- 38 -
- 39 -
respective control). On the other hand, NFT treatment did not increase the level of 8-OHdG in the kidneys of either genotype at either time point. Between the Nrf2-proficient and -deficient mice of each treatment group, the 8-OHdG level was not significantly changed.
In vivo mutation assay of kidneys
The results of the gpt assay of the kidneys of Nrf2-proficient and -deficient mice treated with KBrO3 or NFT are shown in Tables 7 to 12. At both of 4- and 13-week, KBrO3-treated mice showed significant increase or tendencies of increase of gpt MFs compared with those in the respective control groups (Tables 7 and 8). The degree of increase of gpt MFs by 13-week treatment with KBrO3 was as follows: Nrf2+/+, × 2.2;
Nrf2-/-, × 4.4, vs. respective control (Table 8). Specific MFs of deletion mutations were increased in the spectrum analysis of gpt mutants in KBrO3-treated mice (Tables 10 and 11). The frequencies of deletion mutations of more than two base pairs were increased by 13-week treatment with KBrO3 in both genotypes (Table 11).
Furthermore, in both genotypes, gpt MFs were increased by 13-week treatment with NFT, despite no change at 4 weeks (Tables 7 and 9). The degree of increase of gpt MFs by 13-week treatment with NFT was as follows: Nrf2+/+, × 2.1; Nrf2-/-, × 3.3, vs.
respective control (Table 9). In both genotypes, guanine base substitution mutations, including G:C–T:A or G:C–C:G transversion mutations, were increased by 13-week treatment with NFT (Table 12).
The results of the Spi– assay of the kidneys of Nrf2-proficient and -deficient
- 40 -
mice treated with KBrO3 or NFT are shown in Tables 13 to 16. At both time points, KBrO3-treated mice showed significant increase or the tendencies of increase of Spi– MFs compared with those in the respective control groups (Tables 13 and 14). The degree of increase of Spi– MFs by 13-week treatment with KBrO3 was as follows:
Nrf2+/+, × 3.0; Nrf2-/-, × 4.1; vs. respective control (Table 14). In the spectrum analysis of Spi– mutants in KBrO3-treated mice, specific MFs of deletion mutations were increased (Tables 15 and 16), consistent with that of gpt mutants. In both genotypes and at both time points, NFT treatment did not change Spi– MFs (Tables 13 and 14).
- 41 -
Discussion
It is well known that transcriptional upregulation of various antioxidant enzymes, including NQO1 and HO1, is regulated by NRF2, which protect cells from oxidative stress (23, 32). In several studies, Nrf2-/- mice showed higher sensitivity to various toxicants that induced oxidative stress (7, 14, 24, 28, 50, 58, 62). In fact, the mRNA expression level of Nqo1 in the kidneys of vehicle-treated Nrf2-/- mice was significantly lower than that of vehicle-treated Nrf2+/+ mice, and there was no elevation of the level in KBrO3- or NFT-treated Nrf2-/- mice despite the elevation in Nrf2+/+ mice.
Thus, in the present study, Nrf2-/- mice were confirmed to be susceptible to oxidative stress. As previously reported, using this highly oxidative stress-sensitive animal gives us important knowledge about the involvement of oxidative stress in chemical-induced genotoxicity and carcinogenesis (7, 14, 24, 28, 50, 58, 62).
Four or thirteen-week administration of 1,500 ppm KBrO3 in drinking water significantly increased the level of 8-OHdG in the kidneys of both genotypes. At both time points, the degree of 8-OHdG increase was higher in Nrf2-/- mice than in Nrf2+/+
mice. Meanwhile, increases of gpt and Spi– MFs were detected, and the tendencies of the degree of increase of gpt and Spi– MFs at 13-week exposure were the same as those of 8-OHdG. In the spectrum analysis of gpt and Spi– mutants in KBrO3-treated Nrf2 -/-mice, specific MFs of deletion mutations were increased, consistent with a previous study of rats (27), accompanied with the increase of the frequencies of deletion mutations of more than two base pairs. An in vitro report demonstrated that error in the repair process of 8-OHdG induced by KBrO3 treatment caused double-strand breaks
- 42 -
(DSBs) in human cells, and DSBs resulted in a large deletion (33). Considering these mechanisms, the increase in size of deletion mutations might reflect the accumulation of high amounts of 8-OHdG in the nuclei due to KBrO3. These results suggested that the formation of 8-OHdG induced by oxidative stress was directly involved in the increase of deletion mutations in KBrO3-treated animals. It was suspected that the formation of high amounts of 8-OHdG owing to the strong potential of KBrO3 as an oxidizing agent might exceed the repairing capacity of base excision repair enzymes.
