The relationship between 1,2-dimethylhydrazine dose and the induction of colon tumours: tumour development in female SWR mice does not require a K-ras mutational event
Peta E. Jackson1,2,
Donald P. Cooper1,3,
Peter J. O'Connor1 and
Andrew C. Povey1,5
1 CRC Section of Genome Damage and Repair, Paterson Institute for Cancer Research, Manchester M20 9BX, UK
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Abstract
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In this study we have investigated the relationship between the dose of 1,2-dimethylhydrazine (DMH) and the yield (and location) of tumours in a mouse strain susceptible to colon tumour induction. Female SWR mice were injected with 6.8 mg/kg DMH i.p. once a week for 1, 5, 10 and 20 weeks and the animals were followed for almost 2 years. Administration of increasing doses of DMH resulted in a dose-dependent decrease in survival time. Colon tumours developed in 26, 76 and 87% of mice given a total dose of 34, 68 and 136 mg/kg DMH, respectively: no tumours were detected in animals treated with a total dose of 6.8 mg/kg. Most colon tumours (79%) were located in the distal colon with the remainder being found in the mid colon and none were detected in either the proximal colon or small intestine. As mutations in the K-ras gene are thought to be key events in the pathogenesis of human and rodent colon tumours, we determined the frequency of codon 12 and 13 K-ras mutations in these tumours by restriction site mutation analysis and/or DNA sequencing. A total of 50 colon tumour samples were analysed for codon 12 mutations and of these 29 were also screened for codon 13 mutations. No mutations were detected in either of these codons. The mutational activation of the K-ras gene is not an essential step in the development of DMH-induced colon tumours in female SWR mice and if similar considerations apply to humans, then the aetiological role of alkylating agents may be underestimated from the prevalence of K-ras GC
AT transitions in human tumours.
Abbreviations: AOM, azoxymethane; DMH, 1,2-dimethylhydrazine; O6-MeG, O6-methylguanine; NMU, N-methyl-N-nitrosourea; PBS, phosphate-buffered saline; RSM, restriction site mutation.
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Introduction
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The colon carcinogen 1,2-dimethylhydrazine (DMH) has been widely used to study chemically induced colon cancer. Regardless of the mode of administration, DMH specifically induces tumours within the descending colon and the histopathology is similar to that observed for human sporadic colon tumours (13). DMH alkylates DNA and the pro-mutagenic lesion O6-methylguanine (O6-MeG) has been detected in DNA from various rat (4) and mouse (5,6) tissues following exposure to DMH. O6-MeG is known to induce GC
AT transitions in vitro and in vivo (7,8) and this was the only mutation induced in Escherichia coli and Salmonella typhimurium following DMH exposure (9). GC
AT transitions were also preferentially induced in the lacI gene of E.coli recovered from the livers of Swiss mice exposed to DMH or its metabolites (10). In addition, GC
AT mutations have been detected in the K-ras protooncogene in DMH- and azoxymethane (AOM)-induced aberrant crypt foci and tumours of rats (11,12) and AOM-induced aberrant crypt foci of mice (13). Overexpression of the gene for the human DNA repair protein O6-alkylguanine-DNA alkyltransferase has a significant protective effect on the induction of both aberrant crypt foci and K-ras mutations, suggesting that the formation of O6-MeG is a key event in the development of these lesions (13).
The relevance of alkylating agent exposure to human colon cancer, however, remains to be determined. We have shown that O6-MeG is present in DNA from human colorectal tissue (14,15) and others have found that K-ras mutations commonly occur in human colon tumours, with ~40% of tumours harbouring a mutation in the ras gene (16,17). The most common of these alterations is a GC
AT transition at the second base of codon 12 or 13 of the K-ras gene which is consistent with exposure to a chemical carcinogen such as an alkylating agent (18). We have previously found no evidence of an association between current exposure to alkylating agents and the presence of a K-ras GC
AT transition (14) but individuals with low O6-alkylguanine-DNA alkyltransferase activity in normal colorectal tissue were at increased risk of harbouring a transition (GC
AT) (but not a transversion) mutation in the K-ras gene (19). This suggests that alkylating agent exposure may play a role in the aetiology of colorectal tumours containing a GC
AT transition in the K-ras oncogene.
The purpose of this work was thus two-fold: (i) to develop an animal model to examine the relationships between exposure to an alkylating agent (DMH) and colorectal cancer risk; (ii) to determine the extent to which the induced tumours contained K-ras mutations at codon 12 or 13. DMH.2HCl was dissolved in 0.9% saline containing 1 mM EDTA and 10 mM sodium citrate and the pH adjusted to 6.5 using 0.25 M NaOH. Female SWR mice (89 weeks old), obtained from either the Paterson Institute colony or purchased from Harlan Olac (Blackthorn, Bicester, UK) were given 1, 5, 10 or 20 weekly i.p. injections of 15 mg/kg DMH.2HCl (6.8 mg/kg DMH) and body weights were recorded weekly. Animals were housed under normal conditions until they showed signs of discomfort, at which time they were killed by cervical dislocation.
