BACKGROUND
exposure to chemicals like benzopyrene  can lead to structural changes in dna and as a consequence to the development of diseases with a genetic basis  <cit> .
changes in the dna sequence can be induced by exposure to chemicals during life, but may also be inherited via mutations in the spermatogonial stem cells; in that way increasing the risk of developing abnormalities or diseases in the offspring  <cit> .
the mutagenic potential of bp in male germ cells, however, has still not been fully established.
bp related dna damage was observed at all stages of spermatogenesis and in testis  <cit> , but it is largely unknown how germ cells deal with dna damage to protect their genetic material, and to prevent the accumulation of mutations in the germ line.
spermatogenesis is carefully controlled to produce mature spermatozoa from spermatogonial stem cells in three major stages; the mitotic stage, the meiotic stage and the maturation stage.
germ cells are susceptible for the induction of mutations during mitotic and meiotic divisions, because cell turnover is a prerequisite for fixation of dna damage into mutations.
however, it is likely that several processes prevent the occurrence of gene mutations in male germ cells; for example, dna damage can be removed by dna repair mechanisms, of which nucleotide excision repair  is considered to be the most relevant repair mechanism for bulky dna adducts formed by reactive metabolites of bp.
two ner mechanisms have been described: global genome repair  eliminates bulky dna lesions in the entire genome, whereas transcription coupled repair  specifically removes lesions that block rna synthesis  <cit> .
in a previous study, we observed that ggr/ner plays an important role in the removal of bp induced dna adducts in the testis, especially in the first week after exposure  <cit> .
bp induced dna adduct levels in the testis were significantly different between wt and xpc-/- mice, especially at  <dig> days after a single exposure to bp .
therefore, in an attempt to reveal the responses in the testis to this damage, we investigated by using microarrays the changes in gene expression induced by bp in testis from wt and xpc-/- male mice,  <dig> days after exposure to bp.
since xpc-/- mice lack one of the most important protective mechanisms against bp induced dna damage, we expected a different  transcriptional response in these mice as compared to their wt counterparts.
RESULTS
gene expression profiling
two-way anova revealed  <dig> regulated genes that were differentially expressed between treated and untreated mice , of which  <dig> genes were increased and  <dig> genes were decreased by bp exposure.
only  <dig> genes  were differentially expressed between unexposed wt and unexposed xpc-/- mice , and they are all located at the same site of chromosome  <dig>  of these  <dig> genes, xpc, cml <dig> and d6mm5e were decreased in expression in xpc-/- mice as compared to the wt mice, with higher gene expression levels of cml <dig> and d6mm5e than the very low levels of xpc.
on the other hand, 2610209m04rik was increased in xpc-/- mice.
this confirms the absence of a functional xpc gene in the xpc-/- genotype, and shows that closely linked genes have significantly altered expression patterns.
finally, our results show that there is only a modest difference between the gene expression patterns in testis of bp treated wt and xpc-/- mice.
nonetheless, a paired t-test showed that the level in which these bp regulated genes are expressed differs between wt and xpc-/- mice .
the clustering of the combined hits is presented in figure  <dig>
analysis of pathway regulation
the list of the  <dig> genes that were induced by bp suggested, by using david/ease, a significant enrichment for several gene ontology  biological processes and swiss-prot_protein information resource  terms, such as 'cell division' , 'mitosis' , 'chromosome segregation'  and 'spermatogenesis'  .
the  <dig> genes that were down regulated by bp exposure also showed an enrichment for several pathways, including 'oxidative phosphorylation'  .
in addition, two go terms for the cellular components mitochondrial part  and mitochondrion , were enriched among bp suppressed genes, which is in line with the inhibition of the oxidative phosphorylation process.
the combined list of bp  regulated genes showed an enrichment of  <dig> go and sp_pir terms , including cell cycle, translation, chromatin structure and spermatogenesis, which we already found to be enriched among the up regulated genes only.
therefore, we used these  <dig> processes to further select genes and to subsequently compare the two genotypes regarding their gene expression ratios between bp treated vs. control groups.
an overview of the gene expression ratio distribution is given in table  <dig>   <dig> differentially regulated genes were involved in the cell cycle , of which  <dig> genes were induced to a higher extent in xpc-/- mice, whereas only  <dig> of these genes were expressed to a higher extent in wt mice.
regarding the genes that were inhibited in their expression,  <dig> genes were more suppressed in xpc-/- mice, and  <dig> genes in wt mice as compared to animals of the other genotype.
in addition, we found  <dig> genes that are involved in the chromatin structure , of which  <dig> genes were more induced in xpc-/- mice, and  <dig> genes in wt mice.
only  <dig> chromatin structure related genes were more suppressed in wt mice, as compared to  <dig> gene in xpc-/- mice.
among the spermatogenesis related genes , we only found differences between wt and xpc-/- animals among the bp suppressed genes;  <dig> genes were more suppressed in wt mice compared to  <dig> gene in xpc-/- mice as compared to animals of the other genotype.
