Cyclobutane Pyrimidine Dimers Produced with Narrowband UVB Are on Average More Mutagenic than Those with Broadband UVB in Mouse Skin.

Although narrowband UVB (NB-UVB) has replaced broadband UVB (BB-UVB) because of its greater effectiveness in dermatological phototherapy, it is twice as carcinogenic as BB-UVB at an equivalent inflammatory dose. To clarify the basis of the different genotoxicities, we comparatively evaluated the mutagenicities in mouse skin of the two UVB types along with their efficiencies in the formation of cyclobutane pyrimidine dimer (CPD), which is a major mutagenic DNA photolesion specifically produced by UVR. We found that the mutagenicity averaged per single molecule of CPD was 2.5- and 1.8-fold higher in NB-UVB-exposed epidermis and dermis, respectively, which indicates that NB-UVB is more mutagenic for the skin than BB-UVB at doses producing an equimolar amount of CPD. Analysis of effective wavelengths for UV photolesion formation with each UVB source revealed a remarkable difference in the peak effective wavelengths for CPD formation: 15 nm longer for NB-UVB in the epidermis. Although the analysis of mutation profiles showed largely similar UV-specific signatures between the two UVB types, a relatively stronger preference for UVA-specific mutations was observed for NB-UVB. These results suggest that the difference in the effective wavelengths for CPD formation leads to the different mutagenicities and carcinogenicities between the UVB sources.

Seasonal Differences in the UVA/UVB Ratio of Natural Sunlight Influence the Efficiency of the Photoisomerization of (6-4) Photoproducts into their Dewar Valence Isomers.

The UVA and UVB components of sunlight can produce three classes of bipyrimidine DNA photolesions [cyclobutane pyrimidine dimers (CPDs), pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) and related Dewar valence isomers (DewarPPs)]. The UVA/UVB ratio of sunlight is high in winter and low in summer in the Northern Hemisphere. Since UVB radiation produces 6-4PPs and UVA radiation converts them into DewarPPs through photoisomerization, it is expected that there may be differences in the photoisomerization of 6-4PPs between summer and winter, although that has never been documented. To determine that, isolated DNA was exposed to natural sunlight for 8 h in late summer and in winter, and absolute levels of the three classes of photolesions were quantified using calibrated ELISAs. It was found that sunlight produces CPDs and 6-4PPs in DNA at a ratio of about 9:1 and converts approximately 80% of 6-4PPs into DewarPPs within 3 h. Moreover, photoisomerization is more efficient in winter than in late summer after sunlight irradiation for the same duration, at similar solar UV doses and with the same induction level of CPDs. These results demonstrate that seasonal differences in the UVA/UVB ratio influence the efficiency of the photoisomerization of 6-4PPs into DewarPPs.

Author Correction: Nrf2 contributes to the weight gain of mice during space travel.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nrf2 contributes to the weight gain of mice during space travel.

Space flight produces an extreme environment with unique stressors, but little is known about how our body responds to these stresses. While there are many intractable limitations for in-flight space research, some can be overcome by utilizing gene knockout-disease model mice. Here, we report how deletion of Nrf2, a master regulator of stress defense pathways, affects the health of mice transported for a stay in the International Space Station (ISS). After 31 days in the ISS, all flight mice returned safely to Earth. Transcriptome and metabolome analyses revealed that the stresses of space travel evoked ageing-like changes of plasma metabolites and activated the Nrf2 signaling pathway. Especially, Nrf2 was found to be important for maintaining homeostasis of white adipose tissues. This study opens approaches for future space research utilizing murine gene knockout-disease models, and provides insights into mitigating space-induced stresses that limit the further exploration of space by humans.

Wavelength- and Tissue-dependent Variations in the Mutagenicity of Cyclobutane Pyrimidine Dimers in Mouse Skin.

