Sensitivity of the Pig-a assay for detecting gene mutation in rats exposed acutely to strong clastogens

Abstract

Clastogens are potential human carcinogens whose detection by genotoxicity assays is important for safety assessment. Although some endogenous genes are sensitive to the mutagenicity of clastogens, many genes that are used as reporters for in vivo mutation (e.g. transgenes) are not. In this study, we have compared responses in the erythrocyte Pig-a gene mutation assay with responses in a gene mutation assay that is relatively sensitive to clastogens, the lymphocyte Hprt assay, and in the reticulocyte micronucleus (MN) assay, which provides a direct measurement of clastogenicity. Male F344 rats were treated acutely with X-rays, cyclophosphamide (CP) and Cis-platin (Cis-Pt), and the frequency of micronucleated reticulocytes (MN RETs) in peripheral blood was measured 1 or 2 days later. The frequencies of CD59-deficient Pig-a mutant erythrocytes and 6-thioguanine-resistant Hprt mutant T-lymphocytes were measured at several times up to 16 weeks after the exposure. All three clastogens induced strong increases in the frequency of MN RETs, with X-rays and Cis-Pt producing near linear dose responses. The three agents also were positive in the two gene mutation assays although the assays detected them with different efficiencies. The Pig-a assay was more efficient in detecting the effect of Cis-Pt treatment, whereas the Hprt assay was more efficient for X-rays and CP. The results indicate that the erythrocyte Pig-a assay can detect the in vivo mutagenicity of clastogens although its sensitivity is variable in comparison with the lymphocyte Hprt assay.

Fig. 1.

Animal treatment and analyses schedule. Treatment timing is represented by vertical lines and triangles at the top of each graph. Post-treatment timing is relative to the last treatment event. Each grey block represents 1 week. Vertical dashed lines indicate timing for the MN assay, the Pig-a assay or the Hprt assay performed on all or on part of the animals. Vertical ‘Hprt’ label at the end of sequence of 1-week blocks indicate that these animals were euthanised for the Hprt assay. Note that in each experiment there were more animals in vehicle control- and high-dose-treated groups.

Fig. 2.

Reticulocyte micronucleus assay. Left hand vertical scale: bars represent fractions of MN RETs. Average and standard deviations are shown. Right hand vertical scale: solid circles connected with dotted lines show fractions of reticulocytes out of total RBCs (only average values are shown). For X-rays and Cis-platin (Cis-Pt), MN data were determined 24h after the last treatment. For X-rays and cyclophosphamide, the fraction of MN RETs was determined out of the sample of 20 animals (5 animals per each group), although more animals were employed in each experiment. For Cis-Pt, the fraction of MN RETs was determined in all 54 animals. For cyclophosphamide experiment, the data were shown for blood collected 48h after the acute treatment; at 72h, the average fraction of MN RETs was even higher, yet the overall fraction of reticulocytes was severely decreased, so that recommended number of cells could not be processed from individual animals. **different from vehicle control animals (ANOVA, P < 0.05).

Fig. 3.

RBC Pig-a gene mutation assays. Average frequency of CD59-deficient total RBCs (squares) or reticulocytes (circles) are shown for each datapoint. Error bars are not shown in order to avoid congestion. *different than vehicle control (t-test, P < 0.05); **different than vehicle control (ANOVA, P < 0.05). Cis-Pt: at 16-week time point, blood from animals designated for mutant enrichment (Litron’s protocol) also was processed using reticulocyte enrichment protocol; the results produced by the two protocols were similar in terms of the magnitude of the response and the statistical significance (data not shown). Note that in X-rays, cyclophosphamide and Cis-Pt/PIGRET experiments, the number of animals representing average value for the controls and the high-dose-treated animals at different time points is decreasing due to sampling of five vehicle control- and five high-dose-treated animals for the Hprt assay at specific times (see Figure 1). In Cis-Pt experiment, the number of animals analysed using the Litron enrichment protocol remained the same at all time points.

Fig. 4.

Hprt gene mutation assay. Average and standard deviations (as error bars) are shown for each datapoint. X-rays: for 9-week time point, one outlier in the 3 Gy dose group was discarded; for 16-week time point, one outlier from the 3 Gy dose group and one outlier from the 2 Gy dose group were discarded. Cyclophosphamide: for the 16-week time point, one outlier in the 10mg/kg group and one outlier in the 40mg/kg group were discarded. Cis-Pt: for the 16-week time point, one outlier in the 6mg/kg group was discarded. All outliers had much elevated frequency of 6-TG-resistant clones. Note that due to elimination of animals, different number of animals may constitute a datapoint at different time points. *different from the vehicle control (t-test, P < 0.05); **different from vehicle control group (ANOVA, P < 0.05).