Four or thirteen-week administration of 2,500 ppm NFT in diet did not increase the level of 8-OHdG in the kidneys of either genotype. In the previous study, the level of 8-OHdG was increased in the kidneys of Nrf2-/- mice by oral administration of NFT at 70 mg/kg (52). Lower exposure levels of NFT in the present study compared with those of the previous study did not induce the elevation of 8-OHdG levels. On the other hand, 13-week administration of 2,500 ppm NFT in diet significantly increased gpt MFs with guanine base substitution mutations in the kidneys of both genotypes. The degree of increase of gpt MFs was higher in Nrf2-/- mice than in Nrf2+/+ mice. These results implied that the vulnerability to oxidative stress caused by the deficiency of Nrf2 leads to more mutations in NFT-treated mice. Thus, in the genotoxic mechanism of NFT, the formation of 8-OHdG induced by oxidative stress might not be involved in the increase of guanine base substitution mutations. Considering our previous studies, which suggested the involvement of oxidative stress in the chemical structure-related genotoxic mechanism of NFT in rodents (27, 52), further studies are requested to identify oxidative stress markers other than 8-OHdG which might be crucial to the
- 43 -
genotoxicity of NFT, though the present study did not identify them.
In recent years, the level of 8-OHdG has been frequently used as a marker of oxidative stress in human diseases (5, 19). In addition, some reports demonstrated the involvement of oxidative stress in chemical-induced genotoxicity and carcinogenesis using the increase of 8-OHdG level as a parameter of oxidative stress in experimental animals (7, 14, 24, 28, 50, 58, 62). However, the relationship between the formation of 8-OHdG and subsequent mutations, including deletion mutations and G:C–T:A transversions, had not been clarified. The revelation of the relationship between 8-OHdG and several types of mutations induced by KBrO3 or NFT provides new insight into oxidative stress-related in vivo mutagenicity.
The present study demonstrated that the formation of 8-OHdG, which resulted from the oxidizing potential of KBrO3, was directly involved in the increase of deletion mutations; however, oxidative stress-related factors other than 8-OHdG might play a critical role in NFT-induced guanine base substitution mutations. This was the first study to investigate the relationship between 8-OHdG and several types of mutations caused by oxidative stress-inducing chemicals. The accumulation of these detailed examinations such as further research on 8-OHdG about individual chemical substance leads to accurate risk assessment of oxidative stress in carcinogenicity.
- 44 -
Abstract
Oxidative stress is well known as a key factor of chemical carcinogenesis.
However, the actual role of oxidative stress in carcinogenesis such as oxidative stress-related in vivo mutagenicity remains unclear. It has been reported that 8-hydroxydeoxyguanosine (8-OHdG), an oxidized DNA lesion, might contribute to chemical carcinogenesis. Potassium bromate (KBrO3) and nitrofurantoin (NFT) are known as renal carcinogens in rats. Our previous studies showed an increase of mutant frequencies accompanied with an increased level of 8-OHdG in the kidneys of rodents following KBrO3 or NFT exposure. Furthermore, KBrO3 and NFT induced different types of gene mutations. Thus, in the present study, I performed reporter gene mutation assays and 8-OHdG measurements following KBrO3 or NFT exposure using Nrf2-proficient and -deficient mice to clarify the relationship between KBrO3- or NFT-induced oxidative stress and subsequent genotoxicity. The administration of 1,500 ppm of KBrO3 in drinking water resulted in the increase of deletion mutations accompanied with the increase of 8-OHdG level, and the administration of 2,500 ppm of NFT in diet induced the increase of guanine base substitution mutations without elevation of 8-OHdG level in Nrf2-deficient mice. These results demonstrated that the formation of 8-OHdG, which resulted from the oxidizing potential of KBrO3, was directly involved in the increase of deletion mutations, although factors concerning oxidative stress other than 8-OHdG might be crucial for NFT-induced guanine base substitution mutations. The present study provides new insight into oxidative stress-related in vivo mutagenicity.
