Laboratory of Health Effects Research,
1 Laboratory of Pathology and Immunobiology,
2 Laboratory of Organic Chemistry, National Institute of Public Health and the Environment, PO Box 1, 3720 BA Bilthoven,The Netherlands
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Abstract |
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Abbreviations: B[a]P, benzo[a]pyrene; DMBA, 7,12-dimethylbenz-[a]anthracene; HAA, heterocyclic aromatic amine; NER, nucleotide excision repair; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; UV-B, ultraviolet-B radiation.
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Introduction |
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2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant HAA formed during the cooking of meat and fish (3,9). When this compound was administered in the diet of rodents, colon and mammary carcinomas were found in rats (10), whereas in mice lymphomas predominated (11). Lymphoma induction was observed in C57BL/6 and CDF1 mice (11,12), which both have a relatively low spontaneous tumour incidence (1215). The lymphoma induction in mice was observed after long-term exposure. However, after short exposure periods, PhIP has been found to induce mutations in lacI and Dlb-la/b mice predominantly in the intestines (1618). Hence, mutation induction and tumour formation by PhIP do not necessarily have to target the same tissue. We were interested, therefore, to investigate the effects of PhIP in DNA repair deficient mice, because these mice are expected to be more sensitive to mutation induction.
The XPA-knockout mouse was developed as an animal model for the human disease Xeroderma pigmentosum, XP (1921). The mice lack a functional XPA protein, which is one of the components of the nucleotide excision repair (NER) pathway (19,22). Like XP patients, they are extremely sensitive to UV-light and display an increased incidence of tumours at exposed areas of the skin (19,20). Two forms of NER exist: transcription coupled repair (TCR) and global genome repair (GGR). TCR is the most rapid and efficient form of NER, it is involved in the removal of DNA lesions from actively transcribed genes, while lesions in the rest of the genome are removed by global genome repair (GGR) (20,23,24). The XPA protein is involved in both the TCR and GGR pathway, and mutations in the XPA gene, therefore, affect both forms of NER (24,25). In XPA-deficient mice, NER-specific DNA damage will thus remain unrepaired in the actively transcribed genes as well as in the rest of the genome. Upon exposure to relevant genotoxic agents, the biological effects of (enhanced levels of) DNA damage, i.e. toxicity, mutation induction and tumour development might therefore be reinforced in XPA/ mice. In addition, NER deficient XPC-knockout mice (26), which lack GGR but are still able to repair DNA damage in actively transcribed genes, may help to elucidate the relative importance of TCR and GGR in preventing toxicity and carcinogenicity.
Exposure of XPA/ mice to genotoxic agents like UV-B, 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P) has been previously shown to result in an increased tumour-incidence and shorter latency time as compared with wild-type mice (19,21,27). In addition, some cytotoxic effects were observed (19,27). Because PhIP has been shown to introduce DNA lesions that are a substrate for NER (28), it is to be expected that this compound would also evoke an increased response in XPA/ mice. In this paper we describe the effects of dietary administration of PhIP to XPA-deficient mice. PhIP was found to induce severe toxic effects in the small intestines of XPA/ mice that were not observed in wild-type or XPC/ mice. Subsequent analysis of the lacZ mutant frequencies induced by PhIP in the DNA of XPA/.lacZ double transgenic mice suggest that this may be due to increased levels of PhIPDNA adducts in intestinal cells. Furthermore, we studied the carcinogenic response of sub-toxic doses of PhIP in wild-type and XPA/ mice using three different protocols. Depending on the treatment period and observation time used, lymphomas and intestinal tumors were observed in XPA/ mice at very low doses of PhIP.
