Studies of in vivo mutations in rpsL transgene in UVB-irradiated epidermis of XPA-deficient mice

We have established xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair (NER) deficiency, which rapidly developed skin tumors when exposed to a low dose of chronic UV like XP-A patients, confirming that the NER process plays an important role in preventing UVB-induced skin cancer. To examine the in vivo mutation in the UVB-irradiated epidermis, we established XPA (−/−), (+/−) and (+/+) mice carrying the Escherichia coli rpsL transgene with which the mutation frequencies and spectra in the UVB-irradiated epidermal tissue can be examined conveniently. The XPA (−/−) mice showed a higher frequency of UVB-induced mutation in the rpsL transgene with a low dose (150 J/m²) of UVB-irradiation than the XPA (+/−) and (+/+) mice, while, at a high dose (900 J/m²) they showed almost the same frequency of mutation as the XPA (+/−) and (+/+) mice, probably because of cell death in the epidermis of the XPA (−/−) mice. However, CC→TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the XPA (−/−) mice than the XPA (+/−) and (+/+) mice at both doses of UVB. This rpsL/XPA mouse system will be useful for further analyzing the role of NER in the mutagenesis and carcinogenesis induced by various carcinogens.     

Mutagenesis and carcinogenesis in nucleotide excision repair-deficient XPA knock out mice

Mice with a defect in the xeroderma pigmentosum group A (XPA) gene have a complete deficiency in nucleotide excision repair (NER). As such, these mice mimic the human XP phenotype in that they have a >1000-fold higher risk of developing UV-induced skin cancer. Besides being UV-sensitive, XPA^−/− mice also develop internal tumors when they are exposed to chemical carcinogens. To investigate the effect of a total NER deficiency on the induction of gene mutations and tumor development, we crossed XPA^−/− mice with transgenic lacZ/pUR288 mutation-indicator mice. The mice were treated with various agents and chemicals like UV-B, benzo[a]pyrene and 2-aceto-amino-fluorene. Gene mutation induction in several tumor target- and non-target tissues was determined in both the bacterial lacZ reporter gene and in the endogenous Hprt gene. Furthermore, alterations in the p53- and ras genes were determined in UV-induced skin tumors of XPA^−/− mice. In this work, we review these results and discuss the applicability and reliability of enhanced gene mutant frequencies as early indicators of tumorigenesis.     

UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency

Nucleotide excision repair (NER) removes a wide variety of lesions from the genome and is deficient in the genetic disorder, xeroderma pigmentosum (XP). In this paper, an in vitro analysis of the XP group A gene product (XPA protein) is reported. Results of an analysis on the pathogenesis of ultraviolet (UV)-B-induced skin cancer in the XPA gene-knockout mouse are also described: (1) contrary to wild type mice, significant bias of p53 mutations to the transcribed strand and no evident p53 mutational hot spots were detected in the skin tumors of XPA-knockout mice. (2) Skin cancer cell lines from UVB-irradiated XPA-knockout mice had a decreased mismatch repair activity and an abnormal cell cycle checkpoint, suggesting that the downregulation of mismatch repair helps cells escape killing by UVB and that mismatch repair-deficient clones are selected for during the tumorigenic transformation of XPA (-/-) cells. (3) The XPA-knockout mice showed a higher frequency of UVB-induced mutation in the rpsL transgene at a low dose of UVB-irradiation than the wild type mice. CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the rpsL transgene in the XPA-knockout mice than the wild type mice. This rpsL/XPA mouse system will be useful for further analysing the role of NER in the mutagenesis induced by various carcinogens. (4) The UVB-induced immunosuppression was greatly enhanced in the XPA-knockout mice. It is possible that an enhanced impairment of the immune system by UVB irradiation is involved in the high incidence of skin cancer in XP.     

