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Cancer Research 67, 5156, June 1, 2007. doi: 10.1158/0008-5472.CAN-07-0335
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

The Peroxisome Proliferator WY-14,643 Promotes Hepatocarcinogenesis Caused by Endogenously Generated Oxidative DNA Base Modifications in Repair-Deficient Csbm/m/Ogg1–/– Mice

Christian Trapp1, Michael Schwarz2 and Bernd Epe1

1 Institute of Pharmacy, University of Mainz, Mainz, Germany and 2 Department of Toxicology, Institute of Pharmacology and Toxicology, University of Tübingen, Tübingen, Germany

Requests for reprints: Bernd Epe, Institute of Pharmacy, University of Mainz, Staudingerweg 5, D-55099 Mainz, Germany. Phone: 49-6131-39-24309; Fax: 49-6131-39-25521; E-mail: epe{at}uni-mainz.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Basal levels of endogenously generated oxidative DNA modifications such as 7,8-dihydro-8-oxoguanine (8-oxoG) are present in apparently all mammalian cells, but their relevance for the generation of spontaneous cancers remains to be established. Both the 8-oxoG levels and the resulting spontaneous mutations are increased in the livers of Csbm/m/Ogg1–/– mice, which are deficient in the repair of 8-oxoG. In order to determine the consequences of these additional oxidative DNA modifications and mutations and thus assess the tumor initiating potency of this type of endogenous DNA damage, we treated Csbm/m/Ogg1–/– mice and repair-proficient controls with the peroxisome proliferator WY-14,643 (0.025% ad libitum), a potent inducer of liver cell proliferation. The treatment did not generate any additional oxidative DNA damage; the elevated levels of 8-oxoG in the Csbm/m/Ogg1–/– mice even decreased. Also, the spontaneous mutation frequencies observed in the lacI gene of BigBlue Csbm/m/Ogg1–/– mice, which were ~3-fold higher than in the repair-proficient mice, declined by 39% under the treatment, whereas the frequencies in the livers of the repair-proficient animals remained unchanged. Preneoplastic lesions (staining positive or negative for glucose-6-phoshatase) developed in the livers of both wild-type and Csbm/m/Ogg1–/– mice after 30 weeks. Both the numbers and the total volumes of the lesions were ~6-fold higher in the repair-deficient mice than in the wild-type mice. The results indicate that spontaneous mutations generated from endogenous oxidative DNA base damage efficiently translate into increased tumorigenesis when cell proliferation is stimulated. [Cancer Res 2007;67(11):5156–61]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
There is good evidence that reactive oxygen species (ROS) generated during normal cellular oxygen metabolism are an important source of endogenous DNA damage and that the resulting oxidative DNA modifications contribute to the spontaneous mutation rates. Therefore, oxidative DNA modifications have been suspected to contribute to the aging process, several age-related degenerative diseases, and the formation of cancer (1, 2).

The recent generation of mice that are defective in the repair of oxidative DNA modifications has provided a promising strategy to determine the potency of endogenously generated oxidative base DNA modifications to act as initiators of carcinogenesis (3). Thus, Ogg1–/– mice, which are defective in the repair glycosylase OGG1 that removes oxidative purine modifications such as 7,8-dihydro-8-oxoguanine (8-oxoG) from the genome, accumulate higher levels of 8-oxoG in the liver than wild-type mice (4–6). These result in elevated spontaneous mutation frequencies, mostly G:C to T:A transversions characteristic for 8-oxoG (4, 5). The accumulation of the oxidative purine modifications and of the resulting mutations are even more pronounced in Csbm/m/Ogg1–/– mice,3 which additionally lack a back-up repair mechanism for this type of damage mediated by Cockayne syndrome B protein (8, 9). The CSB deficiency alone has no significant effect on the basal levels of oxidative base modifications and on the spontaneous mutation rates (8–10).

In spite of the elevated oxidative damage in Ogg1–/– and Csbm/m/Ogg1–/– mice, no increased incidence of liver tumors was observed; multiple tumor formation in various organs occurred only when the defective removal of 8-oxoG was combined with a defect in MYH, a mismatch repair protein that removes adenine when mispaired with 8-oxoG (11). This could mean that the observed levels of oxidative DNA base damage and the resulting point mutations have a low inherent tumor-initiating potential. Alternatively, the low level of cell proliferation in uncompromised livers could be the decisive factor that prevents tumor formation in repair-deficient animals.

