Assessment of Sprague Dawley rats as biomodel in two antigenotoxicity assays

The discovery of new drugs with antigenotoxic effect constitutes in our days a prioritized line of research. In this work was evaluated the response of Sprague Dawley rats (both sexes) against cyclophosphamide and bleomycin by means of alkaline comet assay of peripheral blood leukocytes and micronuclei in bone marrow cells assay as antigenotoxicy biomodel. Higher induction of the strand breaks (SB) and alkali-labile sites formation on DNA damage were obtained with the use of the cyclophosphamide and bleomycin, both in the design of 48 and 24 hr administration before the euthanasia. In the mironucleis assay was obtained high results of induction the micronucleus formation in bone marrow cells with cyclophosphamide was administered 48 and 24 hr before euthanasia in both sexes. These results are useful in studies of drugs evaluation that they have not been explored in to the in vivo antigenotoxicity environment.


The discovery of new drugs with antigenotoxic
The first is an alquilant agent that forms effect constitutes in our days a prioritized line of monoadducts and crossed connections between chains research.At the same time many researchers develop as consequence of the appearance of ruptures for and validate sensitive biomodels to the damages reparative mechanisms effects; the second induce caused by the classic mutagens.
liability and rupture of the structure of the DNA, to The strand breaks (SB) or alkali-labile sites interchange with the oxygen and the iron, producing formations on DNA are parameters broadly used for free radicals (5). the genotoxicity detection and it has been Both drugs are used with great effectiveness as demonstrated their associations with degenerative antineoplastic.The CF belongs to the cloroethylillnesses, cancer and oxidative stress (1).In 1988, amines group; considered a bifunctional alquilant Singh and collaborators developed the alkaline variant agent, doesn't possess specificity for phase some of the of the electrophoresis of individual cells (comet cellular cycle.The BL is considered inside the group of assay), providing for the first time data that the antibiotic tumorals (6).demonstrate damage on DNA at individual cell level (2).
At the present time the antigenotoxicity The micronuclei assay is easily to reproduce and biomodels has little relevance due to the repetitive its offer clear information on the cellular proliferation absence in the treatment outlines and to the mutagen in bone marrow.These systems allow registering in selection.This bears to that the results of the induction vivo the capacity of the chemical substances to induce of the damage on DNA and the reparative effects of the chromosomics ruptures to interfere with the product that it is evaluated are questionable.For that chromosomes metaphases migration during the mitosis of the somatic cells (3).
the most of the substances with antigenotoxic character in in vivo until the moment have been solely evaluated on The classic mutagens more used on vitro assays.genotoxicity studies are the cyclophosphamide (CF) In this work was evaluated the response of Sprague adjusted by Arencibia et al., 2010 (8).15-20 µL of Dawley rats (both sexes) against cyclophosphamide samples were suspended in 140 µL of low melting (CF) and bleomycin (BL) by means of alkaline comet point agarose to 0,5 %; then previously prepared assay of peripheral blood leukocytes and micronuclei sheets were added with agarose.They dove in lysis in bone marrow cells assay as antigenotoxicy solution to pH 10 for 1,5 hr to 4 ºC and subjected to 20 biomodel.
min of denaturalization in electrophoresis regulatory solution, pH> 13.The electrophoresis was performed