- 45 -
Fig. 6. Experimental design
- 46 -
Fig. 7. Growth curves for Nrf2+/+ or Nrf2-/- mice treated with KBrO3 or NFT for 4 weeks (A) or 13 weeks (B). 4 to 8 mice are used in each group. For both genotypes, there were no significant differences in body weight between treated and untreated mice in either time point.
- 47 -
Fig. 8. Changes in mRNA levels of Nrf2-target gene Nqo1 in the kidneys of Nrf2+/+ or Nrf2-/- mice treated with KBrO3 or NFT for 4 weeks (A) or 13 weeks (B). Data are presented as means ± SD. n=4 or 5/group. **,*: Significantly different (P < 0.01, 0.05) from respective control group. ##: Significantly different (P < 0.01) from respective Nrf2+/+ animals.
- 48 -
Fig. 9. 8-OHdG levels in the kidneys of Nrf2+/+ or Nrf2-/- mice treated with KBrO3 or NFT for 4 weeks (A) or 13 weeks (B). Data are presented as means ± SD. n=3 to 5/group. **, ††: Significantly different (P < 0.01) from respective control group.
49
-27.43±2.59 b 25.70±2.3523.56±2.1526.40±2.1824.05±1.2622.80±1.46 Kidneys (g)0.33±0.030.32±0.040.33±0.040.32±0.030.32±0.040.32±0.04 Kidneys (g%)a1.22±0.111.26±0.091.38±0.061.20±0.101.32±0.161.38±0.13 33.53±3.4529.18±2.4528.28±1.6730.05±2.3824.91±1.8026.55±1.39 Kidneys (g)0.35±0.030.34±0.030.38±0.050.35±0.050.34±0.060.33±0.04 Kidneys (g%)a1.06±0.081.16±0.081.35±0.121.18±0.151.38±0.301.24±0.14 a Kidneys-to-body weight ratios (relative weights) are given as g organ weight/g body weight. b Means ± SD.
Table 6. Final body and kidney weights of male Nrf2+/+ or Nrf2-/- gpt delta mice treated with KBrO3 or NFT for 4 or 13 weeks 8 Final body weights (g)
4 weeks 13 weeks No. of animals88868
Control1,500 ppm KBrO32,500 ppm NFTControl1,500 ppm KBrO32,500 ppm NFT
5 Final body weights (g) Nrf2+/+ Nrf2-/-
Nrf2+/+Nrf2-/-No. of animals45544
Nrf2+/+Nrf2-/- Control1,500 ppm KBrO32,500 ppm NFTControl1,500 ppm KBrO32,500 ppm NFT
50
-GenotypeTreatmentAnimal No.Mean±SD 10114.460.42 10241.2100.24 10320.090.45 10441.0130.320.36±0.09 20132.1300.93 20224.2170.70 20323.7180.76 20421.590.42 20517.6120.680.70±0.19 * 30134.4110.32 30251.4150.29 30338.4180.47 30418.2160.88 30518.1140.770.55±0.27 40140.7140.34 40234.0130.38 40335.4130.37 40418.740.210.33±0.08 50124.5170.69 50231.1220.71 50315.390.59 50429.1110.380.59±0.15 60123.9140.59 60227.7170.61 60329.7170.57 60428.8150.52 60524.3110.450.55±0.06
Table 7. Gpt MFs in the kidneys of Nrf2+/+ or Nrf2-/- gpt delta mice treated with KBrO3 or NFT for 4 weeks * P < 0.05 vs. respective control group CmR , chloramphenicol resistant; 6-TGR , 6-thioguanine resistant; MF, mutant frequency
CmR colonies (× 105 )6-TGR and CmR colonies
MF (× 10-5 ) Control 1,500 ppm KBrO3 2,500 ppm NFT Control 2,500 ppm NFT
Nrf2+/+ Nrf2-/-1,500 ppm KBrO3
51