taken together, these results indicate that gene expression differences between wt and xpc-/- mice are small, and that the induction of genes involved in cell division regulating processes was stronger in xpc-/- than in wt mice.
we did not find relevant differences in average gene expression ratios between xpc-/- and wt mice among translation related genes, that are given in additional file  <dig>
average gene expression ratios for genes involved in significantly regulated pathways are compared between wt and xpc-/- mice.
the table shows the number of genes that are up or down regulated to a higher extent after bp exposure in either genotype.
uw, up regulated to a higher extent in wt mice.
ux, up regulated to a higher extent in xpc-/- mice.
dw, down regulated to a greater extent in wt mice.
dx, down regulated to a greater extent in xpc-/- mice.
<dig> differentially regulated genes regarding the cell cycle process.
results are presented as average gene expression ratios between exposed and control samples.
among the  <dig> go and sp_pir terms that were obtained from the combined list of bp  regulated genes, we again found a few mitochondria related terms, such as the cellular components 'mitochondrial part', 'mitochondrial envelope' and 'mitochondrial membrane', and the sp_pir keyword 'mitochondrion'.
furthermore, we found the sp_pir keyword 'chaperone', which involves genes that are known to be activated during a stress response.
cell cycle phase activity
the setwise average gene expression levels of cell cycle related gene sets were calculated for sets of genes related to specific cell cycle phases, in order to study cell cycle phase activity .
the sets used were based on go functional annotation for the complete cell cycle or genes found specifically expressed in a particular cell cycle phase, reported by both bar-joseph et al.
<cit>  and whitfield et al.
<cit> .
average gene expression levels of all cell cycle genes were lower in xpc-/- mice than in wt mice  in the absence of exposure to bp.
however, after bp exposure gene expression levels of the same cell cycle related genes were increased to comparable levels in both genotypes.
when focusing on specific cell cycle phases, we observed that the average gene expression levels of genes within the cell cycle transition between g <dig> and s was similar in wt and xpc-/- control mice, and again increased after bp exposure to equal levels .
on the other hand, the average level of expression of genes involved in the g2-m phase transition was initially lower in xpc-/- control mice compared to the wt control mice, and gene expression levels were increased by bp exposure to a higher extent in xpc-/- mice compared to the wt mice.
table  <dig> shows the gene expression ratios of the  <dig> genes involved in the g2-m phase transition, indicating that  <dig> out of these  <dig> genes were more induced in xpc-/- mice after bp exposure as compared to wt mice .
although these differences between wt and xpc-/- mice are not significant, they are consistent since the majority of genes respond into the same direction, and represent an observed trend towards a more pronounced response to bp in xpc-/- mice.
within the m-g <dig> phase, we observed a low impact of bp exposure in both genotypes, as compared to the other cell cycle phases.
also within this cell cycle phase, the response to bp was more pronounced in xpc-/- mice than in wt mice, although this was based on the expression of  <dig> genes only .
gene expression ratios are presented of  <dig> differentially regulated genes involved in g <dig> to m phase transition based on microarray results.
these  <dig> genes, of which  <dig> were down regulated and  <dig> were up regulated after bp exposure, showed a trend towards a more pronounced induction in xpc-/- mice as compared to wt mice .
however, a less extensive down regulation is not the same as a greater induction.
therefore, an additional p-value was calculated for the  <dig> up regulated genes, showing that  <dig>  out of these  <dig> up regulated genes are more induced in xpc-/- than in wt mice .
validation with quantitative real-time pcr
quantitative real-time pcr  was performed on  <dig> selected genes that are involved in the g2-m phase transition to validate the gene expression changes in wt vs. xpc-/- mice after bp exposure.
three genes  were selected because their average gene expression ratios were most different between wt and xpc-/- mice based on the microarray results, and asf1b was selected as a control because of the small difference between wt and xpc-/- mice.
after gene expression changes were normalised to the hprt <dig> reference, qpcr data of the  <dig> strongly differentially expressed genes indeed showed a higher induction of gene expression after bp exposure in xpc-/- mice .
however, the effect of bp exposure was smaller on basis of the qpcr expression data.
qpcr data of asf1b showed a down regulation after bp exposure in both wt and xpc-/- mice , and did therefore, not confirm the microarray results.
overall,  <dig> out of  <dig> selected genes confirmed the microarray results found within the g2-m phase, showing that bp induces marginally higher gene expression changes in xpc-/- than in wt mice.
discussion
bp exposure leads to the formation of dna adducts in testis and sperm of exposed male mice and humans  <cit> .
these pro-mutagenic dna adducts are usually removed by dna repair mechanisms to prevent the accumulation of mutations.
in a previous study, we found that there is a difference in dna adduct removal between wtand xpc-/- mouse testes  <cit> .
in the first week after exposure to bp, we observed more dna adducts in testis of xpc-/- mice, and a slower decline of dna adducts over time.