Animals and environmental conditions:
sheets were washed with solution neutralization Rats regulatory using the Tris 0,4 M to pH 7,5 and clarified Sprague Dawley young adults of both sexes was used (6-8 weeks) whose corporal weight oscillated among with distilled water and later they were tinted with silver nitrate to 0,05 % (1, 2).The nucleoids were 180-210 g at the end of the quarantine, they where evaluated using a microscope of light transmission, remained under controlled conditions: temperature Olympus BH-2, for three independent observers, to (25 ± 1 ºC), relative humidity (60 ± 10%) and cycles of establish an average among readings (2,8).The visual light -darkness of 12 hr.The access to the water and analysis includes the quantification of 100 comets per the food (CENPALAB), it was ad libitum.These animal in the gel center (2).The comets were classified characteristics were common for all the experimental chord to the category or degree of corresponding to groups evaluated in both assays.
DNA damage between 0 and 4 category.The Experimental groups: In the experimental groups magnitude of the DNA damage was expressed in the substance were administered in the tomorrow's arbitrary units (UA) (2, 8) starting from possible schedule and the concentrations were adjusted weekly values in an interval of 0-400 (1,2). in function of the corporal weight increase.The The micronucleis assay in bone marrow cells animals were randomly distributed 10 rats/group/sex.
were performed according to the standardized Five experimental groups were formed per sex.The protocols and adjusted by Arencibia et al., 2009 (9).It first was administered with NaCl 0,9 % by was extracted one femur per animal and the medullary intraperitoneal (i.p) route (negative control group), at cavity was washed kindly by flow with 3 mL of fetal dose of 2 ml/kg in two occasions with intervals of 24 bovine serum.The bone marrow cells obtained was hr among both administrations.centrifuged at 200 g for 10 min; after eliminating the The second and third groups were administered liquid it was performed an expansion of the cellular with CF at dose of 50 mg/kg by i.p route.The second button in coverslips (9).After mounted the sheets group was administered in two occasions 48 and 24 hr (minimum: 2/animal) 24 hr stayed to ambient before the euthanasia and the third groups was only temperature for its drying, and to fix in absolute administered 24 hr before the euthanasia, in both methanol during 5 min.They were tinted in Giemsa to groups the CF was dissolved in NaCl to 0,9% at 10 5% during 12-15 min (9).The analysis was performed ml/kg (7).The fourth and fifth groups were for three independent observers, using a microscope administered with BL at dose of 40 mg/kg by i.p route.
Olympus BH-2 (100X with immersion lens).The The fourth group was administered in two occasions presence of polychromatic erythrocytes (PE) and 48 and 24 hr before the euthanasia and the fifth group normochromatic erythrocytes (NE) in 2000 was only administered 24 hr before the euthanasia, in cells/animal were counted.Also, the frequency of PE both groups the BL was dissolved in NaCl to 0,9% at with micronucleus (MN-EP) was calculated in 2000 10 ml/kg (8).

PE/animal. Later on the cytotoxicity index was
Assessments of the comet assay of peripheral calculated (PE/NE) of the total population of blood leukocytes and the micronucleis assay in bone marrow cells: erythrocytes and were counted the number of PE with In both assay the evaluated 1 MN, 2 MN and 2 MN per treatments group (9).cellular product was 24 hr after the second administration of the evaluated substances for the first, The method of euthanasia selected was the second and fourth group.The evaluated cellular cervical dislocation with previous ether atmosphere.
The continue variables were analyzed by ANOVA test product was 24 hr after the only administration for the (p<0.05) and the categorical variables by Chi Squared third and fifth group.
(p<0.01).All the analyses were performed using the The comet assay of peripheral blood leukocytes Statsoft for Windows.StatSoft, Inc. (2003).(alkaline electrophoresis of individual cells) was STATISTICA (data analysis software system), 6 version.performed according to the standardized protocols and Permission of animal ethics committee : During induction on DNA damage and arbitrary units were the experimental process the established ethical principles obtained when administering the CF 48 and 24 hr were respected for the research with laboratory before the euthanasia (Table 1).The CF administered twice induced until up to 3.88-4.72nucleoids per cent animals.The authors declare that this work was made with fourth degree of damage.The two groups treated on the base of good practices of preclinical laboratory with BL also differed significantly.Only with the BL present in the national regulation of protocols approval administration in two doses was possible to induce of research in the Cuban republic.It is also declared on high levels of damage.The values of the fourth level of the part of the authors that it was obtained the consent damage for this group were among 4.33-4.82%.In the of protocol approval and report in writing when this In the micronucleus assay (Table 2), it was other assay differ significantly in both sexes.Bigger       obtained as a result that the CF administered 48 and 24 smaller risk for the personnel and easy treatment of hr before the euthanasia it differed significantly with deactivation of useful polluted during the experiment the other groups.The CF was induced the formation of (14,15), these advantages make that you can implement the CF with more use in this antigenotoxicity assay by 250-260 micronucleus in bone marrow cells (both means of this technique, when it also evaluates sexes).However the results obtained after the unique genotoxicity and oxidative stress effects of new drugs.administration with CF and the two types of treatments The small head of the comet can be observed in (b) and with BL they didn't show differences in the analysed (d) figure; with the great increment of the migration variables in the micronucleus assay.
and bigger quantity of degraded fragments on DNA.