In order to test this assumption and to assess the initiating potential of a defined level of endogenously generated oxidative DNA base damage and the associated mutation frequencies, we treated wild-type and repair-deficient Csbm/m/Ogg1–/– animals with the peroxisome proliferator WY-14,643. In rats and mice, peroxisome proliferators are strong inducers of liver cell proliferation and inhibitors of apoptosis. Chronic administration results in liver hypertrophy, hyperplasia, and tumor formation (12, 13). In two-stage initiation/promotion experiments, WY-14,643 acts as a potent tumor-promoting agent in rodent liver (14, 15). There are some indications that ROS generation is involved in these effects, in particular because the peroxisomal acyl-CoA-oxidase (ACOX1), an enzyme strongly induced by peroxisome proliferators, is known to produce ROS as by-products (16–19). Although no direct mutagenic effects of WY-14,643 were observed (20), ROS have been hypothesized to generate oxidative DNA damage and thus contribute to carcinogenesis (16–18, 21).

Mechanistically, most or all of the biochemical and carcinogenic effects of the peroxisome proliferators have been attributed to a direct activation of the peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), a ligand-activated transcription factor (22). Thus, PPAR{alpha}–/– mice are resistant to WY-14,643–mediated cell proliferation and hepatocarcinogenesis (23).

The results described in this report indicate that the peroxisome proliferator WY-14,643 does not generate oxidative DNA modifications, but promotes the hepatocarcinogenesis of spontaneously initiated cells. Under exposure to WY-14,643, the elevated spontaneous mutation frequency in the livers caused by a relatively low level of additional endogenously generated oxidative DNA base modifications (8-oxoG residues) gives rise to a several-fold higher yield of preneoplastic (enzyme-altered) lesions. This correlation of oxidative DNA damage levels, spontaneous mutations, and early markers of tumorigenesis supports the notion that endogenously generated ROS are quite potent initiators of carcinogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Treatment of mice. C57BL/6 (wild-type) mice were purchased from Charles River. Csbm/m/Ogg1–/– mice were bred from Ogg1–/– mice (4) and Csbm/m mice (7) as described previously (9). The generation of Csbm/m/Ogg1–/– mice carrying the bacterial lacI gene (BigBlue mice) and of repair-proficient comparisons, which were used for mutation analyses, has also been described (8). The mice were housed up to five per cage under a 12 h light and 12 h dark cycle. Water and a special laboratory chow (Altromin), containing 0.025% WY-14,643 [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid] if indicated, were given ad libitum up to 250 days. For short-term treatments (1 or 4 days) WY-14,643 (100 mg/kg of body weight) was administered in an olive oil vehicle by gavage.

Quantification of ACOX1 expression. The expression of the peroxisomal enzyme ACOX1 in Csbm/m/Ogg1–/– mice was monitored using a noncompetitive reverse transcription-PCR as described by Ringhoffer et al. (24). RNA was isolated from untreated (control) and WY-14,643–treated livers using a RNeasy Mini-Kit (Qiagen). Primers used for the amplification of the Acox1 gene with Taq polymerase (Peqlab) were 5'-TGC CGA GCC AGC GTA TCC and 5'-CAC AGC GTT GGG GCG AGT CA. An amplification of the GAPDH gene (primers, 5'-CGT CTT CAC CAC CAT GGA GA and 5'-CGG CCA TCA CGC CAC AGT TT) was carried out for the calibration of the amounts of cDNA. The PCR products were separated in an agarose gel, stained with ethidium bromide, and the color intensity of the products was quantified.

Isolation of hepatocytes. For determination of oxidative DNA modifications, hepatocytes were isolated from untreated and WY-14,643–treated wild-type and Csbm/m/Ogg1–/– mice by a modified two-step collagenase perfusion technique as described previously (25). Briefly, the liver was perfused in situ via the vena portae for 15 min with an EGTA-containing buffer at 37°C. The perfusion was continued with a collagenase buffer for up to 30 min. After perfusion, the liver was removed, the hepatocytes carefully dissociated in a suspension buffer and the suspension filtered through gauze (100 µm pore size). Cells were pelleted by centrifugation (10 min at 15 x g) and resuspended in fresh suspension buffer twice (25). The viability of the hepatocytes was determined by trypan blue (4 g/L) exclusion and ranged from ~60% to 70%, independent of the genotype and treatment with WY-14,643.

