Efficiency of different murine lines for genotoxicity assays

Introduction

In general the mutagenic and genotoxic studies in the experimental toxicology, were performed when it was evaluated all the studies of toxicity including sharp potential of the product to evaluate in two species and for at least two pathways of administration in an independent way (Arencibia et al., 2009). With these studies the risk associated to direct or indirect damages is explored on the genetic material, where a positive response it allows in general the step of the product at a second evaluation level. At the present time it has been described a wide range of in vivo and in vitro assays able to detect the genotoxic damage at the different expression levels, all with a high sensibility and specificity (Loeb and Loeb, 2000).

The main problem is that the researchers use the different mouse and rat genetic lines of existent of the biomodel for convenience, but in the generality of the cases this decision is far from a theoretical-practical basement that justifies the selection, conditioned fundamentally by the lack of studies in this respect. This causes to that in many occasions the results obtained by different research groups that work similar products in research or of the same group, in different moments cannot be comparable, because it is well-known that the genetic differences among the lines of the biomodel, present differentiation in the expression due to the damages at the genome level (Arencibia et al., 2011).

The classic mutagen more used on in vivo genotoxicity studies are the cyclophosphamide (CF), administered by intraperitoneal route (i.p). The CF is an alquilant agent that forms monoadducts and crossed connections between chains as consequence of the appearance of ruptures for reparative mechanisms effects. This drug is used with great effectiveness as an antineoplastic (Arencibia et al., 2011a). The CF belongs to the cloroethylamines group and is considered a bifunctional alquilant agent which does not possess specificity for phase some of the cellular cycle (Arencibia et al., 2011a).

The problem before mentioned makes us think that then at the present time alone those products that are very safe or very genotoxic are truly classified while those capable ones of causing small damages could be not well classified; from the importance of performing a comparative study of the spontaneous and induced rates of genetic damages in the genetic lines of mice and rats available and most used in research worldwide at the different expression levels by means of different cytogenetic methods that it allows to toxicologist that work in the specialty of the genetic toxicology to have a potent tool, endorsed statistically, when selecting the genetic line of mice and rats to perform the mutagenesis and carcinogenesis studies.

The assessment of this research were:

1. Assessment the efficiency in the use of the lines of Balb/c, NMRI, OF-1 and C57/BL6/Cenp mice of both sexes in the alkaline comet assay of peripheral blood leukocytes, micronucleus and chromosomic aberrations in cells of the bone marrow and the sperm head morphology assay.

2. Assessment the efficiency in the use of the lines of rats Sprague Dawley (SD), Lewis and Wistar of both sexes in the alkaline comet assay of peripheral blood leukocytes, micronucleus and chromosomic aberrations in cells of the bone marrow and the sperm head morphology assay.

3. To perform a comparison among the mouse line and rat selected of both sexes in the alkaline comet assay of peripheral blood leukocytes, micronucleus and chromosomic aberrations in cells of the bone marrow and the sperm head morphology assay.

Animals and Experimental Conditions. The used lines of mice were Balb/c, NMRI, OF-1 and C57/BL6/Cenp of both sexes, young mature (8-9 weeks of age) whose corporal weight oscillated among 26-30 g at the end of the quarantine. Which were administered to reason of 5 animals/sex/lines/group for a total of 10 animals in the two performed replics.

Likewise rats of the genetic lines SD, Lewis and Wistar were used, young adults of both sexes (6-8 weeks) whose corporal weight oscillated among 180-210 g at the end of the quarantine. Which were administered to reason of 5 animals/sex/lines/group for a total of 10 animals in the two performed replications.

Experimental Groups. Four experimental groups were formed, group 1 were not use animals tried as negative control. In these animals the technique of gastric intubation was carried out, they were exposed to the same handling conditions as the other groups, during a period of 14 days.

In the experimental group 2 Tween 65 to 2% was used and NaCl to 0.9 % in the experimental group 3, both substances were administered via oral at 2 ml/kg during a period of 14 days, prepared 2 hours before the administration (Shayne, 2007).

In group 4 use as positive control the CF in dose of 50 mg/kg, via intraperitoneally (i.p), (Ledoxina®, Lemery, CORP), which was diluted in saline solution (NaCl) to 0.9 %. The solution was administered immediately after being prepared, 24 than 48 hours and then at the 24 hours before the euthanasia programmed before to the same dose described to reason of 10 ml/kg (Shayne, 2007).