in addition, we showed that spermatogonial stem cells rely on nucleotide excision repair for the complete removal of dna adducts during spermatogenesis  <cit> .
this suggests that ggr/ner plays an important role in mice in protecting the testis and sperm cells from dna damage by bp.
in the present study, we used a microarray approach to study whether these differences in dna adduct removal between wt and xpc-/- mice are accompanied by differences in gene expression, thereby elucidating adaptive responses towards the induction of heritable dna damage in testis.
we chose to analyze gene expression after  <dig> days, since at that time point dna adduct levels differed more between wt and xpc-/- mice compared to day  <dig>  and still differed at day  <dig>  <cit> .
two-way anova indicated that there are only minor expression differences between xpc-/- and wt mice.
no more than  <dig> genes  located in a  <dig> mb locus on chromosome  <dig>  were differentially expressed between the two genotypes.
although the expression levels of cml <dig>  d6mm5e and 2610209m04rik were higher as compared to the very low levels of xpc, the fact that these genes are located in proximity to the xpc gene on chromosome  <dig> suggests that mutating the xpc gene has changed the genomic or regulatory structure at that particular site and therefore the expression of these  <dig> genes.
another explanation could be that these  <dig> genes are of an ola <dig> genetic background, which is used to create the xpc mutant, and are for some unknown reason differently expressed  <cit> .
the main differences were found between the bp exposed and control mice, regardless of genotype.
<dig> genes were differentially regulated after bp exposure and these were enriched for genes involved in cell cycle, translation , chromatin structure and spermatogenesis.
besides these pathways, other more general processes were found to be affected .
regulated processes regarding mitochondrial function , chaperones, and the aforementioned changes in cell cycle seem to indicate a general stress response after exposure to bp in testes of both genotypes.
in addition, the altered expression of mitochondria related genes is indicative for changes in the oxidative phosphorylation; this process was also found to be affected among the bp down regulated genes, and the genes for several atp synthase subunits, nadh dehydrogenase  subunits, cytochrome c and cytochrome c oxidase were differentially regulated .
therefore, the motility and the fertilizing capacity of the sperm cells might have been affected, since mitochondria in sperm are responsible for the energy supply required to propel the flagellum  <cit> .
a change in mitochondrial activity, which leads to less potent sperm cells, may therefore be part of the protective mechanisms against dna damage in the testis and sperm cells.
indeed, litter sizes of female mice that were fertilized by bp exposed xpc-/- male mice were found to be smaller .
bp is a well known mutagen and carcinogen, and mostly studied for its capacity to form bulky dna adducts, which are mainly repaired by ner.
by changing the ner capacity in xpc-/- mice, we expected to find additional mechanisms affected by bp exposure that could possibly protect the testis and male germ cells against the formation of heritable dna damage, as a back up for the lack of ggr/ner.
however, pathways involving dna repair  or apoptosis were not significantly enriched among our regulated genes.
nonetheless, we did find  <dig> differentially regulated genes that are involved in dna repair  , and at least  <dig> out of these  <dig> genes are related to repair of double strand breaks and/or homologous recombination.
this suggests that exposure to bp may cause double strand breaks, for instance by the collapse of the replication fork in replicating cells.
indeed, the number of γh2ax foci, which is a hallmark for double strand break formation, was reported to be increased by exposure to bp  <cit> .
although the pathway involving apoptosis was not significantly enriched among our regulated genes, we found  <dig> apoptosis-related genes to be differentially expressed after bp exposure .
in testes, apoptosis is a common process to discard excessive germ cells, or to remove severely damaged germ cells  <cit> .
from our data it is hard to conclude whether apoptosis is elicited by bp exposure, since we found only a small number of differentially expressed genes after bp exposure and both pro-apoptotic and anti-apoptotic signals were found.
for example, cytochrome c  was up regulated after exposure in both wt and xpc-/- mice, which could be indicative for the initiation of the intrinsic apoptotic pathway.
on the other hand, the expression of the anti-apoptotic gene faim  was also up regulated after bp exposure, indicating an inhibitory effect on the apoptotic pathway  <cit> .
several aspects should be considered for the interpretation of transcriptional data regarding the regulation of apoptosis after bp exposure; 1) the large number of apoptotic cells in normal testis will make it difficult to measure subtle changes in the apoptotic response after a single bp exposure, 2) the apoptotic response after bp exposure is an acute response that is probably hard to assess  <dig> days after exposure, and 3) the activation of the different caspases leading to fragmentation of dna occurs by cleavage of procaspases already stored in the cell, a process that is unlikely to be found at the transcriptional level  <cit> .
it should be noted that this last point may also hold true for other processes such as dna repair, which also relies on activation and transport of already existing proteins.
<dig> differentially regulated genes concerning dna repair.
results are presented as average gene expression ratios between exposed and control samples.