Quantification of oxidative DNA modifications. Oxidative DNA modifications were determined by an alkaline elution protocol (26) with modifications (27, 28). The number of hepatocytes to each filter was 5 x 105, corresponding to ~10 µg of DNA. The sum of modifications sensitive to Fpg protein and single-strand breaks was determined following incubation of the cellular DNA for 60 min at 37°C with 1 µg/mL of Fpg protein immediately after cell lysis. Under these conditions, the incision by the enzyme at sensitive DNA modifications was shown to be completed (28). The number of modifications incised by the Fpg protein was obtained by subtraction of the number of single-strand breaks observed in parallel incubations without enzyme.

Quantification of mutations in the lacI gene of transgenic BigBlue mice. The assay was done with slight modifications according to the manufacturer's protocol (29, 30) as described previously (8). Two Csbm/m/Ogg1–/– animals and two repair-competent controls carrying the Escherichia coli lacI gene as a target for mutagenesis (31) were treated at the age of 5 months with WY-14,643 as described above. Livers were quick-frozen in liquid nitrogen and stored at –70°C. High molecular weight DNA was isolated from the livers with a RecoverEase DNA Isolation Kit (Stratagene) according to the manufacturer's protocol. The lacI gene was then rescued from DNA via in vitro packaging into bacteriophages of whom up to 30,000 (29) were plated overnight with E. coli in the presence of X-gal (5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside). Color control mutants (CM0, CM1, CM2, and CM3) with different color intensities were plated in parallel as an internal control. Per animal, ~300,000 plaque-forming units were plated and mutation frequencies were calculated by dividing the number of mutants by the number of totally screened plaque-forming units.

Enzyme histochemistry and stereological evaluation. Frozen sections were taken from each liver and stained enzyme-histochemically for glucose-6-phosphatase (G-6-Pase) activity (32). G-6-Pase–altered lesions were quantified by means of a computer-assisted digitizer system (33). The volume fraction of enzyme-altered tissue in liver and the number of lesions per unit of liver were calculated using standard stereological techniques.

Statistical analyses. Volume fractions and the number of preneoplastic lesions were compared using the Mann-Whitney U test for non-Gaussian distributions. Mutation frequencies were compared assuming a binomial distribution of mutations. Statistical significance was calculated using Fisher exact test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
WY-14,643 induces liver growth in wild-type and Csbm/m/Ogg1–/– mice. Wild-type and Csbm/m/Ogg1–/– mice aged 5 months at the time of examination were treated with the peroxisome proliferator WY-14,643. For acute treatments (1 or 4 days) the compound was administered by gavage (100 mg/kg body weight per day). For longer treatments, the dose of 0.025% WY-14,643 in the diet (ad libitum) was chosen, which was tolerated for up to 35 weeks in earlier studies (34). In accordance with those findings, the treatment of wild-type and Csbm/m/Ogg1–/– animals up to 250 days did not show a general retardation in weight gain compared with untreated animals (Fig. 1 ).


Figure 1
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  		Figure 1. Influence of WY-14,643 on the body weight of wild-type (top) and repair-deficient (Csbm/m/Ogg1–/–) mice (bottom). The verum group of animals ({circ}) was fed a standard laboratory chow containing 0.025% WY-14,643 ad libitum; the diet of the control group (bullet) was without the additive. Body weights of all animals (seven wild-type and nine Csbm/m/Ogg1–/– in the verum group; two wild-type and three Csbm/m/Ogg1–/– in the control group) were monitored weekly.

 
The potency of WY-14,643 to induce liver growth was measured as a ratio between liver and body weight. The increase in liver weight started directly after the administration of the drug and reached a plateau at an ~3-fold increased liver-to-body weight after 14 days (Fig. 2A ). The two genotypes responded quantitatively very similarly. The determination of the mRNA levels of the peroxisomal ACOX1 using a noncompetitive reverse transcription-PCR revealed a strong increase of the expression of the gene after 1 day of treatment (Fig. 2B). The 7-fold induction observed in the Csbm/m/Ogg1–/– mice is similar to the induction factors previously reported for wild-type mice under the same feeding conditions (35).