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Materials and methods |
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PhIP was synthesized by Dr J.Stavenuiter and M.Hamzink (RIVM, Bilthoven) according to the procedure of Knize and Felton (29), with some important modifications. In short, PhIP was synthesized starting from 5-phenyl-2-pyridinamine (PPA), which was prepared as described by Stavenuiter et al. (30). PPA was reacted with Br2 in a 0.5 M H2SO4 solution at room temperature. After completion, the solution was basified with 25% ammonia, extracted with ethyl acetate, washed with water and dried over anhydrous magnesium sulfate. The dried product was then mixed with 1 g CuSO4 and 180 ml 40% aqueous methylamine and reacted at 220°C in a PTFE-lined pressure bomb for 4 h. The product was extracted with chloroform, evaporated to 10% of the original volume and purified on silica gel. Finally, 2.75 g purified product was dissolved in 180 ml 25% phosphoric acid, 5 g CNBr was added and the mixture was heated in a PTFE-lined pressure bomb for 5 h at 180°C. The final product was poured over ice and extracted with chloroform. The aqueous phase was basified (with 1 M NaOH) and extracted with 1-butanol, after which silica gel was added to the butanol extract. The extract was evaporated and the residue purified further on silica gel. The identity and the purity were checked by NMR analysis and the compound appeared to be over 95% pure.
Mice
Wild-type mice were of strain C57BL/6. The XPA/ mice were generated by gene targeting in embryonic stem cells and backcrossed to C57BL/6 as described (19). The XPC/ mice (C57BL/6 background) were kindly provided by Dr E.Friedberg (University of Texas). pUR288 transgenic mice (line 60) carrying 40 tandemly integrated plasmid copies containing the bacterial lacZ gene in a C57BL/6 genetic background, were obtained from Dr J.Vijg and co-workers (31,32). Genotyping of the different mouse strains occurred by PCR analysis on tail DNA as previously described (19,33). The XPA/ and XPC/ mice used had been backcrossed into a C57BL/6 background for eight and three generations, respectively.
PhIP treatment
Administration of PhIP started when the mice were 79 weeks old. The animals received PhIP daily through the diet (ad libitum). Different PhIP containing diets of 10 (0.001%), 25 (0.0025%), 40 (0.004%), 100 (0.01%) or 200 p.p.m. (0.02%) were prepared by Altromin (Lage, Germany).
Depending on the experiment, treatment with different concentrations of PhIP lasted for 1326 weeks. Groups consisted of equal numbers of male and female mice. During the experiment, food uptake and body weights were recorded once a week. The mice were observed twice a day for any abnormalities in clinical appearance. Moribund mice were killed intercurrently and went for autopsy. Mice that survived the complete treatment period were kept on normal diets for an additional 2 weeks up to 9 months (depending on the protocol) before being killed and examined macroscopically. The following tissues were collected and fixed in 4% neutral buffered formaldehyde: liver, kidney, lung, lymphoid tissue, gastro-intestinal tract and other tissues that showed abnormalities at necropsy.
In the dose-range finding studies, treatment periods were 3 months for the doses of 40, 100 and 200 p.p.m., and 6 months for the doses of 10 and 25 p.p.m. Groups of mice consisted of 1436 animals. In the carcinogenicity studies, we employed three protocols. In the first protocol, the mice were treated during 3 months, after which an observation period was applied of 9 months (total duration time 1 year). Groups of mice consisted of 1214 animals. In the second and third protocol, all mice were treated during 6 months. In the second protocol the groups of mice consisted of approximately 20 animals, which were killed 2 weeks after termination of the treatment. In the third protocol, 714 mice were used in each treatment group, and the mice were killed after an additional observation period of 6 months (total duration time 1 year).
XPC/ mice were administered diets containing 100 p.p.m. PhIP for 13 weeks. This was done in a separate experiment, in which also XPA/ and wild-type animals were included. Each genotype was represented by eight animals (males and females). The mice were observed for changes in clinical appearance, and moribund mice were killed and autopsied intercurrently. Surviving animals were examined macroscopically at the end of the observation period.
For the lacZ mutagenesis studies, treatment periods were sustained to maximally 13 weeks. With exception of the XPA group treated with 100 p.p.m. PhIP, the groups consisted of 12 mice (six males and six females), of which four animals (two males and two females) were killed after 2, 8 or 13 weeks of treatment, respectively. XPA/ mice that were treated with 100 p.p.m. PhIP were killed after an exposure time of 1 or 2 weeks (two males and two females). Tissues from the killed mice were collected, directly frozen into liquid nitrogen and subsequently stored at 80°C.
Histopathological analysis
Formaline-fixed organs were embedded in paraffin wax. Microtomic sections (5 µm) were stained with hematoxylin and eosin and subjected to microscopic analysis.