This is associated with the DNA fragmentation during the necrosis processes and apoptosis induced by both It was demonstrated to induce an acceptable mutagens (16). genotoxic response in the lymphocytes of peripheral
There were opposing statistical differences blood when the groups tried with mutagens differ among the CF administered in two occasions and the significantly with the solvent control group.The administered in unique dose in the genotoxicity and results found in both assays when administering the NaCl to 0,9 % are similar to those obtained by our cytotoxicity indexes in the micronucleus assay.The work group when evaluating the spontaneous damage action of the CF it is potential when being administered in this rat species (10).
in two occasions (15,17).The opposing differences with the CF in two They were differences between CF administered different outlines in both sexes were given to be this in two occasions with the two treatments used in the mutagen an accumulative genotoxic character, being BL in micronucleus assay; this could be for the the variable time is decisive in the increase of their difference of genotoxic mechanisms among mutagens harmful effect on DNA (11).
or for the dependent susceptibility of the species in the On the other hand it is justified to have bigger outlines of used treatments were not observed biodisponibility of the mutagen when differing the two significant differences among the CF and the BL groups tried with BL, being related the variable administered in two occasions (11).For that the number of detected exhibition-damage (12).Since the predominant factor can be the mutagenic effects that BL constitutes a true and direct mutagen (13).
the CF induces linked to its mechanism, being The results obtained with CF affirm that it is detected by this assay with high specificity and possible to use this mutagen in the induction of fidelity, and in smaller measure the species factor (7, 11).damage in the primary structure on DNA (1,9).The This study allowed concluding that it is more feasible to administer the CF in two occasions, if you CF is a cheap mutagen, easy to manipulate, manifesting useful in studies of drugs evaluation that they have not 9.
been explored in to the in vivo antigenotoxicity figure a, b, c and d, typical comets of fourth level of research began.damage for each experimental group are observed Results including the controls group in both sexes (Figure e and f).The two CF treatments in the alkaline comet

Figure. a=
Figure.a= Comet of peripheral blood leukocytes with 4 level of DNA damage, it was treated with cyclophosphamide administered 24 hours before the euthanasia.

Figure. b=
Figure.b= Comet of peripheral blood leukocytes with 4 level of DNA damage, it was treated with cyclophosphamide administered in two occasions, 48 and 24 hours before the euthanasia.

Figure. c=
Figure.c=Comet of peripheral blood leukocytes with 4 level of DNA damage, it was treated with bleomycin administered 24 hours before the euthanasia.

Figure. d=
Figure.d= Comet of peripheral blood leukocytes with 4 level of DNA damage, it was treated with bleomycin administered in two occasions, 48 and 24 hours before the euthanasia.

Figure. e=
Figure.e= Comet of peripheral blood leukocytes with 0 level of DNA damage, negative control group treated with NaCl 0,9 % (female).

Figure. f=
Figure.f= Comet of peripheral blood leukocytes with 0 level of DNA damage, negative control group treated with NaCl 0,9 % (male).

Table - 1. Comet assay in Sprague Dawley rats of both sexes on the induction of DNA damage of peripheral blood leukocytes with Cyclophosphamide and Bleomycin.
. all statistical analysis was carried out using the Mann Whitney U test, p<0.05.(X average; SD Standard Deviation, for the two experimental replics).1.Administration by i.p route.a= it differs with the negative control, b= it differs among the different treatments for the same mutagen and c= it differs among mutagens without keeping in mind the treatment.CF1= Cyclophosphamide administered 24 hours before the euthanasia and CF2= Cyclophosphamide administered 48 and 24 hours before the euthanasia.BL1= Bleomycin administered 24 hours before the euthanasia and BL2= Bleomycin administered 48 and 24 hours before the euthanasia. T

Table - 2. Cytotoxicity and genotoxicity indexes in bone marrow cells of Sprague Dawley rats of both sexes tried with cyclophosphamide and bleomycin.
. All statistical analysis was carried out using the ANOVA test, p<0.05.(X average; SD Standard Deviation, two experimental replics).a. Administration by i.p route.Count in 2000 cells/animal.CF1= Cyclophosphamide administered 48 and 24 hours before the euthanasia and CF2= Cyclophosphamide administered 24 hours before the euthanasia.BL1= Bleomycin administered 48 and 24 hours before the euthanasia and BL2= Bleomycin administered 24 hours before the euthanasia.Different letters (a and b) they differ when comparing the results among mutagens per sex.(A) Results that they differ when comparing the different treatments using the same mutagen per sex.(c) They differ when comparing the results against the negative control (CN).PE= Polychromatic Erythrocytes, NE= Normochromatic Erythrocytes, MN= Micronucleus.

Table - 3. Total micronucleis in bone marrow of Sprague Dawley rats in both sexes tried with cyclophosphamide and bleomycin.
T All statistical analysis was carried out using the ?2 non parametric test, p<0.01.aAdministration by i.p route.Count in 2000 PE/animal.CF1= Cyclophosphamide administered 48 and 24 hours before the euthanasia and CF2= Cyclophosphamide administered 24 hours before the euthanasia.BL1= Bleomycin administered 48 and 24 hours before the euthanasia and BL2= Bleomycin administered 24 hours before the euthanasia.Different letters (a and b) they differ when comparing the results among mutagens per sex.(A) Results that they differ when comparing the different treatments using the same mutagen per sex.(c) They differ when comparing the results against the negative control (CN).MN= Micronucleus, PE= Polychromatic erythrocytes.