Damage, repair, and mutagenesis in nuclear genes after mouse forebrain ischemia-reperfusion

Abstract

To determine whether oxidative stress after cerebral ischemia-reperfusion affects genetic stability in the brain, we studied mutagenesis after forebrain ischemia-reperfusion in Big Blue transgenic mice (male C57BL/6 strain) containing a reporter lacI gene, which allows detection of mutation frequency. The frequency of mutation in this reporter lacI gene increased from 1.5 to 7.7 (per 100,000) in cortical DNA after 30 min of forebrain ischemia and 8 hr of reperfusion and remained elevated at 24 hr reperfusion. Eight DNA lesions that are characteristic of DNA damage mediated by free radicals were detected. Four mutagenic lesions (2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyadenine, 5-hydroxycytosine, and 8-hydroxyguanine) examined by gas chromatography/mass spectrometry and one corresponding 8-hydroxy-2'-deoxyguanosine by a method of HPLC with electrochemical detection increased in cortical DNA two- to fourfold (p < 0.05) during 10-20 min of reperfusion. The damage to gamma-actin and DNA polymerase-beta genes was detected within 20 min of reperfusion based on the presence of formamidopyrimidine DNA N-glycosylase-sensitive sites. These genes became resistant to the glycosylase within 4-6 hr of reperfusion, suggesting a reduction in DNA damage and presence of DNA repair in nuclear genes. These results suggest that nuclear genes could be targets of free radicals.

Fig. 1.

Mutant frequency of a nuclear gene (lacI). The number of phages assayed was 205,430 in three animals with sham operation, and the numbers assayed after 30 min of ischemia were 292,280 (0.25 hr of reperfusion, three animals), 338,875 (8 hr of reperfusion, seven animals), and 130,220 (24 hr of reperfusion, four animals). Two of four mutants from the sham operation have the same mutation at the same location (Table 1) and are regarded as having the same clonal origin. (* denotes statistical significance at p < 0.01 by χ² test; DataMost).

Fig. 2.

SSCP-RNA analysis. A mutant RNA transcript of theE. coli lacI mutant was identified by its altered mobility in 8% nondenaturing PAGE (arrow).

Fig. 3.

The effect of centrifugation on sample preparation in the measurement of DNA lesions after forebrain ischemia–reperfusion. DNA lesions were measured by the HPLC–EC method. Data are expressed as mean ± SEM. The number of animals (n) is indicated at the bottom ofeach bar. The statistical significance (ttest) between total DNA (without centrifugation) and nuclear DNA (with centrifugation) was indicated by the p values within the control (Normal) and ischemic groups.

Fig. 4.

Measurement of damage to total cellular DNA after forebrain ischemia–reperfusion by the HPLC–EC method. Each time point contained samples from at least four animals, except samples from the 360 min reperfusion time point (n = 2), which were not included in statistical analysis but are presented with a bar representing the range of data from two animals. (* denotes significance level at p ≤ 0.05 as compared with the control groups using normal and sham-operated animals).

Fig. 5.

Measurement of DNA lesions in total cellular DNA after forebrain ischemia–reperfusion by the GC/MS-SIM method (see Fig.4 for details). (* denotes significance level at p ≤ 0.05 as compared with the control groups.)

Fig. 6.

Measurement of DNA lesions in total cellular DNA after forebrain ischemia–reperfusion by the GC/MS-SIM method (see Fig.4 for details). (* denotes significance level at p ≤ 0.05 as compared with the control groups.)

Fig. 7.

Measurement of DNA lesions in total cellular DNA after forebrain ischemia–reperfusion by the GC/MS-SIM method (see Fig.4 for details). (* denotes significance level at p ≤ 0.05 as compared with the control groups.)

Fig. 8.

Measurement of DNA lesions in total cellular DNA after forebrain ischemia–reperfusion by the GC/MS-SIM method (see Fig.4 for details). (* denotes significance level at p ≤ 0.05 as compared with the control groups.)

Fig. 9.

DNA damage in nuclear genes after forebrain ischemia–reperfusion. EcoRI-digested DNA with or without additional Fpg protein digestion was resolved in 0.8% neutral agarose gel using field-inversion electrophoresis (see Materials and Methods) and then stained with ethidium bromide (bottom). One of two gels is shown. Three molecular weight markers (lane m) that represent 19, 12, and 8 kb are visible in the bottom panel. A ruler with increments of 10 mm is shown on the left side of the gel. The samples without added Fpg protein were normal (lane N), sham operation (lane S), and 30 min with reperfusion of 0 min (lane 1), 2 min (lane 3), 10 min (lane 5), 20 min (lane 7), 240 min (lane 9), and 360 min (lane 11). The samples with added Fpg protein (thelanes without label, bottom) were on the adjacent lanes to the right of each individual sample. The DNA in the gel was then transferred to a membrane. The membrane was hybridized to³²P-labeled, gel-purified single-stranded cDNA probe. A 2 kb fragment of γ-actin genes was detected in the autoradiogram and presented in the top panel. The radioactivities in the blot were stripped (see Materials and Methods), and the blot was hybridized to a second probe of DNA polymerase-β; a fragment of 6–7 kb was detected (center panel). The label on each lane was the same as in agarose gel except that the DNA sample with Fpg protein treatments was identified with +.

Fig. 10.

DNA fragmentation after forebrain ischemia (30 min). Photographs were taken using a mercury light source with filters [dual exposures: a first exposure with Leica (Deerfield, IL) I3 filter (excitation 450–490 nm; barrier at 510 nm) for fluorescein and a second with N2.1 filter (excitation 515–560 nm; barrier at 580 nm) for PI], and with 1000× magnification in one representative animal of selected time point. A, Layer II nuclei of normal cortex from animal without ischemia. B, C, Layer II nuclei of the cortex and CA 3 nuclei of the hippocampus with 1 d of reperfusion, respectively. D, Caudate putamen nuclei with 3 d of reperfusion. E, F, Nuclei from the cerebellum of normal brain and of brain with ischemia and 3 d of reperfusion, respectively. Nuclei with PI stain appeared red, whereas nuclei with anti-dig-fluorescein conjugates and incorporated dig-dUMP on the DNA fragment appearedbright green (D, TUNEL-positive nuclei). Nuclei with the appearance of orange oryellow fluorescence in B, C, andD suggest a blend of red (PI stain) withslight green (conjugates of anti-dig-fluorescein and dig-dUMP).