The cyclobutane pyrimidine dimer (CPD) is a main mutagenic photolesion in DNA produced by UVR. We previously studied the wavelength-dependent kinetics of mutation induction efficiency using monochromatic UVR sources and transgenic mice developed for mutation assay and established the action spectra of UVR mutagenicity in the mouse epidermis and dermis. Here, we further established the action spectra of CPD and pyrimidine(6-4)pyrimidone photoproduct formation in the same tissues and in naked DNA using the same sources and mouse strain. Quantitative ELISA helped us estimate the photolesion formation efficiencies on a molecule-per-nucleotide basis. Using these action spectra, we confirmed that the UVR mutation mostly depends on CPD formation. Moreover, the mutagenicity of a CPD molecule (CPD mutagenicity) was found to vary by wavelength, peaking at approximately 313 nm in both the epidermis and dermis with similar wavelength-dependent patterns. Thus, the CPD formation efficiency is a main determinant of UVR mutagenicity in mouse skin, whereas a wavelength-dependent variation in the qualitative characteristics of CPD molecules also affects the mutagenic consequences of UVR insults. In addition, the CPD mutagenicity was always higher in the epidermis than in the dermis, suggesting different cellular responses to UVR between the two tissues irrespective of the wavelength.

Roles of the KEAP1-NRF2 system in mammalian skin exposed to UV radiation.

The KEAP1 (Kelch-like ECH-associated protein 1)-NRF2 (NF-E2-related factor 2) system controls the biochemical defense activity against agents toxic to mammals and responds to exogenous and endogenous stressors such as electrophilic and oxidative substances, which can have destructive and genotoxic effects on affected mammalian tissues. Although this system can be activated by various environmental stressors, it remains unclear whether ultraviolet radiation (UVR), which is one of the major environmental agents that has inflammatory and carcinogenic impacts on human skin and eyes, induces NRF2-dependent defense activity. Here, we review the recent progress in the study of the contributions of NRF2 and related factors to protection against UVR. The KEAP1-NRF2 system is not always efficient in responding to UVR, especially to short wavelengths such as UVC/UVB, indicating that UVR is a poor activator of the KEAP1-NRF2 system. However, sustained activation of NRF2 appears to suppress the harmful effects of chronic UVR exposure, such as photoaging of and carcinogenesis in the skin, indicating that NRF2 activation is beneficial for the protection of the skin from the harmful effects of UVR. However, it should be noted that prolonged and strong activation of NRF2 may also have adverse effects on skin, especially in the case of UVR-induced carcinogenesis. We present working models describing mechanisms underlying the involvement of the KEAP1-NRF2 system in skin photoaging and carcinogenesis.

Mechanistic considerations on the wavelength-dependent variations of UVR genotoxicity and mutagenesis in skin: the discrimination of UVA-signature from UV-signature mutation.

Ultraviolet radiation (UVR) predominantly induces UV-signature mutations, C → T and CC → TT base substitutions at dipyrimidine sites, in the cellular and skin genome. I observed in our in vivo mutation studies of mouse skin that these UVR-specific mutations show a wavelength-dependent variation in their sequence-context preference. The C → T mutation occurs most frequently in the 5'-TCG-3' sequence regardless of the UVR wavelength, but is recovered more preferentially there as the wavelength increases, resulting in prominent occurrences exclusively in the TCG sequence in the UVA wavelength range, which I will designate as a "UVA signature" in this review. The preference of the UVB-induced C → T mutation for the sequence contexts shows a mixed pattern of UVC- and UVA-induced mutations, and a similar pattern is also observed for natural sunlight, in which UVB is the most genotoxic component. In addition, the CC → TT mutation hardly occurs at UVA1 wavelengths, although it is detected rarely but constantly in the UVC and UVB ranges. This wavelength-dependent variation in the sequence-context preference of the UVR-specific mutations could be explained by two different photochemical mechanisms of cyclobutane pyrimidine dimer (CPD) formation. The UV-signature mutations observed in the UVC and UVB ranges are known to be caused mainly by CPDs produced through the conventional singlet/triplet excitation of pyrimidine bases after the direct absorption of the UVC/UVB photon energy in those bases. On the other hand, a novel photochemical mechanism through the direct absorption of the UVR energy to double-stranded DNA, which is called "collective excitation", has been proposed for the UVA-induced CPD formation. The UVA photons directly absorbed by DNA produce CPDs with a sequence context preference different from that observed for CPDs caused by the UVC/UVB-mediated singlet/triplet excitation, causing CPD formation preferentially at thymine-containing dipyrimidine sites and probably also preferably at methyl CpG-associated dipyrimidine sites, which include the TCG sequence. In this review, I present a mechanistic consideration on the wavelength-dependent variation of the sequence context preference of the UVR-specific mutations and rationalize the proposition of the UVA-signature mutation, in addition to the UV-signature mutation.