-GenotypeTreatmentAnimal No.Mean±SD 7018.930.34 70213.190.69 7037.4111.48 7047.491.22 7059.540.42 70616.690.54 70715.060.40 70817.570.400.69±0.43 80112.8221.72 80217.1201.17 80317.8261.46 80411.3221.95 80513.6413.01 80610.390.87 80721.8251.15 80817.4221.271.57±0.67** 100115.290.59 100216.380.49 100327.380.29 100424.070.29 100518.4130.710.48±0.18 11019.9222.23 110223.8411.72 11036.6213.17 110410.2262.56 110513.3241.81 110618.0231.28 110714.0191.36 11088.460.711.86±0.78††
1,500 ppm KBrO3 Control 1,500 ppm KBrO3
Nrf2+/+ ** P < 0.01 vs. respective control group, †† P < 0.01 vs. respective control group CmR, chloramphenicol resistant; 6-TGR, 6-thioguanine resistant; MF, mutant frequency
Table 8. Gpt MFs in the kidneys of Nrf2+/+ or Nrf2-/- gpt delta mice treated with KBrO3 for 13 weeks CmR colonies (× 105 )6-TGR and CmR colonies
MF (× 10-5 ) Nrf2-/-
Nrf2-/-Control
52
-GenotypeTreatmentAnimal No.Mean±SD 7018.460.72 7029.250.54 70311.030.27 70413.640.29 70510.640.38 70621.360.28 70719.640.20 70824.590.370.38±0.17 90115.3211.37 90213.5100.74 90316.2120.74 90412.0131.09 90511.0141.27 90624.3240.99 90714.3161.121.04±0.24** 100115.250.33 100213.380.60 100328.3110.39 100424.290.37 100522.960.260.39±0.13 120113.1211.60 120218.8221.17 120321.6221.02 120413.2151.13 120512.3151.22 12067.6121.58 120712.3221.79 120813.6231.691.40±0.30††
Control 2,500 ppm NFT Nrf2-/-
Nrf2-/-Control
Table 9. Gpt MFs in the kidneys of Nrf2+/+ or Nrf2-/- gpt delta mice treated with NFT for 13 weeks ** P < 0.01 vs. respective control group, †† P < 0.01 vs. respective control group CmR , chloramphenicol resistant; 6-TGR , 6-thioguanine resistant; MF, mutant frequency
CmR colonies (× 105)6-TGR and CmR colonies
MF (× 10-5) 2,500 ppm NFT
Nrf2+/+
53
-Base substitution G:C-T:A7 (18.4)0.06±0.0316 (18.6)0.13±0.0416 (21.6)0.12±0.07 G:C-C:G2 (5.3)0.02±0.032 (2.3)0.01±0.027 (9.5)0.05±0.03 A:T-T:A2 (5.3)0.02±0.0210 (11.6)0.08±0.052 (2.7)0.01±0.02 A:T-C:G002 (2.3)0.02±0.034 (5.4)0.03±0.05 G:C-A:T15 (39.5)0.13±0.0217 (19.8)0.14±0.0726 (35.1)0.18±0.07 A:T-G:C2 (5.3)0.02±0.036 (7.0)0.05±0.034 (5.4)0.03±0.02 Single bp5 (13.2)0.05±0.0416 (18.6)0.13±0.0510 (13.5)0.08±0.06 Over 2bp2 (5.3)0.02±0.027 (8.1)0.05±0.043 (4.1)0.02±0.02 1 (2.6)0.01±0.017 (8.1)0.05±0.04 *00 2 (5.3)0.02±0.023 (3.5)0.03±0.052 (2.7)0.02±0.05 Total380.36860.70740.55 Base substitution G:C-T:A7 (15.9)0.06±0.036 (10.2)0.05±0.0419 (25.7)0.14±0.07 G:C-C:G2 (4.5)0.01±0.023 (5.1)0.03±0.035 (6.8)0.04±0.03 A:T-T:A007 (11.9)0.06±0.086 (8.1)0.04±0.05 A:T-C:G2 (4.5)0.02±0.031 (1.7)0.01±0.022 (2.7)0.02±0.02 G:C-A:T20 (45.5)0.14±0.0610 (16.9)0.10±0.0322 (29.7)0.16±0.02 A:T-G:C2 (4.5)0.01±0.025 (8.5)0.05±0.025 (6.8)0.04±0.04 Single bp6 (13.6)0.04±0.0419 (32.2)0.21±0.09 ††11 (14.9)0.08±0.05 Over 2bp1 (2.3)0.01±0.015 (8.5)0.06±0.061 (1.4)0.01±0.02 1 (2.3)0.01±0.012 (3.4)0.02±0.022 (2.7)0.01±0.02 3 (6.8)0.02±0.011 (1.7)0.01±0.021 (1.4)0.01±0.02 Total440.33590.59740.55