We must highlight that for the case of the sperm head morphology assay we used mice and male rats of each evaluated line, forming these same experimental groups to the dose and mentioned previously, the administration of each one of these substances was during 5 serial days and then they were without administering 35 days in the case of the mice (Shayne, 2007) and 52 days in the rats (duration of the spermatic cycle of each species) (Shayne, 2007).

After having concluded the time of administration, we were performed the alkaline electrophoresis of individual cells assay (comet assay), micronucleus assay in bone marrow, chromosomic aberrations assay in bone marrow and the sperm head morphology assay, starting from the standardized protocols by our work group (Arencibia et al., 2009a; Arencibia and Rosario, 2010).

Cytogenetic Technical

Alkaline electrophoresis of individual cells. The comet assay of peripheral blood leukocytes was performed according to the standardized protocols and adjusted by (Arencibia and Rosario, 2010). 15-20 &µL of samples were suspended in 140 &µL of low melting point agarose to 0.5 %; then previously prepared sheets were added with agarose. They dove in lysis solution to pH 10 for 1.5 hr to 4 ºC and subjected to 20 min of denaturalization in electrophoresis regulatory solution, pH> 13. The electrophoresis was performed to 300 mA and 1 V/cm during 18 to 20 min (Collins, 2004). The sheets were washed with solution neutralization regulatory using the Tris 0.4 M to pH 7.5 and clarified with distilled water, later they were tinted with silver nitrate to 0.05 %. The nucleoids were evaluated using a microscope of light transmission, Olympus BH-2, for three independent observers, to establish an average among readings. The visual analysis includes the quantification of 100 comets per animal in the gel center. The comets were classified chord to the category or degree of corresponding to DNA damage between 0 and 4 category (Collins, 2004; Flee and Steinert, 2003). The magnitude of the DNA damage was expressed in arbitrary units (UA) starting from possible values in an interval of 0-400.

The micronucleus assay in bone marrow cells. The micronucleus assay in bone marrow cells were performed according to the standardized protocols and adjusted by (Arencibia et al., 2009a). It was extracted one femur per animal and the medullary cavity was washed generously by flow with 3 mL of fetal bovine serum. The bone marrow cells obtained were centrifuged at 200 g for 10 min; after eliminating the liquid it was performed an expansion of the cellular button in coverslips. After mounted the sheets (minimum: 2/animal) 24 hr stayed to ambient temperature for its drying, and to fix in absolute methanol during 5 min. They were tinted in Giemsa at 5% during 12-15 min. The analysis was performed for three independent observers, using a microscope Olympus BH-2 (100X with immersion lens). The presence of polychromatic erythrocytes (PE) and normochromatic erythrocytes (NE) in 2000 cells/animal were counted (Hayashi et al., 1994). Also, the frequency of PE with micronucleus (MN-EP) was calculated in 2000 PE/animal. Later on the cytotoxicity index was calculated (PE/NE) of the total population of erythrocytes and the number of PE were counted with 1 MN, 2 MN and (2 MN per treatments group (Hayashi et al., 1994).

Chromosomic aberration assay in bone marrow cells. The chromosomic aberration assay in bone marrow cells were performed according to the standardized protocols and adjusted by (Arencibia et al., 2009a). The cellular division in metaphase stopped using colchicine (4 mg/kg, via i.p), in the next day schedule (4 hours before the euthanasia). One femur of each animal was extracted and the medullary cavity was washed with 3 mL of foetal bovine serum (FBS). The cellular suspension was centrifuged, being eliminated the liquid suspension. After the hypotonic treatment of the cells of the button with KCL (0.075 M), it was performed a second centrifugation (Kramer, 2000). The cellular button was fixed in mixture methanol-glacial acetic acid (3:1 proportion) during 15 minutes. Three fixations with successive centrifugations were performed, and extended in humid sheets with previous cooling. The sheets dried off to the air and they were tinted with solution from Giemsa to 10% during 30-35 min. 100 metaphases was counted by animal, being determined the number of cells with aberrations (ruptures and chromosomes exchanges, ruptures and chromatids exchanges) and gaps frequency (Kramer, 2000). Also the mitotic index MI% was calculated (metaphases percentage in 1 000 readable cells) and the number of polyploidy cells in 1 000 readable cells. All the determinations were read by two observers, and then an average among both was established (Arencibia et al., 2009a).

Sperm head morphology assay

When performed this assay for the case of the mice we proceeded to extract both epididymis and we analysed one epididymis for the case of the rats, which decreased to small fragments with the help of sharp scissors when being deposited in Petri dishes that contained 3 mL of isotonic solution of NaCl 0.9% (12). The sample was homogenized with Pasteur pipettes (Arencibia et al., 2009a).