Quantitative analysis of UV photolesions suggests that cyclobutane pyrimidine dimers produced in mouse skin by UVB are more mutagenic than those produced by UVC.

The amount of photolesions produced in DNA after exposure to physiological doses of ultraviolet radiation (UVR) can be estimated with high sensitivity and at low cost through an immunological assay, ELISA, which, however, provides only a relative estimate that cannot be used for comparisons between different photolesions such as cyclobutane pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone photoproduct (64PP) or for analysis of the genotoxicity of photolesions on a molecular basis. To solve this drawback of ELISA, we introduced a set of UVR-exposed, calibration DNA whose photolesion amounts were predetermined and estimated the absolute molecular amounts of CPDs and 64PPs produced in mouse skin exposed to UVC and UVB. We confirmed previously reported observations that UVC induced more photolesions in the skin than UVB at the same dose, and that both types of UVR produced more CPDs than 64PPs. The UVR protection abilities of the cornified and epidermal layers for the lower tissues were also evaluated quantitatively. We noticed that the values of absorbance obtained in ELISA were not always proportional to the molecular amounts of the lesion, especially for CPD, cautioning against the direct use of ELISA absorbance data for estimation of the photolesion amounts. We further estimated the mutagenicity of a CPD produced by UVC and UVB in the epidermis and dermis using the mutation data from our previous studies with mouse skin and found that CPDs produced in the epidermis by UVB were more than two-fold mutagenic than those by UVC, which suggests that the properties of CPDs produced by UVC and UVB might be different. The difference may originate from the wavelength-dependent methyl CpG preference of CPD formation. In addition, the mutagenicity of CPDs in the dermis was lower than that in the epidermis irrespective of the UVR source, suggesting a higher efficiency in the dermis to reduce the genotoxicity of CPDs produced within it. We also estimated the minimum amount of photolesions required to induce the mutation induction suppression (MIS) response in the epidermis to be around 15 64PPs or 100 CPDs per million bases in DNA as the mean estimate from UVC and UVB-induced MIS.

In Vivo Spectrum of UVC-induced Mutation in Mouse Skin Epidermis May Reflect the Cytosine Deamination Propensity of Cyclobutane Pyrimidine Dimers.

Although ultraviolet radiation (UVR) has a genotoxicity for inducing skin cancers, the skin may tolerate UVC component because the epidermal layer prevents this short wavelength range from passing through. Here, UVC genotoxicity for mouse skin was evaluated in terms of DNA damage formation and mutagenicity. UVC induced UVR photolesions and mutations remarkably in the epidermis but poorly in the dermis, confirming the barrier ability of the epidermis against shorter UVR wavelengths. Moreover, the epidermis itself responded to UVC mutagenicity with mutation induction suppression, which suppressed the mutant frequencies to a remarkably low, constant level regardless of UVC dose. The mutation spectrum observed in UVC-exposed epidermis showed a predominance of UV-signature mutation, which occurred frequently in 5'-TCG-3', 5'-TCA-3' and 5'-CCA-3' contexts. Especially, for the former two contexts, the mutations recurred at several sites with more remarkable recurrences at the 5'-TCG-3' sites. Comparison of the UVC mutation spectrum with those observed in longer UVR wavelength ranges led us to a mechanism that explains why the sequence context preference of UV-signature mutation changes according to the wavelength, which is based on the difference in the mCpG preference of cyclobutane pyrimidine dimer (CPD) formation among UVR ranges and the sequence context-dependent cytosine deamination propensity of CPD.

Remarkable induction of UV-signature mutations at the 3'-cytosine of dipyrimidine sites except at 5'-TCG-3' in the UVB-exposed skin epidermis of xeroderma pigmentosum variant model mice.