All mice were examined grossly at necropsy. The entire intestine from the stomach to the anus was removed and the large bowel (from the caecum to the anus) was isolated. It was then opened longitudinally, washed with phosphate-buffered saline (PBS) and the mucosal surface examined for gross pathology. Any lesions detected were measured, their location noted and dissected. A portion was taken for histological examination after fixation in 4% (v/v) formalin in PBS overnight. The remainder of the lesion and a section of macroscopically normal colon tissue was frozen on dry ice and the samples stored at 70°C to await DNA extraction. All other organs were examined for gross pathology and any areas of abnormality were also taken for histological examination. Samples of the liver, kidney, spleen, lung, small intestine and caecum were routinely taken and fixed for histology. These experiments were performed in accordance with the Animals (Scientific Procedures) Act of the UK Parliament, 1986.
Administration of increasing doses of DMH had no effect on the increase in body weight of the mice as compared with untreated animals (data not shown) but resulted in a dose-dependent decrease in survival time (Figure 1A
) and a dose-dependent increase in the number of mice with colon tumours (Table I
) and deaths from causes manifested as rectal bleeding/anal cysts (Figure 1B
). At the highest dose, the average survival time (death from all causes) was, at 320 days, ~60% that of the mice that had received no DMH (546 days; Table I
). The tumours generally appeared to be classic differentiated adenocarcinomas (Figure 2
). Of the animals that received the highest dose (136 mg/kg), 87% developed tumours and this is comparable with an earlier report that 83% of SWR mice treated with a total dose of 300 mg/kg developed colon tumours (20). At a dose of 34 mg/kg DMH, 26% of the mice in this study developed colon tumours whereas it has been reported that no tumours developed in female CF1 mice at a similar dose (30 mg/kg; 21). One control mouse, but none of the mice that received 6.8 mg/kg DMH, developed a colon tumour.

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Fig. 1. Survival analysis for mice treated with increasing doses of DMH. (A) Analysis for all causes of death. (B) KaplanMeier survival curves using death from causes manifested as rectal bleeding/anal cysts as the outcome. Increasing DMH dose resulted in a statistically significant difference in survival times (P < 0.0001, log-rank test).
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Fig. 2. Adenocarcinoma of the distal colon induced in a mouse given a total dose of 136 mg/kg DMH. Magnification x25.
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Other lesions were also apparent in the mouse tissues (Table I
). The frequency of anal cysts increased with increasing DMH dose, suggesting a direct relationship with administered DMH. In contrast, the frequency of lung lesions showed no relationship with DMH dose whereas the frequency of ovarian cysts decreased with dose, possibly because mice treated with no or 6.8 mg/kg DMH lived longer (Table I
), so allowing the ovarian cysts and lung lesions to develop.
DMH-induced tumours were located predominantly in the distal colon (119/151, 79%) although some were observed in the mid-colon (32/151, 21%; Table II
). No tumours were observed in the proximal colon or elsewhere in the intestinal tract. These results are similar to those observed previously in mice by other investigators (2024). Many of the animals had more than one tumour (up to 11 per animal) of various sizes (ranging from ~1 to 8 mm in diameter) extending from mid-way along the colon to the anal margin. Multiple tumours occurred in 85% (22/26) of the tumour-bearing mice given 136 mg/kg DMH (Table II
). The number of distal colon tumours per tumour-bearing animal was strongly associated with DMH dose (P < 0.001) but not the number of mid-colon tumours (Table II
). In the distal colon but not the mid colon, the size of the tumours was also strongly associated with DMH dose (Figure 3
). These observations that the numbers and sizes of distal colon tumours, but not mid colon tumours, were strongly associated with DMH dose may reflect target cell sensitivity. However, a further study with larger numbers of animals would be required to provide a more definitive answer. The specific location of the tumours within the colon, however, provides a model which is similar to that seen following the chronic administration of N-methyl-N'-nitro-N-nitrosoguanidine when tumours are found predominantly within a specific region of the stomach, i.e. the pylorus (25).

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Fig. 3. Variation in size of tumours in the (A) distal colon and (B) mid colon with increasing DMH dose. In the distal colon, the proportion of large tumours increased significantly with increasing DMH dose (P < 0.001 using a 2 test for trend). In the mid colon there was no statistically significant difference between the proportion of tumours with differing sizes at different DMH doses (68 versus 136 mg/kg, P > 0.1).