Count of sperms. The content of the Petri dishes was placed in a graduate tube, to which was added 0.05 mL of trypsin to 0.25%, after of five minutes of enzyme action, more than 2 mL of NaCl 0.9% was added. Then we performed a dilution of the homogenized with trypsin in NaCl-formaldehyde to 1% (1:10) and it was placed in a NewBauer chamber, being both sides from the camera to the microscope Olympus BH-2, at 10x (Wyrobek, 1983; Arencibia et al., 2009a).

Sperm head morphology. To the tube that contained the dilution of the one already homogenized dilution, it was added five eosin drops to 1%, and allowed for 5 minutes achieving their effect. Later on, a drop extended on a dry sheet and the coverslips was placed (Arencibia et al., 2009a). After mounted the sheets (minimum: 2/animal) and 500 sperms were analysed with a microscope Olympus BH-2, at 40x. The observations were performed blindly by two independent observers, and then to establish an average between both. The classification approach was based on normal and abnormal heads that it includes amorphous, banana, without hook and with two tails (Arencibia et al., 2009a).

Euthanasia methods and statistical analysis. The method of euthanasia selected was the cervical dislocation with previous ether atmosphere. All the results were compared against the group negative control and among lines of mice and of rats for the same group and sex. Likewise the results were compared among murine species (Arencibia et al., 2009a; Arencibia and Rosario, 2010). The continue variables were analysed by ANOVA test (p<0.05) and the categorical variables by Chi Squared (p<0.01) (Arencibia et al., 2009a; Arencibia and Rosario, 2010). All the analyses were performed using the Statsoft for Windows. StatSoft, Inc. (2003). STATISTICA (data analysis, software system), version 6.

Permission of animal ethics committee. During the experimental process the established ethical principles were respected for the research with laboratory animals. The authors declare that this work was made on the base of good practices of preclinical laboratory present in the national regulation of protocols approval of research in the Cuban republic. It is also declared on the part of the authors that it was obtained the consent of protocol approval and report in writing when this research began.

Results

When comparing the endogenous and induced results among both murine species, it was obtained as a result that in most of the evaluated assay the line of Balb/c mice turned out to be the ideal biomodel, being the lower spontaneous indexes and induced high to the mutagen used; allowing us to detect in a narrow error margin those substances that are classified of very low genotoxicity. The results obtained in this line of mice differed significantly with those obtained in SD rats in both sexes. Only in the comet assay of peripheral blood leukocytes, was it obtained in rats of the SD line lower endogenous values that in Balb/c mice.

When performed a comparison among lines of mice keeping in mind the results obtained in the alkaline comet assay, it is appreciated that there were significant differences among the Balb/c line in both sexes with the other ones, it didn't seize among the other three evaluated lines. These results were obtained when comparing the% of spontaneous nucleoids and induced with CF in each one of the levels of damage, being in this line the lower spontaneous or basal levels of damage and acceptable induced, standing out their sensibility to mutagenic substances as the CF (Arencibia et al., 2010, 2011a).

From the micronucleus assay it was obtained as a result that the line Balb/c equally differed with the other three lines evaluated in both sexes. The differences were given when keeping in mind the cytotoxicity index obtained of the relationship polychromatic erythrocytes (PE)/normochromatic erythrocytes (NE), the genotoxicity index (% of PE that contain micronucleus), as well as the number of PE with 1, 2 or more than 2 micronucleus, as index of damage severity. In this line we obtained the lower spontaneous results and the highest induced results, being observed a high sensibility of this animal biomodel to detect clastogenic compound (Arencibia et al., 2009d, 2009e, 2010, 2010a, 2011b).

On the other hand, in the chromosomic aberration assay, useful to detect in vivo substances that induce aberrations of structural type in bone marrow cells, met significant differences among the Balb/c line in both sexes with the other evaluated lines. When performed the comparison among lines it was obtained that the Balb/c differed from the other ones, for the fact of having the lower spontaneous indexes and induced intermissions keeping in mind the total cells with aberrations, number of chromosomic aberrations and chromatid aberrations, aberrations of the gaps type, as well as the number of cells with polyploidies and the mitotic index. These last two variables are of the numeric type (Arencibia et al., 2009f, 2010b, 2010c, 2010d). The line of OF-1 mice also differed in significant way of the C57BL/6/cenp, in this last one the highest spontaneous and induced values were obtained, being less efficient and sensitive to the damage determined by this cytogenetic technical (Arencibia et al., 2010b).