Micronucleated erythrocyte frequency in control and azidothymidine-treated Tk+/+, Tk+/- and Tk-/- mice

The first step in the activation of the anti-retroviral nucleoside analogue azidothymidine (AZT) involves its conversion to a 5′-monophosphate. In this study, we have evaluated the role of cytosolic thymidine kinase (Tk), the major enzyme involved in phosphorylating thymidine and its analogues, in the nuclear DNA damage produced by AZT in neonatal mice. Tk^+/+, Tk^+/− and Tk^−/− mice were treated intraperitoneally with 200 mg/kg/day of AZT on postnatal days 1 through 8, and micronuclei were measured in peripheral blood 24 h after the last dose. AZT treatment increased the micronucleus (MN) frequencies to similar extents in both the reticulocytes (RETs) and normochromatic erythrocytes (NCEs) of Tk^+/+ and Tk^+/− mice; AZT did not increase the frequency of micronucleated RETs (MN-RETs) or micronucleated NCEs (MN-NCEs) in Tk^−/− mice. Unexpectedly, neonatal Tk^−/− mice treated with the vehicle had significantly elevated MN frequencies for both RETs and NCEs relative to Tk^+/+ and Tk^+/− mice (e.g., 3.4% MN-RETs and 4.8% MN-NCEs in Tk^−/− mice versus 0.7 and 0.6% MN-RETs and MN-NCEs in neonatal Tk^+/+ mice). Additional assays performed on untreated Tk^−/− mice showed that elevated spontaneous MN frequencies persisted until at least 20 weeks of age, which approaches the average lifespan of Tk^−/− mice. These results indicate that metabolism by Tk is necessary for the genotoxicity of AZT in neonatal mice; however, the genotoxicity of AZT is not altered by reducing the Tk gene dose by half. The elevated spontaneous MN frequencies in Tk^−/− mice suggest the presence of an endogenous genotoxic activity in these mice.     

Characterization of heart size and blood pressure in the vitamin D receptor knockout mouse

Our previous studies showed vitamin D deficiency results in increased cardiac contractility, hypertrophy and fibrosis and has profound effects on heart proteomics, structure and function in rat. In this study we found that the heart in vitamin D receptor knockout (VDR-KO) mice is hypertrophied. Six homozygous VDR knockout (−/−), six wild type (+/+) and six heterozygous (+/−) mice were fed a diet containing 2% Ca, 1.25% P and 20% lactose to maintain normal blood calcium and phosphate levels for 12 months. Tail-cuff blood pressure was performed on all mice. Blood pressure determinations showed no differences in systolic or mean blood pressure in WT (+/+), KO (−/−) or HETERO (+/−) mice at 3 and 6 months. However, decreased systolic BP in the KO mice relative to WT at 9 months of age was observed. ECG analysis showed no significant differences in the intact KO, HETERO or WT mice. The mice were killed at 12 months. Heart weight/body weight ratio was 41% (P < .003) greater in the KO mice versus WT and HETERO was 19% (P < .05) increased versus WT. Other VDR-KO tissues did not display hypertrophy. Cross sectional and longitudinal analysis of the heart myofibrils showed highly significant cellular hypertrophy in VDR-KO mice. Trichrome staining of heart tissue showed marked increase in fibrotic lesions in the KO mice. Analysis of plasma renin activity, angiotensin II (AII) and aldosterone levels showed elevated but not significantly different renin activity in KO versus WT and no significant differences in AII or aldosterone levels. Our data do not support the concept that the renin-angiotensin system or hypertension are the factors that elicit these changes. Data presented here reveal that ablation of the VDR signaling system results in profound changes in heart structure. We propose that calcitriol acts directly on the heart as a tranquilizer by blunting cardiomyocyte hypertrophy.     