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The molecular mechanisms involved in DMH-induced murine tumours have yet to be elucidated. However, as it has been reported that mutations in K-ras codon 12/13 have been detected in lung tumours induced in SWR mice by 3-methylcholanthrene or 3-methylcholanthrene and butylated hydroxytoluene (26), we quantified the frequency of K-ras codon 12 and 13 mutations in mouse colon tumours that developed following DMH administration. High molecular weight genomic DNA was prepared from tumour and normal tissue by conventional phenol/chloroform extraction and ethanol precipitation. Tumour tissue was obtained by macroscopic dissection of the tumour from surrounding normal tissue: to reduce the possibility of contamination with normal tissue only larger tumours that were protruding into the lumen were analysed. K-ras mutations in the DNA from tumours or normal tissue were detected by restriction site mutation (RSM) analysis as described previously (14) with the exception that a different 3' primer was used and the PCR conditions were therefore modified. The sequences of the primers employed were 5'-GA ATATAAACTTGTGGT CCATGGAGCT-3' (5' primer, K-ras exon 1, codons 311, bold denotes mismatches with the mouse sequence) and 5'-GTCCTGAGAAGCAGCGTTAC-3' (3' primer, K-ras exon 1, codons 3945). The 5' primer has three mismatches designed to create a BstXI site (CCAN6TGG involving codon 12) and an XcmI site (CCAN9TGG) involving codon 13. An additional mismatch was present towards the 5'-end of the primer. Amplification was performed for 30 cycles at 94, 55 and 72°C for 30 s in a buffer containing 50 mM KCl, 10 mM TrisHCl, pH 8.8, 5 mM MgCl2 and 0.1% Triton X-100 using Taq polymerase (Promega, Madison WI). Restriction endonuclease digestion of PCR-amplified material was carried out using either BstXI (Promega) or XcmI (New England Biolabs, Beverly, MA) as previously described (14) and each sample was analysed twice. Such RSM procedures can detect one mutant allele in the presence of at least 100 normal alleles (27). DNA was sequenced using a Sequenase v.2.0 kit (Amersham International, Aylesbury, UK) and after initiation using a primer (5'-CTCTATCGTAGGGTC-3') which annealed at a site towards the 3'-end of the template.
DNA extracted from colon tumours isolated from mice treated with 136 mg/kg DMH was then analysed for mutations in codons 12 and 13 of the K-ras gene by RSM analysis. No mutations were detected in codon 12 of the K-ras gene in 41 colon tumour samples and, similarly, no mutations were detected in codon 13 in 20 samples. In addition, macroscopically normal colon samples from 13 animals were analysed for K-ras mutations and none were detected. As these results were uniformly negative, the DNA sequence of exon 1 of the K-ras gene of 19 colon tumour samples (including 10 previously screened by RSM analysis) was determined and no base changes were identified. As mutations in the H-ras gene are also implicated in some human and animal tumours (16,18), we also examined six tumour samples for codon 61 mutations by sequencing PCR products generated by amplification using primers and conditions described previously (28). Again, no mutations were detected.
Our results thus show unambiguously that mutational activation of the K-ras protooncogene is not a critical factor in the development of DMH-induced colonic neoplasms in female SWR mice. Mutational inactivation of the p53 gene also does not appear to be of critical importance as a previous study reported that only two out of 19 tumours contained mutations (29). In contrast, the K-ras gene has been found to be mutated in rats following treatment with DMH, AOM or N-methyl-N-nitrosourea (NMU) at frequencies ranging from 29 to 66% (Table III
) and no p53 mutations have as yet been identified (30,31). Alkylating agents can induce tumours in experimental animals, therefore, through mechanisms other than the formation of K-ras or p53 mutations; other potential mechanisms may involve mutations in the mismatch repair genes or the APC gene (32).
Humans are unavoidably exposed to methylating agents and we ourselves have shown that the human colon contains detectable levels of O6-MeG (14,15) but whether such agents cause human cancer is unknown. The presence of a specific mutational fingerprint would therefore help to determine whether alkylating agents are specific risk factors for certain cancers. K-ras codon 12/13 GC
AT mutations are generally thought to be a specific marker of alkylating agent exposure. However, the absence of these mutations in this mouse model indicates that this mutational event is not necessarily a sensitive marker of exposure. Furthermore, it has been shown that the prevalence of K-ras mutations in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-initiated lung tumours decreases when these tumours are promoted by butylated hydroxytoluene in the diet (37). If similar considerations apply to humans, then the aetiological role of alkylating agents may well be underestimated from the prevalence of K-ras GC
AT transitions in human tumours.
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Acknowledgments
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This work was supported by the Cancer Research Campaign, UK.
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Notes
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2 Present addresses: Department of Environmental Health Sciences, School of Hygiene and Public Health, Baltimore, MD 21205, USA and 
3 Micromass UK Ltd, Floats Road, Wythenshawe, Manchester M23 9LZ, UK 
5 To whom correspondence should be addressed at: School of Epidemiology and Health Sciences, Medical School, University of Manchester, Oxford Road, Manchester M13 9PT, UK 
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Received June 22, 1998;
revised November 26, 1998;
accepted December 1, 1998.