Table 10. Mutation spectra of gpt mutant colonies in the kidneys of Nrf2+/+ or Nrf2-/-gpt delta mice treated with KBrO3 or NFT for 4 weeks * P < 0.05 vs. respective control group, †† P < 0.01 vs. respective control group
Deletion Insertion Complex Insertion Complex
1,500 ppm KBrO32,500 ppm NFT Number (%)Mutation frequencies (10-5)Number (%)Mutation frequencies (10-5)Number (%)Mutation frequencies (10-5)
ControlNrf2-/-
ControlNrf2-/-Nrf2+/+ Transversions Transitions Deletion
Number (%)Mutation frequencies (10-5 )Number (%)Mutation frequencies (10-5 )Number (%)Mutation frequencies (10-5 )
1,500 ppm KBrO32,500 ppm NFTControl Transversions Transitions
54
-Base substitution G:C-T:A15 (25.9)0.18±0.1620 (10.7)0.16±0.1113 (28.9)0.13±0.0615 (8.2)0.15±0.11 G:C-C:G2 (3.4)0.03±0.065 (2.7)0.04±0.051 (2.2)0.01±0.022 (1.1)0.02±0.03 A:T-T:A0011 (5.9)0.10±0.11*3 (6.7)0.03±0.0328 (15.4)0.27±0.16† A:T-C:G0010 (5.3)0.07±0.06**001 (0.5)0.01±0.03 G:C-A:T19 (32.8)0.22±0.2133 (17.6)0.27±0.1319 (42.2)0.20±0.0827 (14.8)0.29±0.16 A:T-G:C4 (6.9)0.05±0.087 (3.7)0.06±0.093 (6.7)0.04±0.034 (2.2)0.05±0.06 Single bp11 (19.0)0.12±0.1169 (36.9)0.60±0.34**3 (6.7)0.03±0.0362 (34.1)0.65±0.41†† Over 2bp2 (3.4)0.03±0.0520 (10.7)0.17±0.07**1 (2.2)0.01±0.0329 (15.9)0.30±0.13†† 4 (6.9)0.04±0.078 (4.3)0.07±0.071 (2.2)0.01±0.026 (3.3)0.06±0.10 1 (1.7)0.01±0.024 (2.1)0.03±0.041 (2.2)0.01±0.038 (4.4)0.07±0.05† Total580.691871.57450.481821.86
Number (%) **, *P < 0.01, 0.05 vs. respective control group, ††, †P < 0.01, 0.05 vs. respective control group
Number (%)Mutation frequencies (10-5 ) Transversions Transitions
Nrf2+/+ Nrf2-/- 1,500 ppm KBrO3 Number (%)Mutation frequencies (10-5 )
Table 11. Mutation spectra of gpt mutant colonies in the kidneys of Nrf2+/+ or Nrf2-/- gpt delta mice treated with KBrO3 for 13 weeks Deletion Insertion Complex
Number (%)Mutation frequencies (10-5 )Mutation frequencies (10-5 ) 1,500 ppm KBrO3ControlControl
55
-Base substitution G:C-T:A9 (22.0)0.08±0.0731 (28.2)0.29±0.05**8 (20.5)0.07±0.0547 (30.9)0.43±0.15†† G:C-C:G4 (9.8)0.04±0.0528 (25.5)0.26±0.06**3 (7.7)0.02±0.0338 (25.0)0.37±0.17†† A:T-T:A005 (4.5)0.05±0.071 (2.6)0.01±0.027 (4.6)0.07±0.06† A:T-C:G1 (2.4)0.01±0.022 (1.8)0.02±0.04003 (2.0)0.02±0.03 G:C-A:T19 (46.3)0.18±0.1026 (23.6)0.24±0.1216 (41.0)0.17±0.0727 (17.8)0.25±0.09 A:T-G:C1 (2.4)0.01±0.024 (3.6)0.04±0.04003 (2.0)0.03±0.04 Single bp4 (9.8)0.04±0.047 (6.4)0.06±0.059 (23.1)0.09±0.0910 (6.6)0.09±0.05 Over 2bp2 (4.9)0.03±0.052 (1.8)0.02±0.031 (2.6)0.02±0.033 (2.0)0.03±0.04 001 (0.9)0.01±0.021 (2.6)0.01±0.025 (3.3)0.04±0.06 1 (2.4)0.01±0.034 (3.6)0.04±0.07009 (5.9)0.08±0.10 Total410.381101.04390.391521.40