Figure 2
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  		Figure 2. A, influence of WY-14,643 treatment on the liver weight of wild-type and Csbm/m/Ogg1–/– mice. WY-14,643 was given to mice for 1 or 4 d by gavage (100 mg/kg body weight) or administered for longer time periods with the laboratory chow (0.025%) ad libitum. At necroscopy, the body and liver weights of the animals were determined and the liver weight relative to the body weight (%) was calculated. B, influence of WY-14,643 treatment on gene expression of peroxisomal ACOX1 in livers of Csbm/m/Ogg1–/– mice. WY-14,643 (100 mg/kg body weight) was given to mice for 1 or 4 d. mRNA levels were determined by semiquantitative reverse transcription-PCR. For determination of the induction factor (relative increase compared with untreated control animals; right), the ACOX1 mRNA levels were normalized for equal expression of GAPDH.

 
The serum variables observed after 250 days of treatment with WY-14,643 and in untreated control mice are summarized in Table 1 . The basal levels in the untreated wild-type and Csbm/m/Ogg1–/– mice were similar, and very similar responses to the chronic WY-14,643 exposure were observed in wild-type and Csbm/m/Ogg1–/– mice. The serum concentrations of liver enzymes (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and glutamate lactate dehydrogenase) were strongly increased, whereas concentrations of bilirubin and serum protein were unchanged. The influence of the peroxisome proliferator on the lipid metabolism is reflected by a moderate increase of total serum cholesterol and a decrease of triglycerides.


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  		Table 1. Serum concentrations of various biological markers in untreated and WY-14,643–treated (250 d) wild-type and Csbm/m/Ogg1–/– mice

 
WY-14,643 does not generate oxidative DNA damage. We quantified the levels of oxidative purine modifications (8-oxoG) in the DNA of primary hepatocytes isolated by liver perfusion from treated and untreated animals with a modified alkaline elution technique in combination with the bacterial Fpg protein as a probe. The latter enzyme is the functional bacterial analogue of the mammalian OGG1 protein and recognizes 8-oxoG and some other oxidized purine bases, e.g., formamidopyrimidines, in DNA (36). According to present knowledge, all modifications recognized by OGG1 (and therefore accumulating in consequence of an Ogg1 deficiency) are also substrates of Fpg.

The levels of oxidative DNA modifications sensitive to Fpg in untreated and WY-14,643–treated mice are shown in Fig. 3A . In accordance with previous observations (9), the steady-state levels of oxidative DNA modifications in the livers of untreated Csbm/m/Ogg1–/– mice were 2-fold higher than in those from wild-type animals. During treatment with WY-14,643, no changes in the levels of oxidative DNA modifications were observed in wild-type animals. In the repair-deficient Csbm/m/Ogg1–/– mice, a time-dependent decrease of the steady-state levels of oxidative DNA modifications was detected. The decrease may reflect a dilution of the accumulated oxidative base damage by cell proliferation, assuming that newly synthesized DNA contains only few oxidized purine residues, due to the action of the murine MTH1 protein that hydrolyses oxidized 8-oxo-dGTP into 8-oxo-dGMP in the nucleotide pool. No such dilution effect is observed in the repair-proficient wild-type cells, probably because the repair rate in these cells is much higher than the proliferation rate, so that the reduction of the damage level by dilution has little influence (9).


Figure 3
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  		Figure 3. A, Fpg-sensitive DNA modifications in primary hepatocytes isolated from untreated and WY-14,643–treated (1–112 d) wild-type and Csbm/m/Ogg1–/– mice aged 5 mo at the time of analysis. The oxidative DNA modifications were quantified by alkaline elution. Numbers above the columns indicate the numbers of independent experiments. B, mutation frequencies observed in the transgenic lacI gene in the livers of wild-type and Csbm/m/Ogg1–/– BigBlue mice, without treatment and after treatment with WY-14,643 for 21 d. DNA from two animals was isolated and ~300,000 plaque-forming units per animal were analyzed for mutations. Values for untreated animals are taken from Trapp et al. (8).