Effect of p53 genotype on gene expression profiles in murine liver

The tumor suppressor protein p53 is a key regulatory element in the cell and is regarded as the “guardian of the genome”. Much of the present knowledge of p53 function has come from studies of transgenic mice in which the p53 gene has undergone a targeted deletion. In order to provide additional insight into the impact on the cellular regulatory networks associated with the loss of this gene, microarray technology was utilized to assess gene expression in tissues from both the p53^−/− and p53^+/− mice. Six male mice from each genotype (p53^+/+, p53^+/−, and p53^−/−) were humanely killed and the tissues processed for microarray analysis. The initial studies have been performed in the liver for which the Dunnett test revealed 1406 genes to be differentially expressed between p53^+/+ and p53^+/− or between p53^+/+ and p53^−/− at the level of p ≤ 0.05. Both genes with increased expression and decreased expression were identified in p53^+/− and in p53^−/− mice. Most notable in the gene list derived from the p53^+/− mice was the significant reduction in p53 mRNA. In the p53^−/− mice, not only was there reduced expression of the p53 genes on the array, but genes associated with DNA repair, apoptosis, and cell proliferation were differentially expressed, as expected. However, altered expression was noted for many genes in the Cdc42-GTPase pathways that influence cell proliferation. This may indicate that alternate pathways are brought into play in the unperturbed liver when loss or reduction in p53 levels occurs.     

Genotoxic effects of inhaled ethylene oxide, propylene oxide and butylene oxide on germ cells: sensitivity of genetic endpoints in relation to dose and repair status

We report here results on forward mutation induction (recessive lethal mutations, RL) in Drosophila spermatozoa and spermatids by the three 1,2-alkyl-epoxides ethylene oxide (EO), propylene oxide (PO) and butylene oxide (BO), at doses ranging from 47 to 24,000 ppm h for EO, 375 to 48,000 ppm h for PO, and 24,000 to 91,200 ppm h for BO. The results indicate for EO mutation induction at doses 500-fold below the LD₅₀. In crosses of mutagenized NER⁺ males with NER⁺ females, the 500-fold increase in EO dose from 47 ppm h to 24,000 ppm h resulted in no more than a 17-fold enhanced mutant frequency in spermatozoa. This flat dose–response relationship is primarily the result of efficient repair of EO-induced DNA adducts in the fertilized egg, as was evident from the up to 40-fold or 240-fold increased mutant frequencies above NER⁻ or NER⁺ background levels, respectively, in crosses with NER⁻ females. With decreasing dose, / ratios decreased from 9 to 14 at high doses down to ≈1 at the two lowest doses, indicating that a small fraction of premutagenic lesions induced by EO cannot be repaired by the NER system of Drosophila. Linear extrapolation from high to low EO exposure led to an underestimation of the mutation frequency actually observed at low doses. The pattern of EO-induced ring chromosome loss (CL) differed in two respects from that observed for forward mutations: (a) an increase in CL frequencies was observed only at the two highest EO exposure levels, and (b) inactivation of the NER pathway by the mus201 mutant had no measurable effect on the occurrence of CL. The absence of a potentiating effect of mus201 on EO-induced clastogenicity suggests the formation of clastogenic DNA lesions not causing point mutations, and which are not repaired by NER. Consistent with an inversed correlation of reactivities towards N7-guanine and chain length of 1,2-alkyl-epoxides, the relative mutagenic efficiencies of EO:PO:BO are 100:7.2:1.8 for the NER⁺ groups, and 100:20:0.7 in the absence of NER. Although in Drosophila germ cells EO is also more effective as a clastogen than PO, the difference (EO:PO=100:58) is much smaller than for recessive mutations. These results provide another argument that DNA lesions generating base substitutions as opposed to those causing clastogenic damage may not be the same for these agents.     

Mutagenic effects of poly (ADP-ribose) polymerase-1 deficiency in transgenic mice