2,500 ppm NFT Transitions
ControlNrf2-/- 2,500 ppm NFT Number (%)Mutation frequencies (10-5 )Number (%)Mutation frequencies (10-5 )Number (%)Mutation frequencies (10-5 ) Nrf2+/+ Control
Table 12. Mutation spectra of gpt mutant colonies in the kidneys of Nrf2+/+ or Nrf2-/- gpt delta mice treated with NFT for 13 weeks ** P < 0.01 vs. respective control group, ††, †P < 0.01, 0.05 vs. respective control group
Deletion Insertion Complex
Number (%)Mutation frequencies (10-5 ) Transversions
56
-GenotypeTreatmentAnimal No.Mean±SD 10124.680.33 10243.740.09 10316.930.18 10424.290.370.24±0.13 20133.5220.66 20230.2120.40 20331.7130.41 20438.980.21 20521.4100.470.43±0.16 30138.470.18 30244.1150.34 30333.8120.36 30427.2130.48 30519.930.150.30±0.13 40164.5110.17 40224.730.12 40329.070.24 40419.840.200.18±0.05 50119.590.46 50236.590.25 50319.260.31 50423.3100.430.36±0.10 60125.660.23 60234.470.20 60330.160.20 60427.8100.36 60529.490.310.26±0.07 Nrf2-/-1,500 ppm KBrO3
Plaques within XL-1 Blue MRA (× 105 )
Plaques within XL-1 Blue MRA (P2)MF (× 10-5 ) Control 1,500 ppm KBrO3
Table 13. Spi– MFs in the kidneys of Nrf2+/+ or Nrf2-/- gpt delta mice treated with KBrO3 or NFT for 4 weeks MF: mutant frequency2,500 ppm NFT Control 2,500 ppm NFT
Nrf2+/+
- 57 -
Genotype Treatment Animal No. Mean ± SD
701 31.0 13 0.42
702 37.5 11 0.29
703 23.7 5 0.21
704 26.5 9 0.34
705 20.8 6 0.29
706 27.9 9 0.32
707 22.9 7 0.31
708 46.9 13 0.28 0.31 ± 0.06
801 49.8 45 0.90
802 16.1 16 0.99
803 42.0 32 0.76
804 25.0 26 1.04
805 29.6 30 1.01
806 16.6 12 0.72
807 12.9 10 0.78
808 26.0 16 0.62 0.85 ± 0.16 **
901 40.5 14 0.35
902 6.5 2 0.31
903 24.3 8 0.33
904 16.5 4 0.24
905 18.3 7 0.31
906 22.7 4 0.32
907 12.3 8 0.46 0.33 ± 0.07
1001 32.2 8 0.25
1002 33.2 7 0.21
1003 43.6 14 0.32
1004 22.2 8 0.36
1005 31.7 9 0.28 0.28 ± 0.06
1101 26.9 18 0.67
1102 35.6 45 1.26
1103 15.9 18 1.13
1104 30.0 18 0.60
1105 20.6 21 1.02
1106 14.3 14 0.98
1107 29.3 16 0.55
1108 6.0 7 1.16 0.92 ± 0.28 ††
1201 31.2 12 0.38
1202 22.0 6 0.27
1203 35.5 14 0.39
1204 23.5 4 0.17
1205 19.7 11 0.56
1206 17.3 7 0.41
1207 21.3 11 0.52
1208 28.4 6 0.21 0.36 ± 0.14
Control
1,500 ppm KBrO3
MF, mutant frequency
** P < 0.01 vs. respective control group, †† P < 0.01 vs. respective control group
Table 14. Spi– MFs in the kidneys fof Nrf2+/+ or Nrf2-/- gpt delta mice treated with KBrO3 or NFT for 13 weeks
2,500 ppm NFT
Control
2,500 ppm NFT Nrf2+/+
Nrf2
-/-1,500 ppm KBrO3
Plaques within XL-1 Blue MRA
(× 105)
Plaques within XL-1 Blue MRA (P2)
MF (× 10-5)
58