The human POLH gene is responsible for the variant form of xeroderma pigmentosum (XP-V), a genetic disease highly susceptible to cancer on sun-exposed skin areas, and encodes DNA polymerase η (polη), which is specialized for translesion DNA synthesis (TLS) of UV-induced DNA photolesions. We constructed polη-deficient mice transgenic with lacZ mutational reporter genes to study the effect of Polh null mutation (Polh(-/-)) on mutagenesis in the skin after UVB irradiation. UVB induced lacZ mutations with remarkably higher frequency in the Polh(-/-) epidermis and dermis than in the wild-type (Polh(+/+)) and heterozygote. DNA sequences of a hundred lacZ mutants isolated from the epidermis of four UVB-exposed Polh(-/-) mice were determined and compared with mutant sequences from irradiated Polh(+)(/)(+) mice. The spectra of the mutations in the two genotypes were both highly UV-specific and dominated by C→T transitions at dipyrimidines, namely UV-signature mutations. However, sequence preferences of the occurrence of UV-signature mutations were quite different between the two genotypes: the mutations occurred at a higher frequency preferentially at the 5'-TCG-3' sequence context than at the other dipyrimidine contexts in the Polh(+/+) epidermis, whereas the mutations were induced remarkably and exclusively at the 3'-cytosine of almost all dipyrimidine contexts with no preference for 5'-TCG-3' in the Polh(-/-) epidermis. In addition, in Polh(-/-) mice, a small but remarkable fraction of G→T transversions was also observed exclusively at the 3'-cytosine of dipyrimidine sites, strongly suggesting that these transversions resulted not from oxidative damage but from UV photolesions. These results would reflect the characteristics of the error-prone TLS functioning in the bypass of UV photolesions in the absence of polη, which would be mediated by mechanisms based on the two-step model of TLS. On the other hand, the deamination model would explain well the mutation spectrum in the Polh(+/+) genotype.

Skin can control solar UVR-induced mutations through the epidermis-specific response of mutation induction suppression.

Skin exposure to solar ultraviolet radiation (UVR) has been a major public concern because of its genotoxicity. We established recently three action spectra of UVR biological effects using inflammation, mutagenicity, and mutation induction suppression (MIS) as indicators to evaluate UVR risk for mammalian skin. MIS is an antigenotoxic epidermis-specific response by which the increase of the mutant frequency (MF) levels off above a certain UVR dose. Here, based on these spectra, the mutation load of the skin after sunlight exposure was evaluated utilizing the spectral solar-UVR intensity data which had been measured at Tsukuba, Japan by the Japan Meteorological Agency. We estimated the daily variation of the solar-UVR effectiveness (effect per second) for the three indicators, and revealed that the effectiveness efficiency (effect per dose) of midday sunlight is 3-4-fold higher than those in the early morning and late afternoon. Based on the daily variations of mutagenicity and MIS effectiveness, we further estimated MFs induced after every one-hour sunlight exposure and reached a remarkable prediction that MFs should be suppressed to a constant level during 9:00-15:00 by MIS. The estimates agreed well with the equivalent values directly determined at Sendai, a site close to Tsukuba, although a small difference was detected for the epidermis at the dose range where the suppressed MFs were predicted. We propose the use of observed minimum inflammation/erythema doses to improve the difference. Our method could provide reliable estimates of sunlight genotoxicity to evaluate skin cancer probabilities.

Solar-UV-signature mutation prefers TCG to CCG: extrapolative consideration from UVA1-induced mutation spectra in mouse skin.