Poly (ADP-ribose) polymerase-1 (Parp1) plays a central role in the maintenance of genomic integrity and has been unequivocally associated to DNA base excision repair (BER) but its involvement in double-strand break (DSB) repair pathways remains unclear. In this work, using transgenic Parp1-deficient mice harbouring the lacZ reporter gene, we provide in vivo evidence that Parp1 contributes to the prevention of deletions/insertions in testis following an alkylation insult. In response to N-Methyl-N-Nitrosurea (MNU) treatment no significant difference in the mutant frequency (MF) in the liver and testis could be attributed to Parp1 status, given that both Parp1^+/+ and Parp1^−/− mice showed a similar significant increase in the overall MF. However, restriction analysis of MNU-induced mutants evidenced a shift in the distribution of mutations between deletions/insertions and point mutations in testis, but not in the liver, dependent on the Parp1 status. A significant higher frequency of deletions/insertions was observed in testis from Parp1^−/− in comparison to Parp1^+/+ mice, whereas point mutations were not significantly affected. Overall, our findings show that Parp1 participates in the prevention of deletions/insertions induced by methylating agents and that organ-specific factors may influence its capacity to protect against genotoxic damage.     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

Induction of specific-locus and dominant lethal mutations in male mice by 1-methyl-1-nitrosourea (MNU)

1-Methyl-1-nitrosourea (MNU) induced specific-locus mutations in mice in all spermatogenic stages except spermatozoa. After intraperitoneal injection of 70 mg/kg body weight of MNU a high yield of specific-locus mutations was observed in spermatids (21.8 × 10⁻⁵ mutations per locus per gamete). The highest mutational yield was induced in differentiating spermatogonia. In 1954 offspring we observed 5 specific-locus mutants (44.8 × 10⁻ mutations per locus per gamete). In addition, 2 mosaics were recovered, which gave a combined mutation rate of 62.7 × 10⁻⁵. In A_s spermatogonia the mutation rate was 3.9 × 10⁻⁵. The same dose of 70 mg/kg of MNU induced dominant lethal mutations 5–48 days post treatment, mainly due to post-implantation loss in spermatids and spermatocytes. It is interesting to compare the induction pattern of mutations by MNU with methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and ethylnitrosourea (ENU). Based on the different spermatogenic response of the induction of specific-locus mutations we can characterize the 4 mutagens in the following way: EMS = MMS ≠ MNU ≠ ENU.     

Effect of target gene CpG content on spontaneous mutation in transgenic mice

Transgenic mutation assays utilizing bacterial target genes display a high frequency of spontaneous mutation at CpG sequences. This is believed to result from the fact that: (1) the prokaryotic genes currently being used as transgenic mutation targets have a high CpG content and (2) these sequences are methylated by mammalian cells to produce 5-methylcytosine (5MC), a known promutagenic base. To study the effect of CpG content on the frequency and type of spontaneous mutation, we have synthesized an analogue of the bacterial lacI target gene (mrkII) that contains a reduced number of CpG sequences. This gene was inserted into a lambda vector and used to construct trangenic mice that undergo vector rescue from genomic DNA upon in vitro packaging. Results on spontaneous mutation frequency and spectrum have been collected and compared to those observed at the lacI gene in Big Blue™ transgenic mice. Spontaneous mutations at the mrkII gene occurred at a frequency in the mid-10⁻⁵ range and were predominantly base pair substitutions, similar to results seen in Big Blue™. However, mrkII mutations were distributed toward the carboxyl end of the gene instead of the bias toward the amino terminus seen in lacI. Unexpectedly, 23% of the spontaneous mrkII mutations were GC → AT transitions at CpG sequences (compared to 32% in lacI), despite the reduction in CpG number from 95 in lacI to only 13 in mrkII. Nine of the CpG bases undergoing transition mutations in mrkII have not been recorded previously as spontaneous sites in Big Blue™. Therefore, substantial reduction of the number of CpG sequences in the lacI transgene did not significantly reduce the rate of spontaneous mutation or alter the contribution of CpG-related events. This suggests that other factors are also operating to establish frequency and composition of spontaneous mutations in transgenic targets.     

