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The rat peripheral blood micronucleus (MN) assay is not considered to be a sufficient biomarker of genotoxin exposure due to the selective elimination of micronucleated cells from peripheral circulation by the spleen. However, several recent reports suggest that peripheral blood reticulocytes of rats may represent a suitable cell population for use in the MN assay. The MN assay in rats with prior bleeding was thus conducted to determine the sensitivity of the bioassay. Hirai et al. reported that prior bleeding enhances the sensitivity of the in vivo MN test in mice. Based on these findings, the rat was used as a model to see the effect of prior bleeding on the sensitivity of the peripheral blood and bone marrow MN assays. In the present experiment, young male Sprague-Dawley (SD) rats ranging in age from 21 to 24 days were used. However, for the comparison of strain-specific induction of MN, Wistar rats were used. The kinetics of MN formation were investigated in adult, young bled and non-bled SD rats treated with cyclophosphamide (CP). For the MN kinetic study, CP was administered intraperitoneally 2 h after bleeding and sampling was done at intervals of 12, 24, 36, 48 and 96 h after chemical administration. Significant increases in MN induction activity in both bone marrow and peripheral blood were observed with prior bleeding. To further validate the influence of prior bleeding in the induction of MN frequency, two other known genotoxins (chlorambucil and mitomycin C) were used. It was concluded that prior bleeding can significantly increase the sensitivity of MN induction in both bone marrow and peripheral blood of rats compared with non-bled animals. Once validated, this model may be suitable for detecting different genotoxins, especially weak and marginally active clastogens.

The chromosome aberration assay (CA) in bone marrow cells and the micronucleus test (MNT) in both bone marrow and peripheral blood erythrocytes have been carried out for the evaluation of the clastogenic potential of acephate (asataf) in a chick in vivo test system. Technical grade acephate was administered to evaluate dose-responses (25, 50 and 100 mg/kg), route-responses (i.p. and p.o.) and time-responses (6, 24 and 48 h). A comparison of CA frequencies after acute and chronic dosing was also performed. Only 50 mg/kg of acephate induced significant bone marrow chromosome aberrations after 24 h exposure while all three doses induced significant increases in micronuclei in both bone marrow and peripheral blood erythrocytes in i.p. route only. The presented data confirm our earlier reports that the neonatal chicken test system is a convenient model and can be used as an alternative to mammalian systems for screening some environmental contaminants for genotoxicity.

Adult rats are generally not considered as a suitable model for the peripheral blood micronucleus (PBMN) assay in regulatory consideration, owing to the splenic removal of the micronucleated cells from circulation. Although prior bleeding (PrB) increases the sensitivity of the PBMN assay in young rats, the volume of bleeding and the associated stress caused are major concerns for its possible use in genotoxicity studies. The present study was aimed to overcome these limitations in using pre-bled young rats in genotoxicity studies. The bleeding volume was reduced by the simultaneous use of erythropoietin (EPO) to increase the sensitivity of PBMN assay. Young Sprague-Dawley (SD, 26 days) rats were used in the study. The kinetics of RETs-to-ERTs ratio was determined in response to EPO (10-3000 IU/kg) or PrB (0.1-1.0 ml) at different time points (0, 6, 12, 24, 36, 48, 72 and 96 h). Injection of EPO (30 IU/kg) and PrB (0.5 ml) led to a significant increase in the MN frequency in the PBMN assay in response to cyclophosphamide and zidovudine. The effect of EPO treatment and/or PrB on cell viability and proliferation in the bone marrow (BM) was examined. The results of the present study clearly demonstrate that the simultaneous use of both EPO and PrB enhances the sensitivity of the PBMN assay in young rats due to increased cellular proliferation in the BM. This may provide a useful experimental model for the evaluation of marginally active genotoxicants.