*genes associated with double strand breaks and/or homologous recombination
<dig> differentially regulated genes concerning apoptosis.
results are presented as average gene expression ratios between exposed and control samples.
to have a better understanding of the changes in cell cycle regulation, we compared setwise average expression levels of genes related to specific cell cycle phases between the  <dig> experimental groups.
genes involved in the g <dig> to s phase transition and the g <dig> to m phase transition were induced in animals of both genotypes after bp exposure, suggesting that cells may accumulate in both g <dig> and g <dig>  cell cycle arrest after bp exposure was previously found, using mcf- <dig>  and hepg <dig>  cells, and identified altered expression of genes that affect cell cycle regulation  <cit> .
moreover, cell cycle phases assessed by facs analysis indeed showed an accumulation of cells in the g <dig> and g <dig> phase after bp exposure  <cit> .
it has also been shown that benzo pyrene- <dig> -dihydrodiol  and benzopyrene-diol-epoxide , both metabolites of bp, can induce a g2/m accumulation  <cit> .
all these data are in line with our findings in testis of bp exposed male mice.
the bp induced g1-s arrest did not differ between wt and xpc-/- mice .
however, expression of g2-m phase genes showed a trend towards a higher induction of these genes in testes of xpc-/- mice after bp exposure .
therefore, the expression ratios of  <dig> g2-m phase genes were validated by qpcr, and results of  <dig> out of these  <dig> genes confirmed that bp induces slightly higher gene expression changes in xpc-/- mice than in wt mice.
however, the level of induction by bp exposure was higher on basis of microarray analysis, than by qpcr analysis.
qpcr could not confirm the induction of asf1b gene expression by bp as shown by microarray analysis, although the difference in gene expression ratios between wt and xpc-/- mice remained small.
such a small difference in reaction may be explained by the fact that xpc-/- mice are less capable of removing bp induced damage in testis  <cit> , and therefore more damaged cells are arrested .
this was also observed in a study using uvb irradiated xpc-deficient epidermal cells  <cit> ; xpc-deficient cells were blocked in the late s phase due to incomplete dna replication at sites of damaged dna  <cit> .
as a result, the regulation of the g <dig> phase genes might differ slightly between xpc-/- and wt mice, as found in this study.
unrepaired dna damage blocks the cell cycle.
however, specialized y-family dna polymerases are capable of extending replication beyond lesions in an error-free or error-prone way  <cit> .
for example the expression of dna polymerase κ  is under the control of the bp activated arylhydrocarbon receptor , and more highly expressed in testes than in other tissues  <cit> .
it bypasses bpde-n2-dg in an efficient and mostly error-free way  <cit> .
if we assume that dna repair deficient xpc-/- cells that are present in testes , in contrast to dna repair proficient wt cells, are blocked in the late s phase due to unrepaired damage, then this damage could be passed in an error-free way with progression of the cell cycle and spermatogenesis, but without the actual removal of the blocking lesion.
although polκ was not present among the differentially expressed genes by using two-way anova, we found, however, that the expression of polκ was significantly higher in xpc-/- than in wt mice after bp exposure as assessed by a simple t-test .
however, this differential expression between wt and xpc-/- mice after bp exposure was found only after correction for the slight initial expression differences found in their unexposed counterparts.
progression of the cell cycle and spermatogenesis, without the actual removal of the dna lesion is in line with the differences in dna damage kinetics between xpc-/- and wt mice found in our previous study  <cit> .
therefore, we suggest that xpc-/- mice are not per se more sensitive to mutation induction in the testis than wt mice after bp exposure, and thereby could have equal risks for heritable gene mutations.
however, future studies analyzing mutation induction should elucidate the mutagenic potency of bp in this reproductive organ.
CONCLUSIONS
from this study, we can conclude that gene expression in testis of untreated xpc-/- and wt mice are similar, and this is also the case after exposure to bp.
bp exposure, which formed a substantial amount of dna damage, differentially regulated  <dig> genes that were enriched for cell cycle, translation, chromatin structure and spermatogenesis.
overall, bp-induced gene expression changes in xpc-/- mice differed from their exposed wild type counterparts , which was also found for stage specific cell cycle gene sets.
these gene expression changes in testis may represent a cell cycle block due to the presence of unrepaired damage.
specialized dna polymerases that are highly expressed in testis may still bypass bp induced dna damage in an error-free way, and cell cycle/spermatogenesis can thus progress in the presence of dna lesions.