LacZ mutation analysis
Genomic DNA was isolated essentially as described by Dollé et al. (32); tissues were homogenized in a buffer of 10 mM TrisHCl, pH 8.0, 150 mM NaCl and 10 mM EDTA. Then, SDS was added to a final concentration of 1% and the samples were treated with proteinase K and RNAse A. The samples were extracted with 25:24:1 phenol:chloroform:isoamyl alcohol. The aqueous phase was supplemented with potassium acetate (1.3 M final concentration) and extracted again with chloroform only. Subsequently, DNA was precipitated with ethanol and washed three times with 70% ethanol. Between 20 and 50 µg DNA was taken up into 73 µl with binding buffer added to a final concentration of 10 mM TrisHCl, 1 mM EDTA, 10 mM MgCl2, 5% glycerol, pH 7.5. In order to release the plasmid DNA, 2 µl (a total of 40 U) HindIII and 60 µl suspension containing lacIlacZ coated magnetic beads (prepared from 1 ml magnetic beads, 300 µg mouse anti-ß-galactosidase and 100 µl LacILacZ fusion protein) were added. The mixture was incubated for 60 min at 37°C, after which the beads plus the plasmid DNA were pelleted using a magnetic particle concentrator, washed and resuspended in 75 µl elution buffer (10 mM TrisHCl, 1 mM EDTA, 125 mM NaCl, pH 7.5). A 5 µl aliquot of 25 mg/ml IPTG solution was added to elute the plasmid DNA from the beads, and a second HindIII digestion was carried out. After inactivation of HindIII, the plasmids were recircularized by adding T4 DNA ligase, precipitated and electroporated into Escherichia coli C (lacZ/galE)indicator cells (34). [For a detailed description see Dollé et al. (32) and Vijg and Douglas (35).]
After recovery of the E.coli cells, 1/1000 part of the suspension was plated on LB medium containing X-gal to determine the rescue efficiency. The remainder was plated on medium containing P-gal to select for mutant clones. The mutation frequencies were established as (no. colonies on P-gal plate)/(no. colonies on X-gal-platex1000).
Statistical analysis
Differences in body weights and mutation frequencies were compared by standard t-tests, and differences in tumour incidence by using the Fisher's exact probability test, comparing the treated groups with the concurrent control groups.
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Results |
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The relative body weight curves of this dose-range finding study are depicted in Figure 1. For both sexes, untreated XPA/ mice showed a body weight gain comparable with wild-type mice (Figure 1a
). Also XPA/ mice treated with 10 p.p.m. PhIP (Figure 1b
) showed the same body weight gain as 10 p.p.m. PhIP treated wild-type mice, and the observed body weight gains, moreover, were similar to the untreated mice. However, XPA/ mice that were given 100 and 200 p.p.m. PhIP (Figure 1e and f
) lost weight from the beginning of the experiment, and all the mice died rapidly. Besides the loss in body weight, the animals displayed no abnormalities in clinical appearance until a few days before death. Up to this moment, the food and water intake was normal (data not shown). The loss of body weight of XPA/ mice that were administered 40 p.p.m. PhIP (Figure 1d
) started at a later stage, after they had initially gained weight. Compared with wild-type mice, however, this initial weight gain was reduced for the males, and all seven XPA/ males ultimately died. In the group of 40 p.p.m. PhIP treated XPA/ females the initial body weight gain was normal until week 8. Thereafter it also dropped and ultimately four out of seven females died within the treatment period of 13 weeks. Strikingly, the three female mice that had survived the treatment started to gain weight again after a few weeks, their body weights fully recovering to normal levels. Finally, a limited effect on body weight gain (10% reduction compared with treated wild-type mice) was still observed with male XPA/ mice that received a dose of 25 p.p.m. PhIP (Figure 1c
). Two males of this dose group also displayed intestinal abnormalities at the end of the treatment period (see next section).
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In order to investigate the PhIP-related toxicity in XPA/ mice in more detail, microscopic slides of the intestines were examined for the presence of histopathological changes. In some instances, we observed mucosal oedema of the villi and signs of increased cell division and hyperplasia in the crypts of XPA/ animals that had received 25 or 40 p.p.m. PhIP. However, obvious degenerative lesions were absent.