When performed a comparison among these 4 lines in male mice, in the sperm head morphology assay, it was obtained that the most efficient line again was the Balb/c (Arencibia et al., 2009c, 2009e, 2010). This line differed in an evaluated significant way of the other ones, keeping in mind that in this line the highest values in spontaneous spermatic concentration were obtained as cytotoxicity index and the lowest values in anomalies in the sperm head as indicator of genotoxic damage (Arencibia et al., 2009e, 2010, 2010d; Arencibia, 2010). Of equal forms it was the line that better responded to the evaluated mutagen (Arencibia et al., 2009e, Arencibia and Rosario, 2011). The spontaneous results obtained in Balb/c mice are lower than those obtained by us in the negative control group (not administered), in a study where they were used OF-1 mice (Arencibia et al., 2009g). These results demonstrate that genetically the line of Balb/c mice in both sexes is more stable than the other ones evaluated, besides demonstrating that it presents an acceptable response to the action of the mutagenic substances.

In the study of the comparison among lines of rat"s smaller number of damage it was observed at the basal DNA in the line of SD rats in both sexes when being evaluated in the alkaline comet assay of individual cells (Arencibia and Rosario, 2010a; Arencibia et al., 2010e, 2011c). The induction results obtained with the CF did not differ among lines, result that it was manifest in both sexes.

The CF differed again with the other groups in the three lines of rats evaluated in the micronucleus assay. The smallest basal result of cytotoxicity given by the relationship PE/NE was obtained in the line of SD rats in both sexes (Arencibia and Rosario, 2010b, 2010c; Arencibia et al., 2011c, 2011d). Equally the response of this rat line to the CF was high, but smaller to the clastogenic results obtained in the Lewis and Wistar lines in both sexes. On the other hand the CF induced a considerable number of MN in this line of rats but the biggest clastogenic effect it was in the line of Wistar rats in the male"s sex.

The results of the chromosomic aberrations assay, demonstrated once again the use of SD rats as more efficient biomodels in the genotoxicity assay. Again the analysed variables keeping in mind the aberrations of structural type and the mitotic index differed among the SD rats in both sexes when being compared with the Lewis and Wistar lines. We obtained the lowest basal results in cells with aberrations in SD rats (Arencibia and Rosario, 2010b, 2010d). But the biggest effect in the CF it was obtained in the line of Lewis rats.

The male SD rats overcame the other evaluated lines in the number of normal basal sperms. Of the other two evaluated lines the one where bigger results of basal anomalous sperms obtained was the Lewis. On the other hand in the three evaluated lines were obtained a genotoxic ambient in the germinal cells with the use of the CF, being obtained bigger results of induction of anomalous sperms in the line of Lewis rats.

The fact that SD rats differed in a significant way in the variables of damage measured by these four assay with the other two evaluated lines, it reports a great utility, since the SD rats constitutes a heterogeneous line, with very similar response to that of the human populations, justifying its use in most of the pharmacological and toxicological preclinical studies (Arencibia and Rosario, 2009, 2010e; Arencibia et al., 2010f).

Once selected the best mouse and rat as biomodels in genotoxicity assay we performed a comparison among both murine species among the endogenous rates and induced with cyclophosphamide keeping in mind the cytogenetic technical of the alkaline comet assay of peripheral blood leukocytes, micronucleus and chromosomic aberrations in bone marrow cells assay and lastly the sperm head morphology assay starting from samples of epididymis sperm.

The comet assay it showed that the best biomodel in both sexes was the SD rats differing significantly with the results obtained in Balb/c mice keeping in mind the spontaneous and induced values of damage to the DNA (Arencibia et al., 2011e). The SD rats constitutes a heterogeneous line, reason why it will mimic with more degree of trust the possible effects that can achieve a drug evaluated by means of this assay in humans. Not being this way the mice of the Balb/c line, this mice lines is isogenic and it experiences smaller degree of genetic variability (Arencibia et al., 2011e).

In the micronucleus assay, the ideal biomodel was the Balb/c mouse, keeping in mind the lowest values in spontaneous micronucleus and the induced higher values, being more susceptible than the SD rats to the CF (Arencibia et al., 2011f). Demonstrating that the Balb/c mice by means of the mechanism of endogenous formation of micronucleus in erythrocytes of bone marrow cells are more stable genetically that the SD rats (Arencibia et al., 2011g), being a strong predominant factor in this study again the fact that mouse line is it is isogenic, obtaining low rates of genetic variations and epigenetics among individuals (Arencibia et al., 2011f).