 
WY-14,643 does not increase the mutation frequency in vivo. To analyze the influence of the peroxisome proliferator on the mutation frequencies in the livers, Csbm/m/Ogg1–/– mice and repair-proficient (heterozygous) controls carrying the bacterial lacI gene (BigBlue mice) were treated for 3 weeks with the peroxisome proliferator WY-14,643, and ~300,000 plaque-forming units from multiple sample preparations were analyzed for mutations.

As shown in Fig. 3B, the mutation frequency in the livers of untreated Csbm/m/Ogg1–/– mice was 3-fold higher than in the repair-proficient controls (P < 0.005; ref. 8). Although no significant change of the mutation frequency was observed after 3 weeks of WY-14,643 treatment in the wild-type mice, the mutation frequency in the livers of Csbm/m/Ogg1–/– was reduced by ~39%. The decrease is statistically of only moderate significance (P = 0.074). Because mutations cannot disappear, a decrease would indicate that the induced cell proliferation predominantly affects a subgroup of hepatocytes (e.g., hepatocellular stem cells) with lower than average levels of accumulated mutations.

WY-14,643 induces preneoplastic lesions in Csbm/m/Ogg1–/– mice more efficiently than in wild-type mice. As an early and sensitive indicator for tumor formation, preneoplastic lesions (foci of cells staining positive or negative for G-6-Pase) were quantified in liver cross-sections at various time points after starting the treatment with WY-14,643 (37). The fraction of liver tissue occupied by G-6-Pase–deficient lesions was determined. In addition, the number of lesions per cubic centimeter of liver tissue was calculated by standard stereology from data on the size distribution of two-dimensional transections through G-6-Pase–deficient lesions (33). Although the livers of both wild-type and Csbm/m/Ogg1–/– mice were virtually free of enzyme-altered foci until day 176 of treatment, lesions staining either positive or negative for G-6-Pase (Fig. 4 ) were observed at day 250 in three out of seven wild-type animals and by day 211 in two out of three Csbm/m/Ogg1–/– mice. When the majority of the animals was sacrificed at day 250, the number of liver lesions per volume unit was much higher in the Csbm/m/Ogg1–/– mice than in the repair-proficient controls (Fig. 5A ; significance levels were P = 0.013 when calculated with the Mann-Whitney U test for non-Gaussian distributions and 0.012 when calculated with Student's t test). The total volume fraction occupied by the foci (Fig. 5B) was also higher in the repair-deficient mice, although the difference was less significant (P = 0.04 at day 211 and P = 0.186 at day 250 according to the Mann-Whitney U test). In both genotypes, a minority of the islets stained positive (13% in the wild-type and 11% in the Csbm/m/Ogg1–/– mice), the remaining stained negative for G-6-Pase. One very large basophilic tumor was detected in one of the wild-type animals, which was excluded from subsequent calculations because of its different biology.


Figure 4
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  		Figure 4. Liver sections from a Csbm/m/Ogg1–/– mouse showing two representative examples of liver lesions staining negative (A) or positive (B) for G-6-Pase.

 

Figure 5
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  		Figure 5. Numbers per volume unit (A) and volume fractions (B) of enzyme-altered (preneoplastic) foci observed in cross-sections from the livers of wild-type and Csbm/m/Ogg1–/– mice treated for various periods with WY-14,643. Frozen liver sections were stained enzyme-histochemically for G-6-Pase activity. The volume fractions were determined by standard stereology. Points, results for single animals; bars, medians for animals of the same genotype treated for identical times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
First, the results contribute to our mechanistic understanding of tumor formation by peroxisome proliferators. In spite of a strong induction of the peroxisomal ACOX1 (Fig. 2B), an established source of hydrogen peroxide, treatment with WY-14,643 neither generated oxidative DNA damage nor mutations (Fig. 3), which would be detectable in Csbm/m/Ogg1–/– mice with high sensitivity. ROS, either generated by ACOX1 in the peroxisomes or by other mechanisms such as by stimulation of neighboring Kupffer cells (38), apparently do not reach the nuclear DNA. Tumors occurring during peroxisome proliferator treatment, therefore, most probably result from promotional (epigenetic) effects on spontaneously initiated cells. Whether ROS play a role for these epigenetic effects remains to be established.

