obtaining additional data on toxicity of using pharmacological substances.

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