 exposure to chemicals like benzopyrene can lead to structural changes in dna and as a consequence to the development of diseases with a genetic basis. changes in the dna sequence can be induced by exposure to chemicals during life, but may also be inherited via mutations in the spermatogonial stem cells; in that way increasing the risk of developing abnormalities or diseases in the offspring. the mutagenic potential of bp in male germ cells, however, has still not been fully established. bp related dna damage was observed at all stages of spermatogenesis and in testis, but it is largely unknown how germ cells deal with dna damage to protect their genetic material, and to prevent the accumulation of mutations in the germ line. spermatogenesis is carefully controlled to produce mature spermatozoa from spermatogonial stem cells in three major stages; the mitotic stage, the meiotic stage and the maturation stage. germ cells are susceptible for the induction of mutations during mitotic and meiotic divisions, because cell turnover is a prerequisite for fixation of dna damage into mutations. however, it is likely that several processes prevent the occurrence of gene mutations in male germ cells; for example, dna damage can be removed by dna repair mechanisms, of which nucleotide excision repair is considered to be the most relevant repair mechanism for bulky dna adducts formed by reactive metabolites of bp. two ner mechanisms have been described: global genome repair eliminates bulky dna lesions in the entire genome, whereas transcription coupled repair specifically removes lesions that block rna synthesis. in a previous study, we observed that ggr/ner plays an important role in the removal of bp induced dna adducts in the testis, especially in the first week after exposure. bp induced dna adduct levels in the testis were significantly different between wt and xpc-/- mice, especially at days after a single exposure to bp. therefore, in an attempt to reveal the responses in the testis to this damage, we investigated by using microarrays the changes in gene expression induced by bp in testis from wt and xpc-/- male mice, days after exposure to bp. since xpc-/- mice lack one of the most important protective mechanisms against bp induced dna damage, we expected a different transcriptional response in these mice as compared to their wt counterparts.  gene expression profiling two-way anova revealed regulated genes that were differentially expressed between treated and untreated mice, of which genes were increased and genes were decreased by bp exposure. only genes were differentially expressed between unexposed wt and unexposed xpc-/- mice, and they are all located at the same site of chromosome of these genes, xpc, cml and d6mm5e were decreased in expression in xpc-/- mice as compared to the wt mice, with higher gene expression levels of cml and d6mm5e than the very low levels of xpc. on the other hand, 2610209m04rik was increased in xpc-/- mice. this confirms the absence of a functional xpc gene in the xpc-/- genotype, and shows that closely linked genes have significantly altered expression patterns. finally, our results show that there is only a modest difference between the gene expression patterns in testis of bp treated wt and xpc-/- mice. nonetheless, a paired t-test showed that the level in which these bp regulated genes are expressed differs between wt and xpc-/- mice. the clustering of the combined hits is presented in figure analysis of pathway regulation the list of the genes that were induced by bp suggested, by using david/ease, a significant enrichment for several gene ontology biological processes and swiss-prot_protein information resource terms, such as'cell division','mitosis','chromosome segregation' and'spermatogenesis'. the genes that were down regulated by bp exposure also showed an enrichment for several pathways, including'oxidative phosphorylation'. in addition, two go terms for the cellular components mitochondrial part and mitochondrion, were enriched among bp suppressed genes, which is in line with the inhibition of the oxidative phosphorylation process. the combined list of bp regulated genes showed an enrichment of go and sp_pir terms, including cell cycle, translation, chromatin structure and spermatogenesis, which we already found to be enriched among the up regulated genes only. therefore, we used these processes to further select genes and to subsequently compare the two genotypes regarding their gene expression ratios between bp treated vs. control groups. an overview of the gene expression ratio distribution is given in table differentially regulated genes were involved in the cell cycle, of which genes were induced to a higher extent in xpc-/- mice, whereas only of these genes were expressed to a higher extent in wt mice. regarding the genes that were inhibited in their expression, genes were more suppressed in xpc-/- mice, and genes in wt mice as compared to animals of the other genotype. in addition, we found genes that are involved in the chromatin structure, of which genes were more induced in xpc-/- mice, and genes in wt mice. only chromatin structure related genes were more suppressed in wt mice, as compared to gene in xpc-/- mice. among the spermatogenesis related genes, we only found differences between wt and xpc-/- animals among the bp suppressed genes; genes were more suppressed in wt mice compared to gene in xpc-/- mice as compared to animals of the other genotype. taken together, these results indicate that gene expression differences between wt and xpc-/- mice are small, and that the induction of genes involved in cell division regulating processes was stronger in xpc-/- than in wt mice. we did not find relevant differences in average gene expression ratios between xpc-/- and wt mice among translation related genes, that are given in additional file average gene expression ratios for genes involved in significantly regulated pathways are compared between wt and xpc-/- mice. the table shows the number of genes that are up or down regulated to a higher extent after bp exposure in either genotype. uw, up regulated to a higher extent in wt mice. ux, up regulated to a higher extent in xpc-/- mice. dw, down regulated to a greater extent in wt mice. dx, down