Impaired spermatogenesis and elevated spontaneous tumorigenesis in xeroderma pigmentosum group A gene (Xpa)-deficient mice

We have reported that xeroderma pigmentosum group A (Xpa) gene-knockout mice [Xpa (−/−) mice] are deficient in nucleotide excision repair (NER) and highly sensitive to UV-induced skin carcinogenesis. Although xeroderma pigmentosum group A patients show growth retardation, immature sexual development, and neurological abnormalities as well as a high incidence of UV-induced skin tumors, Xpa (−/−) mice were physiologically and behaviorally normal. In the present study, we kept Xpa (−/−) mice for 2 years under specific pathogen-free (SPF) conditions and found that the testis diminished in an age-dependent manner, and degenerating seminiferous tubules and no spermatozoa were detected in the 24-month-old Xpa (−/−) mice. In addition, a higher incidence of spontaneous tumorigenesis was observed in the 24-month-old Xpa (−/−) mice compared to Xpa (+/+) controls. Xpa (−/−) mice provide a useful model for investigating the aging and internal tumor formation in XPA patients.     

Use of genetically modified mice to examine the skeletal anabolic activity of vitamin D

We employed genetically modified mice to examine the role of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] on skeletal and calcium homeostasis. In mice expressing the null mutation for 25-hydroxyvitamin D 1 hydroxylase (1OHase^−/−), or the vitamin D receptor (VDR^−/−), 1,25(OH)₂D₃ and calcium were both required for optimal epiphyseal growth plate development, serum calcium and phosphorus alone were sufficient to mineralize skeletal tissue independent of 1,25(OH)₂D₃ and the VDR, and endogenous 1,25(OH)₂D₃ and the VDR were essential for baseline bone formation. In 2-week-old 1OHase^−/− mice and in 2-week-old mice homozygous for the PTH null mutation(PTH^−/−), PTH and 1,25(OH)₂D₃ were each found to exert independent and complementary effects on skeletal anabolism, with PTH predominantly affecting appositional trabecular bone growth and 1,25(OH)₂D₃ influencing both endochondral bone formation and appositional bone growth. Endogenous 1,25(OH)₂D₃ maintained serum calcium homeostasis predominantly by modifying intestinal and renal calcium transporters but not by producing net bone resorption. Administration of exogenous 1,25(OH)₂D₃ to double mutant PTH^−/−1OHase^−/− mice produced skeletal effects consistent with the actions of endogenous 1,25(OH)₂D₃. These studies reveal an important skeletal anabolic role for both endogenous and exogenous 1,25(OH)₂D₃ and point to a potential role for 1,25(OH)₂D₃ analogs in the treatment of disorders of bone loss.     

High somatic mutation frequencies in a LacZ transgene integrated on the mouse X-chromosome

To study spontaneous and induced mutagenesis in vivo we recently constructed a series of transgenic mice harboring different numbers of bacteriophage lambda shuttle vectors, provided with a LacZ mutational target gene, integrated in their genome. The transgenic mice enabled analysis of spontaneous and induced mutation frequencies in postmitotic tissues like liver and brain. The obtained data indicated spontaneous mutation frequencies in the order of ⁻⁵ - 10⁻⁶. Here we report a 25–100 times higher spontaneous mutation frequency in liver and brain DNA of mice from strain 35.5, with the lambda-gt10LacZ concatemer integrated on the X-chromosome. These results indicate the presence of a mutational ‘hot spot’ in the mammalian somatic genome in vivo.     

Role of endogenous prostacyclin in gastric ulcerogenic and healing responses—a study using IP-receptor knockout mice