UVA1 exerts its genotoxicity on mammalian skin by producing cyclobutane pyrimidine dimers (CPDs) in DNA and preferentially inducing solar-UV-signature mutations, C → T base substitution mutations at methylated CpG-associated dipyrimidine (Py-mCpG) sites, as demonstrated previously using a 364 nm laser as a UVA1 source and lacZ-transgenic mice that utilize the transgene as a mutational reporter. In the present study, we confirmed that a broadband UVA1 source induced the same mutation profiles in mouse epidermis as the UVA1 laser, generalizing the previous result from a single 364 nm to a wider wavelength range of UVA1 (340-400 nm). Combined with our previous data on the mutation spectra induced in mouse epidermis by UVB, UVA2 and solar UVR, we proved that the solar-UV-signature mutation is commonly observed in the wavelength range from UVB to UVA, and found that UVA1 induces this mutation more preferentially than the other shorter wavelength ranges. This finding indicates that the solar-UV-signature mutation-causing CPDs, which are known to prefer Py-mCpG sites, could be produced with the energy provided by the longer wavelength region of UVR, suggesting a photochemical reaction through the excitation of pyrimidine bases to energy states that can be accomplished by absorption of even low-energy UVR. On the other hand, the lower proportions of solar-UV-signature mutations observed in the mutation spectra for UVB and solar UVR indicate that the direct photochemical reaction through excited singlet state of pyrimidine bases, which can be accomplished only by high-energy UVR, is also involved in the mutation induction at those shorter wavelengths of UVR. We also found that the solar-UV signature prefers 5'-TCG-3' to 5'-CCG-3' as mutational target sites, consistent with the fact that UVA induces CPDs selectively at thymine-containing dipyrimidine sites and that solar UVR induces them preferably at Py-mCpG sites. However, the mutation spectrum in human p53 gene from non-melanoma skin cancers shows the opposite preference for 5'-CCG-3' sites. This apparent discrepancy in the site preference seems to result from the lack of 5'-TCG-3' sites mutable to missense mutations on the nontranscribed strand of human p53 gene, which should be evolutionally acquired under selective pressure from the sun.

Action spectrum analysis of UVR genotoxicity for skin: the border wavelengths between UVA and UVB can bring serious mutation loads to skin.

UVR causes erythema, which has been used as a standardized index to evaluate the risk of UVR for human skin. However, the genotoxic significance of erythema has not been elucidated clearly. Here, we characterized the wavelength dependence of the genotoxic and erythematic effects of UVR for the skin by analyzing the induction kinetics of mutation and inflammation in mouse skin using lacZ-transgenic mice and monochromatic UVR sources. We determined their action spectra and found a close correlation between erythema and an epidermis-specific antigenotoxic response, mutation induction suppression (MIS), which suppressed the mutant frequencies (MFs) to a constant plateau level only 2-3-fold higher than the background MF at the cost of apoptotic cell death, suggesting that erythema may represent the threshold beyond which the antigenotoxic but tissue-destructive MIS response commences. However, we unexpectedly found that MIS attenuates remarkably at the border wavelengths between UVA and UVB around 315 nm, elevating the MF plateaus up to levels ∼40-fold higher than the background level. Thus, these border wavelengths can bring heavier mutation loads to the skin than the otherwise more mutagenic and erythematic shorter wavelengths, suggesting that erythema-based UVR risk evaluation should be reconsidered.

Role of the Msh2 gene in genome maintenance and development in mouse fetuses.

In an attempt to evaluate the roles of the mismatch repair gene Msh2 in genome maintenance and in development during the fetal stage, spontaneous mutations and several developmental indices were studied in Msh2-deficient lacZ-transgenic mouse fetuses. Mutation levels in fetuses were elevated at 9.5 dpc (days post coitum) when compared to wild-type mice, and the level of mutations continued to increase until the fetuses reached the newborn stage. The mutation levels in 4 different tissues of newborns showed similar magnitudes to those in the whole body. The levels remained similar after birth until 6 months of age. The molecular nature of the mutations examined in 12.5 dpc fetuses of Msh2(+/+) and Msh2(-/-) revealed unique spectra which reflect errors produced during the DNA replication process, and those corrected by a mismatch repair system. Most base substitutions and simple deletions were reduced by the presence of the Msh2 gene, whereas G:C to A:T changes at CpG sequences were not affected, suggesting that the latter change was not influenced by mismatch repair. On the other hand, analysis of developmental indices revealed that there was very little effect, including the presence of malformations, resulting from Msh2-deficiencies. These results indicate that elevated mutation levels have little effect on the development of the fetus, even if a mutator phenotype appears at the organogenesis stage.

Fully functional global genome repair of (6-4) photoproducts and compromised transcription-coupled repair of cyclobutane pyrimidine dimers in condensed mitotic chromatin.