regulated to a greater extent in xpc-/- mice. differentially regulated genes regarding the cell cycle process. results are presented as average gene expression ratios between exposed and control samples. among the go and sp_pir terms that were obtained from the combined list of bp regulated genes, we again found a few mitochondria related terms, such as the cellular components'mitochondrial part','mitochondrial envelope' and'mitochondrial membrane', and the sp_pir keyword'mitochondrion'. furthermore, we found the sp_pir keyword'chaperone', which involves genes that are known to be activated during a stress response. cell cycle phase activity the setwise average gene expression levels of cell cycle related gene sets were calculated for sets of genes related to specific cell cycle phases, in order to study cell cycle phase activity. the sets used were based on go functional annotation for the complete cell cycle or genes found specifically expressed in a particular cell cycle phase, reported by both bar-joseph et al. and whitfield et al.. average gene expression levels of all cell cycle genes were lower in xpc-/- mice than in wt mice in the absence of exposure to bp. however, after bp exposure gene expression levels of the same cell cycle related genes were increased to comparable levels in both genotypes. when focusing on specific cell cycle phases, we observed that the average gene expression levels of genes within the cell cycle transition between g and s was similar in wt and xpc-/- control mice, and again increased after bp exposure to equal levels. on the other hand, the average level of expression of genes involved in the g2-m phase transition was initially lower in xpc-/- control mice compared to the wt control mice, and gene expression levels were increased by bp exposure to a higher extent in xpc-/- mice compared to the wt mice. table shows the gene expression ratios of the genes involved in the g2-m phase transition, indicating that out of these genes were more induced in xpc-/- mice after bp exposure as compared to wt mice. although these differences between wt and xpc-/- mice are not significant, they are consistent since the majority of genes respond into the same direction, and represent an observed trend towards a more pronounced response to bp in xpc-/- mice. within the m-g phase, we observed a low impact of bp exposure in both genotypes, as compared to the other cell cycle phases. also within this cell cycle phase, the response to bp was more pronounced in xpc-/- mice than in wt mice, although this was based on the expression of genes only. gene expression ratios are presented of differentially regulated genes involved in g to m phase transition based on microarray results. these genes, of which were down regulated and were up regulated after bp exposure, showed a trend towards a more pronounced induction in xpc-/- mice as compared to wt mice. however, a less extensive down regulation is not the same as a greater induction. therefore, an additional p-value was calculated for the up regulated genes, showing that out of these up regulated genes are more induced in xpc-/- than in wt mice. validation with quantitative real-time pcr quantitative real-time pcr was performed on selected genes that are involved in the g2-m phase transition to validate the gene expression changes in wt vs. xpc-/- mice after bp exposure. three genes were selected because their average gene expression ratios were most different between wt and xpc-/- mice based on the microarray results, and asf1b was selected as a control because of the small difference between wt and xpc-/- mice. after gene expression changes were normalised to the hprt reference, qpcr data of the strongly differentially expressed genes indeed showed a higher induction of gene expression after bp exposure in xpc-/- mice. however, the effect of bp exposure was smaller on basis of the qpcr expression data. qpcr data of asf1b showed a down regulation after bp exposure in both wt and xpc-/- mice, and did therefore, not confirm the microarray results. overall, out of selected genes confirmed the microarray results found within the g2-m phase, showing that bp induces marginally higher gene expression changes in xpc-/- than in wt mice. discussion bp exposure leads to the formation of dna adducts in testis and sperm of exposed male mice and humans. these pro-mutagenic dna adducts are usually removed by dna repair mechanisms to prevent the accumulation of mutations. in a previous study, we found that there is a difference in dna adduct removal between wtand xpc-/- mouse testes. in the first week after exposure to bp, we observed more dna adducts in testis of xpc-/- mice, and a slower decline of dna adducts over time. in addition, we showed that spermatogonial stem cells rely on nucleotide excision repair for the complete removal of dna adducts during spermatogenesis. this suggests that ggr/ner plays an important role in mice in protecting the testis and sperm cells from dna damage by bp. in the present study, we used a microarray approach to study whether these differences in dna adduct removal between wt and xpc-/- mice are accompanied by differences in gene expression, thereby elucidating adaptive responses towards the induction of heritable dna damage in testis. we chose to analyze gene expression after days, since at that time point dna adduct levels differed more between wt and xpc-/- mice compared to day and still differed at day. two-way anova indicated that there are only minor expression differences between xpc-/- and wt mice. no more than genes located in a mb locus on chromosome were differentially expressed between the two genotypes. although the expression levels of cml d6mm5e and 2610209m04rik were higher as compared to the very low levels of xpc, the fact that these genes are located in proximity to the xpc gene on chromosome suggests that mutating the xpc gene has changed the genomic or regulatory structure at that particular site and therefore the expression of these genes. another explanation could be that these genes are of an ola genetic background, which is used to create the xpc mutant, and are for some unknown reason differently expressed. the main differences were found between the bp exposed and control mice, regardless of