Endogenous prostaglandins (PGs) play an important role in the cytoprotective and healing responses in the stomach, by altering various functions, i.e., an increase of the mucosal blood flow, yet the role of prostacyclin (PGI₂) and its receptor (IP-receptor) in these responses remains unclarified. In the present study, we used IP-receptor knockout mice [IP (−/−)] and examined the importance of IP-receptors in gastric ulcerogenic, cytoprotective and healing responses in these animals. The studies included the ulcerogenic response to cold-restraint stress, the cytoprotective response to a mild irritant (20 mM taurocholate: TC) and capsaicin, and the healing response of chronic gastric ulcers induced by thermo-cauterization. We first checked the absence of IP-receptors by examining the effect of cicaprost (a PGI₂ agonist, topical mucosal application) on gastric mucosal blood flow and found that this agent increased the mucosal blood flow in wild-type [WT (+/+)] mice but not in IP (+/−) mice. Cold-restraint stress (4 h) induced gastric lesions in both groups of mice, but the severity of damage was significantly greater in IP (−/−) mice. Prior p.o. administration of both TC and capsaicin exhibited a marked cytoprotection against HCl/ethanol-induced gastric damage in WT (+/+) mice, both responses being significantly mitigated in the presence of indomethacin. The adaptive cytoprotection induced by TC was similarly observed in IP (−/−) mice, while the capsaicin protection was totally attenuated in the animals lacking IP receptors. On the other hand, the healing of gastric ulcers was significantly delayed by daily administration of indomethacin in WT (+/+) mice. However, this process was not altered in IP (−/−) mice. These results suggest that endogenous PGI₂ is involved in the gastric ulcerogenic response to stress, but not in the healing of pre-existing gastric ulcers. In addition, PGI₂ and its receptors may play a crucial role in capsaicin-induced gastric protection but not in the adaptive cytoprotection-induced by mild irritants.     

Transpiration rates of the understory annual Impatiens capensis (Balsaminaceae) in response to the autumnal changes in canopy leaf area

Leaf transpiration rates of Impatiens capensis were measured beneath a broadleaved deciduous forest canopy over successive growing seasons using a steady-state porometer. The transpiration measurements, which continued into early autumn, provided a framework for assessing whether I. capensis exhibits stomatal opening in response to the autumnal increase in available direct-beam radiation reaching the forest floor. The deciduous canopy LAI (leaf area index) decreased from a growing season maximum of 3.94 m² m⁻², while the understory I. capensis population located along a stream channel maintained LAI values ranging from 0.58 to 1.05 m² m⁻² late into the growing season. Late morning and early afternoon leaf transpiration rates during the months of June and July averaged about 8 μg cm⁻² s⁻¹, with a mean stomatal conductance of 0.5 cm s⁻¹. In August, leaf transpiration averaged almost 12 μg cm⁻² s⁻¹, with stomatal conductance exceeding 1.5 cm s⁻¹. However, beginning in early to mid-September, before canopy leaf-fall, the persistent green leaves of I. capensis exhibited a sharp decline in transpiration, possibly a result of decreasing vapor pressure deficits or non-lethal physiological damage induced by cold stress. This physiological decline offsets any advantage that could have been gained by the increased exposure to direct-beam radiation after canopy leaf-fall in mid-October. Although green leaf area and seed-bearing capsules may persist until the first frost in October or early November, there is no evidence of stomatal opening suggestive of carbon assimilation for enhanced seed development during this early autumn period. We conclude that the persistent green leaf area of I. capensis fails to exploit the increase in available direct-beam radiation in the final stage of its life cycle.     

Suppressed apoptosis in the inflamed gastric mucosa of Helicobacter pylori-colonized iNOS-knockout mice