Effects of NER deficiency on mutation induction
Because of the DNA repair defect in the XPA/ mice, we hypothesized that the observed effects in the intestines of XPA/ mice might be related to the accumulation of PhIP-induced lesions in the intestinal DNA of these mice. LacZ mutant frequencies in intestinal, spleen and liver DNA were therefore determined in double transgenic XPA/.lacZ mice, which also carried a bacterial lacZ-reporter gene (31). The results obtained for males and females were combined because no differences in mutant frequencies were observed between both sexes at a given concentration, time point and genotype (Figure 3). The XPA/ mice displayed a dose-dependent increase in the lacZ mutant frequencies in all tissues examined at dose levels of 25 and 40 p.p.m. PhIP; however, the administration of 100 p.p.m. PhIP did not result in a further increase. The lacZ mutant frequencies in XPA/ mice were higher in intestinal DNA than in spleen or liver DNA, and were also higher than those found in wild-type mice. This seems consistent with the acute toxic effects of PhIP observed in the small intestines. The levels of lacZ mutant frequencies in spleen and liver DNA of the XPA-deficient mice were approximately similar. Induction of mutations in spleen and liver DNA of wild-type mice was much lower or even absent. The lacZ mutant frequencies in the small intestines of wild-type mice seemed to be slightly higher than those found in the spleen and liver, although the differences were not significant.
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Carcinogenicity of PhIP in XPA deficient and wild type mice
The carcinogenicity of PhIP in XPA/ mice was tested in several experiments using three different protocols. In the first experiment, the mice were treated with PhIP for 3 months and, in order to allow tumors to develop, the mice were subsequently kept on a normal diet for another 9 months. XPA knockout mice were exposed to PhIP concentrations of 10 and 25 p.p.m., since higher concentrations would have been too toxic (see above). Wild-type C57BL/6 mice were exposed to dietary levels of 25, 100 and 200 p.p.m. PhIP. The percentages of tumour bearing animals and the data on tumour types are summarized in Table I(a). Untreated XPA/ and wild-type mice display a similar spontaneous tumour incidence after the additional observation period of 9 months. The spontaneously occurring tumours observed were one lymphoma (in wild-type females) and one benign bronchiolo-alveolar adenoma (in XPA/ females). These two tumour types are the major background tumours in XPA/ and wild-type mice at around 11 months of age, as demonstrated in other studies in our laboratory (data not shown). A very low occurrence of the same two tumour types was observed in XPA/ and wild-type animals treated with 10 or 25 p.p.m. PhIP. Despite the additional two animals in the 25 p.p.m. groups (one XPA/ and one wild type), which carried a benign small intestinal adenoma, all observed tumour incidences were not statistically significantly different from the untreated controls. It was concluded, therefore, that a negative carcinogenic response was obtained in the XPA/ mice. The most overt effect was observed in wild-type C57BL/6 mice treated with PhIP doses of 100 or 200 p.p.m., which clearly evoked increased tumour incidence in males. In this case, the number of tumour bearing animals as compared with the untreated controls was statistically significant (P < 0.05) for the 100 p.p.m. group or approached statistical significance (P = 0.07) for the 200 p.p.m. PhIP group. The majority of the tumours induced were found to be lymphomas. It should furthermore be noted that by combining incidences of lymphoma-bearing male plus female mice the data are statistical significant (P
0.05) also for the 200 p.p.m. PhIP group. The lymphomas were either found in the mesenteric lymph nodes or in the Peyers Patches in the small intestines, or were multicentric, affecting the mesenteric lymph nodes, spleen and other lymphoid tissues.
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Discussion |
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While PhIP has been reported to induce lymphomas in wild-type mice at dietary concentrations of 300 to 400 p.p.m. (11,12), our results show that this compound causes acute and severe toxic effects in XPA/ mice at dietary concentrations of 40200 p.p.m. These effects consist of body weight reduction, starvation and mortality. Enhanced toxicity of PhIP in XPA/ mice has also been recently reported by Imaida et al. (36). However, these authors reported that they could not detect a specific cause of death after administration of (toxic) dietary PhIP concentrations (4080 p.p.m.), nor could they detect any carcinogenic responses. From our experiments presented in this paper, it appeared that PhIP presumably impairs proper functioning of the small intestines of XPA/ mice. At necropsy this was manifested as obstipation and dilatation of the small intestines and caecum.