genotype. genes were differentially regulated after bp exposure and these were enriched for genes involved in cell cycle, translation, chromatin structure and spermatogenesis. besides these pathways, other more general processes were found to be affected. regulated processes regarding mitochondrial function, chaperones, and the aforementioned changes in cell cycle seem to indicate a general stress response after exposure to bp in testes of both genotypes. in addition, the altered expression of mitochondria related genes is indicative for changes in the oxidative phosphorylation; this process was also found to be affected among the bp down regulated genes, and the genes for several atp synthase subunits, nadh dehydrogenase subunits, cytochrome c and cytochrome c oxidase were differentially regulated. therefore, the motility and the fertilizing capacity of the sperm cells might have been affected, since mitochondria in sperm are responsible for the energy supply required to propel the flagellum. a change in mitochondrial activity, which leads to less potent sperm cells, may therefore be part of the protective mechanisms against dna damage in the testis and sperm cells. indeed, litter sizes of female mice that were fertilized by bp exposed xpc-/- male mice were found to be smaller. bp is a well known mutagen and carcinogen, and mostly studied for its capacity to form bulky dna adducts, which are mainly repaired by ner. by changing the ner capacity in xpc-/- mice, we expected to find additional mechanisms affected by bp exposure that could possibly protect the testis and male germ cells against the formation of heritable dna damage, as a back up for the lack of ggr/ner. however, pathways involving dna repair or apoptosis were not significantly enriched among our regulated genes. nonetheless, we did find differentially regulated genes that are involved in dna repair, and at least out of these genes are related to repair of double strand breaks and/or homologous recombination. this suggests that exposure to bp may cause double strand breaks, for instance by the collapse of the replication fork in replicating cells. indeed, the number of γh2ax foci, which is a hallmark for double strand break formation, was reported to be increased by exposure to bp. although the pathway involving apoptosis was not significantly enriched among our regulated genes, we found apoptosis-related genes to be differentially expressed after bp exposure. in testes, apoptosis is a common process to discard excessive germ cells, or to remove severely damaged germ cells. from our data it is hard to conclude whether apoptosis is elicited by bp exposure, since we found only a small number of differentially expressed genes after bp exposure and both pro-apoptotic and anti-apoptotic signals were found. for example, cytochrome c was up regulated after exposure in both wt and xpc-/- mice, which could be indicative for the initiation of the intrinsic apoptotic pathway. on the other hand, the expression of the anti-apoptotic gene faim was also up regulated after bp exposure, indicating an inhibitory effect on the apoptotic pathway. several aspects should be considered for the interpretation of transcriptional data regarding the regulation of apoptosis after bp exposure; 1) the large number of apoptotic cells in normal testis will make it difficult to measure subtle changes in the apoptotic response after a single bp exposure, 2) the apoptotic response after bp exposure is an acute response that is probably hard to assess days after exposure, and 3) the activation of the different caspases leading to fragmentation of dna occurs by cleavage of procaspases already stored in the cell, a process that is unlikely to be found at the transcriptional level. it should be noted that this last point may also hold true for other processes such as dna repair, which also relies on activation and transport of already existing proteins. differentially regulated genes concerning dna repair. results are presented as average gene expression ratios between exposed and control samples. *genes associated with double strand breaks and/or homologous recombination differentially regulated genes concerning apoptosis. results are presented as average gene expression ratios between exposed and control samples. to have a better understanding of the changes in cell cycle regulation, we compared setwise average expression levels of genes related to specific cell cycle phases between the experimental groups. genes involved in the g to s phase transition and the g to m phase transition were induced in animals of both genotypes after bp exposure, suggesting that cells may accumulate in both g and g cell cycle arrest after bp exposure was previously found, using mcf- and hepg cells, and identified altered expression of genes that affect cell cycle regulation. moreover, cell cycle phases assessed by facs analysis indeed showed an accumulation of cells in the g and g phase after bp exposure. it has also been shown that benzo pyrene- -dihydrodiol and benzopyrene-diol-epoxide, both metabolites of bp, can induce a g2/m accumulation. all these data are in line with our findings in testis of bp exposed male mice. the bp induced g1-s arrest did not differ between wt and xpc-/- mice. however, expression of g2-m phase genes showed a trend towards a higher induction of these genes in testes of xpc-/- mice after bp exposure. therefore, the expression ratios of g2-m phase genes were validated by qpcr, and results of out of these genes confirmed that bp induces slightly higher gene expression changes in xpc-/- mice than in wt mice. however, the level of induction by bp exposure was higher on basis of microarray analysis, than by qpcr analysis. qpcr could not confirm the induction of asf1b gene expression by bp as shown by microarray analysis, although the difference in gene expression ratios between wt and xpc-/- mice remained small. such a small difference in reaction may be explained by the fact that xpc-/- mice are less capable of removing bp induced damage in testis, and therefore more damaged cells are arrested. this was also observed in a study using uvb irradiated xpc-deficient epidermal cells; xpc-deficient cells were blocked in the late s phase due to incomplete dna replication at sites of