Deregulated cell turnover in Helicobacter pylori (H. pylori)-colonized gastric mucosa has been suggested to be linked to the gastric carcinogenesis pathway. We previously reported attenuation of apoptosis and enhancement of cellular proliferation in the H. pylori-colonized gastric mucosa of Mongolian gerbils as compared to that in mice, which might reflect a specific link between H. pylori colonization and carcinogenesis in the Mongolian gerbils; the difference between the two strains could be attributable to differences in the host genetic background. Inducible-type nitric oxide synthase (iNOS) is thought to participate in not only the inflammatory response, but also in the regulation of gastric mucosal cell turnover in H. pylori-colonized gastric mucosa. Thus, the present study was designed to examine gastric leukocyte activation and epithelial cell apoptosis in the gastric mucosa following H. pylori inoculation in iNOS-knockout mice. Methods: iNOS-knockout mice (iNOS^−/−) and their iNOS^+/+ littermates were orally inoculated with the Sydney strain of H. pylori (SS1, 10⁸ colony-forming units [CFU]). H. pylori infection was confirmed by microaerobic bacterial culture. The stomach of each mouse was evaluated 14 weeks and 30 weeks after the inoculation. Gastric mucosal accumulation of polymorphonuclear leukocytes (PMN) was assessed by determining the myeloperoxidase (MPO) activity and histological score based on the updated Sydney system. The level of apoptosis was determined by estimation of the cytoplasmic levels of mono- and oligonucleosomes and by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method. Results: The SS1-inoculated mice showed persistent H. pylori colonization for 12 weeks. While gastric mucosal PMN infiltration increased following SS1 inoculation in both iNOS^+/+ and iNOS^−/−strains, enhanced DNA fragmentation was observed in only SS1-colonized iNOS^+/+ mice, and not in the iNOS^−/− mice. In conclusion, although the recruitment of PMN in response to H. pylori was evoked even in the gastric mucosa of iNOS^−/− mice, epithelial cell apoptosis induced by H. pylori was attenuated in this strain. These data suggest that iNOS may play an important role in promoting apoptosis in the H. pylori-infected inflamed gastric mucosa, and that persistent inflammation without apoptosis in iNOS^−/− mice with H. pylori infection may be linked to preneoplastic transformation.     

Mutagenicity of 8-methoxypsoralen and long-wave ultraviolet irradiation in V-79 Chinese hamster cells : A first approach to a risk estimate in photochemotheraphy

The effect of 8-methoxypsoralen (8-MOP) and long-wave ultraviolet irradiation (UVA) on cell killing and mutation induction was studied in V-79 Chinese hamster cells. No effect was observed after treatment with 8-MOP alone (50 μg/ml, 4 h), UVA alone (9000 J/m²), or 8-MOP metabolized by rat-livermicrosomes. Combined treatment with 8-MOP and UVA induced both cell killing and mutation. This was also observed under conditions approaching patient treatment with PUVA photochemotherapy with respect to the concentration of 8-MOP in the skin and the amount of UVA received by the epidermal cells. A simple relation proved to apply for mutation induction under different treatment conditions: 5.5 × 10⁻⁸ per J/m² per μg 8-MOP/ml. On this basis the mutation induction in dividing cells per session of PUVA-photochemotherapy amounts to 12.4 × 10⁻⁵, which is probably an over-estimation.     

Additive roles of XPA and MSH2 genes in UVB-induced skin tumorigenesis in mice

We have made xeroderma pigmentosum group A gene (XPA)-knockout mice (XPA(-/-) mice). The XPA(-/-) mice had no detectable activity for nucleotide excision repair (NER) and showed a high incidence of UVB-induced skin tumorigenesis. We have also found that cell lines derived from skin cancers in UVB-irradiated XPA(-/-) mice become tolerant to UV-irradiation and showed abnormal UV-induced cell cycle checkpoints and decreased mismatch repair (MMR) activity. These results suggested that the MMR-downregulation may help cells escape killing by UV-irradiation and thus MMR-deficient clones are selected for during the tumorigenic transformation of XPA(-/-) cells. In this report, we examined whether the incidence of UVB-induced skin tumorigenesis is enhanced in XPA(-/-)MSH2(-/-), XPA(-/-) and MSH2(-/-) mice when compared with that in wild-type mice. Our results indicate that the MSH2-deficiency caused a high incidence of spontaneous and UVB-induced skin tumorigenesis and the XPA and MSH2 genes have additive roles in the UV-induced skin tumorigenesis.     

A model for the involvement of Okazaki fragments maturation in the expansion of short tandem repeats

Mutations were accumulated with a wide variety in the p53 pseudogene of various wild mouse species and subspecies captured at different localities, as extensively observed in the exon 4 – exon 5 region. The rate of mutation accumulation in the mouse p53 pseudogene was estimated to be 1.4–2.1×10⁻⁸ mutations/bp/year, which is 20–30 times faster than that of the functional p53 and makes the dating possible for the time range of 10⁶ years or more. From comparison of the mutation spectrum, the origin of laboratory mice was identified to one of two M. m. domesticus groups.