On the other hand in the chromosomic aberrations assay the best experimental biomodel as for the spontaneous indexes was the Balb/c mice, experienced a smaller number of cells with aberrations, but the rats SD demonstrated to be more susceptible to the CF, inducing bigger number of structural chromosomic aberrations (Arencibia et al., 2011f). Then SD rats could be used in this assay to determine genotoxic activity of new drugs that induce damage to the DNA by alquilant effect, as chemical with clastogenic effects (Hayashi et al., 1994; Higashikuni and Sutou, 1995), allowing the determination of the mechanism of damage of new drugs.

When comparing the basal and induced results in the sperm head morphology assay among both species it was obtained that the line of Balb/c mice differs with the SD rats in all the analysed variables (Arencibia et al., 2011k). Experiencing this line of mice the lower spontaneous values as for the analysis of the genotoxic variables, but in turn this line experiment bigger spermatic concentration that the SD rats (Arencibia et al., 2011k). When analysing the results of the induced variables it is observed that equally in the case of the morphology of the sperm head the Balb/c mice is more susceptible to the CF (Arencibia et al., 2011i), but when measuring the cytotoxicity by means of the spermatic concentration they turned out to be more resistant to the damage induced by the CF than the SD rats (Arencibia et al., 2011k). In this study we obtained as a final result that the best experimental biomodel to be the Balb/c mice (Arencibia et al., 2011i), differing significantly with the results obtained in the SD rats, keeping in mind the spontaneous and induced values in the spermatic concentration and the frequency of morphological anomalous sperms in epididymis (Arencibia et al., 2011k).

The opposing differences as for the expression of the genotoxic damage caused by the CF could be since in response to different levels of expression of the genes that code for the cytochrome P-4501A1 enzyme in the liver, the mice and rats differ genetic and epigenetically when keeping in mind this fundamental hepatic enzyme in the I phase of the xenobiotic metabolism that participates in the metabolism of the CF in the liver when being used as cytostatic drug (Amri et al., 1986; Bell et al., 1993; Jana et al., 1998).

For this drug to be metabolized, it is necessary the activation in the hepatic microsomal, in a first step to hydroxide-cyclophosphamide, transforming spontaneously to aldo-phosphamide, and later on in the target cells it becomes mustard phosphor amide, of which four metabolites arises: -mustard phosphor amide (activate), achroleine, carboxyl- phosphamide and 4-cecophosphamide that are barely active. The CF is inactivated for microsomal and hepatic enzymes with active participation of the P-450 cytochrome (Arencibia et al., 2010f).

For the first time it was performed a deep characterization, endorsed statistically by several biomodels keeping in mind their efficiency (Arencibia et al., 2011, 2011h, 2011j). Besides not existing until the moment a harmonization as for the best biomodel able to detect substances with low or little genotoxic effect, that which when suggesting the use of a species and line in particular will allow to these assays a bigger sensibility and robustness (Arencibia et al., 2011, 2011h, 2011j). This study allowed the obtention of a good characterization from the point of view of the endogenous damage to the DNA for several genotoxicity mechanisms, allowing a bigger knowledge of the biomodels that markets our country, being of importance to support in equal forms the export toward other countries interested in evaluating its products using our biomodels.

Acknowledgment

We wanted to thank to the researchers of the biochemistry department of the Centre for the evaluations and biological investigations CIEB-IFAL (Cuba). We can never cease to express gratitude as well to all the persons who made it possible to conclude this basic investigation in genotoxicity in vivo assays, and also to the excellent student Stacy Olivia James (IFAL-UH) to consent to help us with the translation of this research.

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Autor:

Daniel Francisco Arencibia Arrebola1*

MSc, Luis Alfredo Rosario Fernández2

MSc, Yolanda Emilia Suárez Fernández3

PhD, Alexis Vidal Novoa4

PhD, Livan Delgado Roche5 MSc.

1Finlay Institute. Havana. Cuba.

2Institute of Pharmacy and Food Science. Havana. Cuba.

3Agrarian University of Havana. Veterinary Faculty. Havana. Cuba.

4University of Havana. Biology Faculty. Havana. Cuba.

5Center of Study for the Research and Biological Evaluations (CEIEB-U.H). Havana. Cuba.

*Correspondency to: Finlay Institute, Research Vicepresidency. 17 avenue between 198 and 200 street, Atabey Repart, Playa Municipality, Post Office 16017, Postal Code 11600, Havana, Cuba.