During mitosis, chromatin is highly condensed, and activities such as transcription and semiconservative replication do not occur. Consequently, the condensed condition of mitotic chromatin is assumed to inhibit DNA metabolism by impeding the access of DNA-transacting proteins. However, about 40 years ago, several researchers observed unscheduled DNA synthesis in UV-irradiated mitotic chromosomes, suggesting the presence of excision repair. We re-examined this subject by directly measuring the removal of UV-induced DNA lesions by an ELISA and by a Southern-based technique in HeLa cells arrested at mitosis. We observed that the removal of (6-4) photoproducts from the overall genome in mitotic cells was as efficient as in interphase cells. This suggests that global genome repair of (6-4) photoproducts is fully functional during mitosis, and that the DNA in mitotic chromatin is accessible to proteins involved in this mode of DNA repair. Nevertheless, not all modes of DNA repair seem fully functional during mitosis. We also observed that the removal of cyclobutane pyrimidine dimers from the dihydrofolate reductase and c-MYC genes in mitotic cells was very slow. This suggests that transcription-coupled repair of cyclobutane pyrimidine dimers is compromised or non-functional during mitosis, which is probably the consequence of mitotic transcriptional repression.

The mechanisms of UV mutagenesis.

Ultraviolet (UV) light induces specific mutations in the cellular and skin genome such as UV-signature and triplet mutations, the mechanism of which has been thought to involve translesion DNA synthesis (TLS) over UV-induced DNA base damage. Two models have been proposed: "error-free" bypass of deaminated cytosine-containing cyclobutane pyrimidine dimers (CPDs) by DNA polymerase η, and error-prone bypass of CPDs and other UV-induced photolesions by combinations of TLS and replicative DNA polymerases--the latter model has also been known as the two-step model, in which the cooperation of two (or more) DNA polymerases as misinserters and (mis)extenders is assumed. Daylight UV induces a characteristic UV-specific mutation, a UV-signature mutation occurring preferentially at methyl-CpG sites, which is also observed frequently after exposure to either UVB or UVA, but not to UVC. The wavelengths relevant to the mutation are so consistent with the composition of daylight UV that the mutation is called solar-UV signature, highlighting the importance of this type of mutation for creatures with the cytosine-methylated genome that are exposed to the sun in the natural environment. UVA has also been suggested to induce oxidative types of mutation, which would be caused by oxidative DNA damage produced through the oxidative stress after the irradiation. Indeed, UVA produces oxidative DNA damage not only in cells but also in skin, which, however, does not seem sufficient to induce mutations in the normal skin genome. In contrast, it has been demonstrated that UVA exclusively induces the solar-UV signature mutations in vivo through CPD formation.

Effects of calorie restriction on the age-dependent accumulation of mutations in the small intestine of lacZ-transgenic mice.

To understand the effect of calorie restriction on genome maintenance systems, the age-dependent accumulation of mutations in animals maintained on high and low calorie diets was examined using lacZ-transgenic mice. Mice were fed a diet of 95 kcal/w or 65 kcal/w from 2 to 17 months of age. The mutation frequencies in the lacZ gene in epithelial tissues from the small intestine were examined at 12 and 17 months. Mutation frequencies were found to be lower in mice fed with a low calorie diet than in mice fed with a high calorie diet at the two age points. The molecular nature of the mutations was examined with DNA sequencing. It showed a predominance of transversions from G:C to T:A, and this is a typical type of mutation induced by reactive oxygen species. The fraction of this type of mutation among the different types of mutations detected was not affected by calorie restriction. The percentage of the other types of mutation was not influenced either. These results suggest that calorie restriction reduces the age-dependent accumulation of mutations by stimulating or inducing various types of DNA protection and repair systems rather than protecting cells against any specific type of DNA alteration.

Antigenotoxic effects of p53 on spontaneous and ultraviolet light B--induced deletions in the epidermis of gpt delta transgenic mice.