-Single bp deletion SimpleG or C6 (25.0)0.06±0.046 (9.2)0.04±0.056 (12.0)0.03±0.03 A or T005 (7.7)0.03±0.041 (2.0)0.01±0.01 In runG or C4 (16.7)0.04±0.0317 (26.2)0.12±0.0815 (30.0)0.09±0.09 A or T5 (20.8)0.05±0.0318 (27.7)0.11±0.0913 (26.0)0.08±0.04 2 to 1 kb deletion005 (7.7)0.03±0.0400 Over 1 kb deletion8 (33.3)0.08±0.1012 (18.5)0.08±0.1112 (24.0)0.07±0.07 Complex1 (4.2)0.01±0.022 (3.1)0.01±0.031 (2.0)0.01±0.01 Insertion00001 (2.0)0.01±0.02 Base substitution00001 (2.0)0.00±0.01 Total240.24650.43500.30 Single bp deletion SimpleG or C007 (20.6)0.08±0.053 (7.9)0.02±0.02 A or T2 (8.0)0.01±0.022 (5.9)0.02±0.0200 In runG or C7 (28.0)0.04±0.039 (26.5)0.10±0.0912 (31.6)0.08±0.05 A or T8 (32.0)0.06±0.088 (23.5)0.09±0.0611 (28.9)0.08±0.05 2 to 1 kb deletion003 (8.8)0.03±0.0400 Over 1 kb deletion8 (32.0)0.07±0.064 (11.8)0.04±0.0412 (31.6)0.08±0.05 Complex001 (2.9)0.01±0.0100 Insertion000000 Base substitution000000 Total250.18340.36380.26
Mutation frequencies (10-5 ) Mutation frequencies (10-5 )Number (%)
Table 15. Mutation spectra of Spi– plaques in the kidneys of Nrf2+/+ or Nrf2-/- gpt delta mice treated with KBrO3 or NFT for 4 weeks Nrf2-/-
Nrf2-/-Nrf2+/+ Number (%)Mutation frequencies (10-5 )Number (%)Mutation frequencies (10-5 )Number (%) Mutation frequencies (10-5 )
Control
1,500 ppm KBrO32,500 ppm NFTControl 1,500 ppm KBrO32,500 ppm NFT Number (%)Mutation frequencies (10-5 )Number (%)
59
-Single bp deletion SimpleG or C10 (13.7)0.04±0.0324 (12.8)0.11±0.07*6 (12.8)0.05±0.05 A or T2 (2.7)0.01±0.0213 (7.0)0.05±0.04*2 (4.3)0.01±0.02 In runG or C20 (27.4)0.09±0.0560 (32.1)0.27±0.09**22 (46.8)0.16±0.10 A or T28 (38.4)0.12±0.0663 (33.7)0.29±0.10**10 (21.3)0.06±0.06 2 to 1 kb deletion1 (1.4)0.00±0.0112 (6.4)0.05±0.05*00 Over 1 kb deletion12 (16.4)0.05±0.0315 (8.0)0.08±0.064 (8.5)0.02±0.03 Complex00001 (2.1)0.00±0.01 Insertion000000 Base substitution00002 (4.3)0.01±0.03 Total730.311870.85470.33 Single bp deletion SimpleG or C13 (28.3)0.09±0.0717 (10.8)0.10±0.095 (7.0)0.03±0.05 A or T1 (2.2)0.00±0.0115 (9.6)0.10±0.11†3 (4.2)0.02±0.02 In runG or C6 (13.0)0.04±0.0344 (28.0)0.23±0.09††24 (33.8)0.12±0.07† A or T22 (47.8)0.13±0.0750 (31.8)0.31±0.13†20 (28.2)0.10±0.06 2 to 1 kb deletion0019 (12.1)0.13±0.09††3 (4.2)0.02±0.03 Over 1 kb deletion4 (8.7)0.02±0.0411 (7.0)0.05±0.0714 (19.7)0.08±0.07 Complex000000 Insertion00001 (1.4)0.01±0.02 Base substitution001 (0.6)0.00±0.011 (1.4)0.00±0.01 Total460.281570.92710.36 Number (%)Mutation frequencies (10-5 )
Number (%)Mutation frequencies (10-5 )
Table 16. Mutation spectra of Spi– plaques in the kidneys of Nrf2+/+ or Nrf2-/- gpt delta mice treated with KBrO3 or NFT for 13 weeks **, *P < 0.01, 0.05 vs. respective control group, ††, †P < 0.01, 0.05 vs. respective control group
1,500 ppm KBrO32,500 ppm NFT Number (%)Mutation frequencies (10-5 )Number (%)Mutation frequencies (10-5 )
1,500 ppm KBrO32,500 ppm NFT ControlNrf2-/-
ControlNrf2-/-Nrf2+/+ Number (%)Mutation frequencies (10-5 )Number (%)
Control Mutation frequencies (10-5 )
- 60 -