damaged dna. as a result, the regulation of the g phase genes might differ slightly between xpc-/- and wt mice, as found in this study. unrepaired dna damage blocks the cell cycle. however, specialized y-family dna polymerases are capable of extending replication beyond lesions in an error-free or error-prone way. for example the expression of dna polymerase κ is under the control of the bp activated arylhydrocarbon receptor, and more highly expressed in testes than in other tissues. it bypasses bpde-n2-dg in an efficient and mostly error-free way. if we assume that dna repair deficient xpc-/- cells that are present in testes, in contrast to dna repair proficient wt cells, are blocked in the late s phase due to unrepaired damage, then this damage could be passed in an error-free way with progression of the cell cycle and spermatogenesis, but without the actual removal of the blocking lesion. although polκ was not present among the differentially expressed genes by using two-way anova, we found, however, that the expression of polκ was significantly higher in xpc-/- than in wt mice after bp exposure as assessed by a simple t-test. however, this differential expression between wt and xpc-/- mice after bp exposure was found only after correction for the slight initial expression differences found in their unexposed counterparts. progression of the cell cycle and spermatogenesis, without the actual removal of the dna lesion is in line with the differences in dna damage kinetics between xpc-/- and wt mice found in our previous study. therefore, we suggest that xpc-/- mice are not per se more sensitive to mutation induction in the testis than wt mice after bp exposure, and thereby could have equal risks for heritable gene mutations. however, future studies analyzing mutation induction should elucidate the mutagenic potency of bp in this reproductive organ.  from this study, we can conclude that gene expression in testis of untreated xpc-/- and wt mice are similar, and this is also the case after exposure to bp. bp exposure, which formed a substantial amount of dna damage, differentially regulated genes that were enriched for cell cycle, translation, chromatin structure and spermatogenesis. overall, bp-induced gene expression changes in xpc-/- mice differed from their exposed wild type counterparts, which was also found for stage specific cell cycle gene sets. these gene expression changes in testis may represent a cell cycle block due to the presence of unrepaired damage. specialized dna polymerases that are highly expressed in testis may still bypass bp induced dna damage in an error-free way, and cell cycle/spermatogenesis can thus progress in the presence of dna lesions. 
in this study, we investigated by using microarray technology whether compromised dna repair in xpc-/- mice may lead to a transcriptional reaction of the testis to cope with increased levels of bp induced dna damage. exposure to benzopyrene affected the expression of genes that are involved in cell cycle regulation in both genotypes, indicating that the presence of unrepaired dna damage in testis blocks cell proliferation to protect dna integrity in both dna repair proficient and deficient animals. however, the level in which these bp regulated genes are expressed differs between wt and xpc-/- mice, and were predominantly involved in the regulation of cell cycle, translation, chromatin structure and spermatogenesis, indicating a general stress response. two-way anova revealed only genes differentially expressed between wild type and xpc-/- mice, and genes between testes of bp treated and untreated mice irrespective of the mouse genotype. in addition, analysis of cell cycle phase dependent gene expression revealed that expression of genes involved in g1-s and g2-m phase arrest was increased after bp exposure in both genotypes. other processes that were expected to have changed by exposure, like apoptosis and dna repair, were not found to be modulated at the level of gene expression. benzo pyrene exposure induces dna adducts at all stages of spermatogenesis and in testis, and removal of these lesions is less efficient in nucleotide excision repair deficient xpc-/- mice than in wild type mice. a slightly higher induction of average gene expression was observed at the g2-m checkpoint in xpc-/- mice, but this did not reach statistical significance. gene expression in testis of untreated xpc-/- and wild type mice were very similar, with only genes differentially expressed.
 benzo pyrene exposure induces dna adducts at all stages of spermatogenesis and in testis, and removal of these lesions is less efficient in nucleotide excision repair deficient xpc-/- mice than in wild type mice. in this study, we investigated by using microarray technology whether compromised dna repair in xpc-/- mice may lead to a transcriptional reaction of the testis to cope with increased levels of bp induced dna damage.  two-way anova revealed only genes differentially expressed between wild type and xpc-/- mice, and genes between testes of bp treated and untreated mice irrespective of the mouse genotype. however, the level in which these bp regulated genes are expressed differs between wt and xpc-/- mice, and were predominantly involved in the regulation of cell cycle, translation, chromatin structure and spermatogenesis, indicating a general stress response. in addition, analysis of cell cycle phase dependent gene expression revealed that expression of genes involved in g1-s and g2-m phase arrest was increased after bp exposure in both genotypes. a slightly higher induction of average gene expression was observed at the g2-m checkpoint in xpc-/- mice, but this did not reach statistical significance. other processes that were expected to have changed by exposure, like apoptosis and dna repair, were not found to be modulated at the level of gene expression.  gene expression in testis of untreated xpc-/- and wild type mice were very similar, with only genes differentially expressed. exposure to benzopyrene affected the expression of genes that are involved in cell cycle regulation in both genotypes, indicating that the presence of unrepaired dna damage in testis blocks cell proliferation to protect dna integrity in both dna repair proficient and deficient animals. 