PhIP is taken up by the small intestines, and there are indications that the intestinal cells of mice are able to metabolize heterocyclic aromatic amines. In any case, these compounds are transported to the liver where they are converted into their N-OH intermediates, which are released (via the bile) back into the gut. Subsequently, these intermediates will be converted into ultimate mutagens/carcinogens, capable of binding to the DNA in intestinal cells (3742). In this way, it is possible that the intestines will be one of the first target tissues in a row to acquire increased levels of PhIPDNA adducts, which cannot be removed in XPA/ mice. High levels of PhIPDNA adducts may induce cell death (apoptosis), which might be counteracted by renewal of lost cells via division of stem cells (in the crypts). However, proliferating cells that are still carrying DNA lesions will acquire mutations into their genome. Therefore, both the induction of cell death and the accumulation of gene mutations may ultimately impair the proper functioning of the intestinal cells. Morphological changes that are indicative for degenerative processes, however, were not observed. Also, the biological relevance of the oedematous villi in the XPA/ mice is not clear.
Evidence that the toxic effects were indeed caused by PhIP-induced DNA damage was obtained from the lacZ gene mutation analysis of the DNA from three different tissues. For this purpose, the XPA-deficient mice were crossed with transgenic mice carrying the bacterial lacZ reporter gene. LacZ mutant induction is assumed to take place when the DNA lesions present are being replicated during cell division. As shown in Figure 3, lacZ mutant induction appeared to be selectively increased in the intestinal DNA of XPA knockout mice that had received 25 to 100 p.p.m. PhIP. The increase in LacZ mutant frequencies was higher in the intestines than in the liver or spleen of these mice, and furthermore appeared to be dose-dependent. This is consistent with literature data which showed that PhIP also induced the highest mutation frequencies in the intestines of Dlb-1a/b mice or the Muta mouse (43,44). Although both mouse strains are NER-proficient, elevated mutation frequencies could still be detected shortly after oral administration of high (20 mg/kg bw = 400 p.p.m.) doses of PhIP. These doses were 4-fold higher than the maximum dose we used (100 p.p.m.), which might explain the absence of a detectable lacZ mutant induction in the tissues of wild-type mice we have examined.
The results of Figure 4 indicate that the PhIP-induced toxic effects in XPA-deficient mice might be linked to the absence of transcription-coupled repair, since such effects were not found in XPC knockout mice. This suggests that a defect in the removal of PhIPDNA lesions from actively transcribed genes (needed for house-keeping or tissue-specific functions) plays a crucial role in the observed intestinal toxicity. The stalled RNA polymerases on active genes could have induced cytotoxic or apoptotic reactions. However, the observed distension of the intestines in the absence of degenerative lesions is more indicative for an impaired function (probably related to motility) of the intestines.
We also investigated the carcinogenic response at sub-toxic PhIP doses in XPA-deficient mice and compared this to the response in wild-type C57BL/6 mice. In wild-type mice, treatment with doses of 100 and 200 p.p.m. PhIP for a period of 3 months was found to increase the lymphoma incidence in the males after an additional observation period of 9 months [Table I(a)]. This is consistent with literature reports for wild-type C57BL/6 male mice treated with 300 p.p.m. PhIP for longer periods of time (12,15). Under the same conditions (3 months exposure, 9 months observation), doses of 10 and 25 p.p.m. PhIP did not result in a statistically significant increase in tumour incidence in XPA/ mice. However, when the mice were treated for 6 months with an additional observation period of 6 months, then the same dose levels were found to enhance the carcinogenic response in XPA/ mice [Table I(c)
]. This is in contrast to the previously reported lack of tumorigenicity of PhIP in XPA/ mice (36), and may be due to the differences in dose regimens employed. Moreover, we also found benign adenomas and malignant adenocarcinomas in the intestines of XPA/ mice at the dose level of 25 p.p.m. PhIP. These intestinal tumours were not found in the untreated controls, and are also relatively rare in untreated mice of the same strain, as observed in related experiments in our laboratory. Therefore, the formation of this type of tumour seems related to the PhIP exposure and to the dose regimen used.
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Notes |
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Acknowledgments |
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References |
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