The absence of oxidative DNA damage generated by WY-14,643 in mice also means that the reported absence of ACOX1 induction in humans treated with peroxisome proliferators (fibrates) for hyperlipidemia (39) does not automatically exclude an associated tumor risk. The induction of hyperplasia, which is mechanistically poorly understood, seems to be more relevant for carcinogenesis under the influence of peroxisome proliferators. Although both the therapeutic effects and the stimulation of cell proliferation seem to be mediated by the activation of the PPAR{alpha}-receptor in the liver, there are indications that the latter effect is less pronounced in humans than in rodents (40).

Second, the results allow a correlation between elevated levels of endogenously generated oxidative DNA base modification, increased spontaneous mutation frequencies, and the initiation of carcinogenesis. Thus, <0.4 additional oxidative purine modifications per million base pairs in the livers of mice (Fig. 3A) are associated with a 3-fold increased spontaneous mutation frequency in the (nontranscribed) lacI gene (Fig. 3B) and translate into a 6-fold higher number of preneoplastic foci after treatment with a tumor promoter (Fig. 5). Thus, the tumor-initiating potency of the additionally accumulated oxidative mutations (mostly GC to TA transversions) seems to be as high as that of the mostly other types of spontaneous mutations, which are responsible for the preneoplastic lesions in the repair-proficient animals.

It seems unlikely that the recently shown effects of CSB on gene expression observed after UV-C irradiation (41) play a significant role for the tumor promotion by the peroxisome proliferator because the liver weight gain indicates a very similar response of Csbm/m/Ogg1–/– and wild-type mice (Fig. 2A).

According to the current knowledge of the substrate specificity of the repair glycosylase OGG1, the accumulating endogenous DNA modifications could be 8-oxoG and Fapy-G, although the recognition of other (unknown) types of oxidative purine modifications by OGG1 cannot be completely excluded. The spontaneous mutation spectrum observed in the Ogg1–/– and Csbm/m/Ogg1–/– mice (4, 5, 8) suggests that only 8-oxoG is relevant as accumulating premutagenic lesion, other substrates of OGG1 are either not generated or lack a mutagenic potential or are rapidly repaired by back-up mechanisms. The level of additional oxidative purine modifications that is actually responsible for the increased mutation frequency and the elevated levels of preneoplastic foci is probably even lower than the level of Fpg-sensitive modifications determined in the analysis because the levels have been shown to increase with age (6), and most of the mutations might have been generated early in development when the liver cell proliferation was still high. Several other factors have to be considered when the three end points are related to each other. Thus, the measured increase of oxidative purine modifications is an average for the whole genome and could be different from that in the lacI gene and the genes relevant for malignant transformation. Also, potential sources of errors in the determination of absolute levels of damage by alkaline elution must be considered, as discussed previously (28, 42). The factor by which mutation frequencies increase in response to a given number of additional unrepaired 8-oxoG residues may also differ within the genome. It is also not clear whether a given number of unrepaired 8-oxoG residues causes the same number of mutations in wild-type and in Csbm/m/Ogg1–/– mice, as would be required for an extrapolation of the observed consequences to the basal levels of Fpg-sensitive modifications observed in wild-type mice.

Despite these limitations, the results indicate a pronounced tumor-initiating potential of spontaneous mutations that result from unrepaired endogenous oxidative base modifications, in particular, 8-oxoG. Our results confirm the classic notion that additional mutations inevitably result in an increased tumor risk if promotion (in particular cell proliferation) is sufficient. Further studies may allow a comparison with other types of endogenous DNA modifications as well as with exogenously generated modifications and mutations.


   Acknowledgments
 
Grant support: Deutsche Forschungsgemeinschaft (Ep11/5). The excellent technical assistance of Karin Pauly, Lydia Weidenfeller, and Elke Zabinski is acknowledged.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
3 The defect in the Csb gene of the mice used in this study is indicated as Csbm/m rather than Csb–/– because Csb is severely truncated, but not completely missing (7). Back

Received 1/25/07. Revised 3/22/07. Accepted 3/26/07.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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