Tumor development in the skin may be a multistep process where multiple genetic alterations occur successively. The p53 gene is involved in genome stability and thus is referred to as "the guardian of the genome." To better understand the antigenotoxic effects of p53 in ultraviolet light B (UVB)-induced mutagenesis, mutations were measured in the epidermis of UVB-irradiated p53(+/+) and p53(-/-) gpt delta mice. In the mouse model, point mutations and deletions are separately identified by the gpt and Spi(-) assays, respectively. The mice were exposed to UVB at single doses of 0.5, 1.0, or 2.0 kJ/m(2) . The mutant frequencies (MFs) were determined 4 weeks after the irradiation. All doses of UVB irradiation enhanced gpt MFs by about 10 times than that of unirradiated mice. There were no significant differences in gpt MFs and the mutation spectra between p53(+/+) and p53(-/-) mice. The predominant mutations induced by UVB irradiation were G:C to A:T transitions at dipyrimidines. In contrast, in unirradiated p53(-/-) mice, the frequencies of Spi(-) large deletions of more than 1 kb and complex-type deletions with rearrangements were significantly higher than those of the Spi(-) large deletions in p53(+/+) counterparts. The specific Spi(-) mutation frequency of more than 1 kb deletions and complex types increased in a dose-dependent manner in the p53(+/+) mice. However, no increase of such large deletions was observed in irradiated p53(-/-) mice. These results suggest that the antigenotoxic effects of p53 may be specific to deletions and complex-type mutations induced by double-strand breaks in DNA.

Influences of p53 deficiency on the apoptotic response, DNA damage removal and mutagenesis in UVB-exposed mouse skin.

p53 suppresses the genomic instability provoked by genotoxic agents. Ultraviolet (UV) B induces skin cancers by producing DNA damage and mutations in the skin genome, whereas the skin tissue responds to the UVB insult with cell cycle arrest and apoptosis as well as damage exclusion by DNA repair. To address the p53 contribution to these skin responses in vivo, we analyzed the time course of DNA damage removal, apoptosis induction and hyperplasia in the skin after UVB irradiation in p53-knockout mice. We also examined UVB-induced mutations in the skin. We found that p53 deficiency does not abolish the UVB-induced apoptotic response in the epidermis but delays the process and the following hyperplasia 12-24 h. Regardless of the p53 genotype, 1 kJ/m(2) UVB induced a total replacement of the epidermal layer by destroying the damaged epidermis by apoptosis and rebuilding a new one through hyperplasia. We failed to detect a clear defect in removal of UVB-induced DNA photolesions from the genome of the p53-deficient skin except for a delay in the epidermis, which seemed to result from the delay in the apoptotic response. However, we found that p53 deficiency enhanced UVB-induced mutagenesis. Furthermore, in a genetic study using Xpa-knockout mice, we showed that the enhanced mutagenic response depends on the activity of nucleotide excision repair (NER), which was also supported by the mutation spectrum observed in the UVB-exposed p53-knockout mice. These results indicate that p53 protects the skin genome from the UVB genotoxicity by facilitating NER, whereas its contribution to the UVB-induced apoptosis is limited.

XPC is involved in genome maintenance through multiple pathways in different tissues.

In an attempt to evaluate the role of the Xpc gene in maintaining genomic stability in vivo under normal conditions, the age-dependent accumulation of spontaneous mutations in different tissues was analyzed in Xpc-deficient lacZ-transgenic mice. Brain, testis, and small intestine revealed no effects from the Xpc-deficiency, whereas liver, spleen, heart, and lung showed an enhanced age-related accumulation of mutations in Xpc-deficient mice. In the spleen, the effect was not obvious at 2 and 12 months of age, but became apparent at 23 months. The magnitude of the observed effect at an advanced age was similar in the liver, spleen and heart, but was comparatively smaller in the lung. Haploinsufficiency was observed in liver and spleen but not in heart and lung. Analysis of DNA sequences in the mutants revealed that the frequency of G:C to T:A changes were elevated in the liver and heart of Xpc-deficient aged mice, supporting the possible involvement of XPC in base excision repair of oxidized guanine. The occurrence of two or more mutations within a single lacZ gene was termed a multiple mutation and was also elevated in old Xpc-deficient mice. Among the clones examined, two mutant clones showed as many as four mutations within a short stretch of DNA. This is the first demonstration to support suggestions for the existence of a role for XPC in the suppression of multiple mutations. These multiple mutations could conceivably be generated by error-prone trans-lesional DNA synthesis. Overall, these results indicate that there may be diverse roles or mechanisms through which XPC participates in genome maintenance in different tissues.