FABAD J. Pharm. Sci., 24, 67-73, 1999
RESEARCH ARTICLES /BİLİMSEL ARAŞTIRMALAR
Analysis of 7,12-dimethylbenz[a]anthracene Induced Mutations by the Use of Restriction Site Mutation Technique in the Rat
Sinan SÜZEN°*, James M. PARRY**
Analysis of7,12-dimethylbenz[a]anthracene Induced Mutations by the Use of Restriction Site Mutation
Technique in the Rat
Summary : The restriction site nıutation (RSM) technique was applied as a nıethodology for the detection of base changes in restriction enzyme recognition sequences. Rats were exposed to 7,12-dimethlybenz[a]anthracene (DMBA) in two different doses. Mutations were analyzed in a variety of restriction enzyme recognition sequences of the rat p53 and ras genes. Mutations were detected in the Bsll recogni- tion sequences (CCTGTCCTGGG) in the high dose DMBA treated rats. DNA sequences aııalyses revealed that the in- duction of nuıtation was G--7A transition at the tenth hase of the CCTGTCCTGGG sequences. The developed method al- lows the detection of base pair substitutions, snıall insertions or deletions within the restriction enzyme recognition site in any gene far which the DNA sequence is known without se-
!ection of mutanı phenotype.
Key words: Restriction site nıutation assay, DMBA, ras, p53.
Received 6.1.1998 Revised : 21.04.1998
INTRODUCTION
Polycyclic aromatic hydrocarbons (PAH) represent a very important class of chemical carcinogens. The history of the detection, synthesis and biological study of PAH is closely related to the establishment of the carcinogenicity of coal tar. The PAHs have long been a concern as a potential human health hazard, since many members of this class are tu- mour initiators or promoters or mutagenic and/ or tumorigenic in vivoand in vitro1~3.
7,12~dimetilbenz[a]antrasen ile İndüklenmiş
Mutasyonların Sınırlı Bölge Mutasyon Tekniği Kullanılarak Analizi
Özet : Sınırlı bölge mutasyon (RSM) tekniği, restriksiyon enzim tanıma dizilimlerindeki baz değişimlerinin sap-
tanmasında bir metod olarak uygulandı. Sıçanlar iki farklı
dozda 7,12-dimetilbenz[a}antrasene (DMBA) nıaruz bı
rakıldılar. Mutasyonlar sıçan p53 ve ras genlerinde ÇCf'itli restriksiyon enzim tanıma dizilinılerinde analiz edildi. Yük- sek dozda DMBA'ne maruz bırakılmış sıçanlardaki Bsll ta-
nıma diziliminde mutasyonlar saptandı. DNA diziliş analizi
indüklenmiş mutasyonun CCTGTCCTGGG dizilimindeki onuncu bazda G--,\A düz baz değişimi olduğunu gösterdi.
Geliştirilen metod, DNA dizilimi bilinen herhangi bir gende mutant fenotip seçimine gerek duyulmadan restriksiyon enzim tanıma bölgesindeki baz değişimlerinin, az sayıda baz eklenme/erinin veya çıkmasının saptanmasına olanak sağ
lar.
Anahtar kelimeler: Sınırlı bölge mutasyon, DMBA, p53, ras.
The major routes of environmental exposure to PAHs are via the gut after ingestion of contaminated food or water, through the lungs by inhalation of aerosols and/ or hydrocarbon-absorbed particles and through the skin by direct contact with the hy- drocarbons. Combustion, pyrolysis and pyrosyn- thesis of organic matter result in the formation of PAHs. Such thermal degradation products have been found in trace amounts in tobacco and mar- ijuana smoke, polluted air, some food and drinking water4 .
* Department of Toxicology, Faculty of Pharmacy, Ankara University, Tandoğan-06100, Ankara, Turkey.
** School ofBiological Sciences, University ofWales Swansea, Swansea, UK.
° Correspondence
The principal interest in hydrocarbon metabolism arose from the realization !hal hydrocarbons, like many environmental carcinogens, were chemically unreactive and that their adverse biological effects were probably mediated by electrophilic me- tabolites capable of covalent interaction with DNA5,6. Formation of these reactive metabolites from PAH requires metabolism by mixed-function oxidases and related enzyınes. These enzymes have been classified into two broad categories (phase 1 and phase JI enzyrnes) on the hasis of the types of reaction they catalyze7. PAH carcinogens are be- lieved to initiate the carcinogenic process through a mutagenic mechanism dependent upon interactions of dihydrodiol epoxide rnetabolites with DNA.
7,12-dimethlybenz[a]anthracene (DMBA) is one of most patent carcinogenic polycyclic arornatic hydro- carbons, and can induce skin, lung and mammary tumours in experimental animals819. This carcinogen, like other P AHs, requires metabolic activation by the mixed-function oxidases and related enzymes be- fore it can induce malignant cell transformation and mutagenic activities8,ID,n DMBA is rnetabolized in mammalian cells through several steps involving both the cytochrome P450 mono-oxygenases and epoxide hydrase to highly reactive, mutagenic and tumorigenic diol epoxide derivatives12.
The restriction site mutation (RSM) assay or re- striction fragment length polymorphism / poly- merase chain reaction (RFLP /PCR) is a mutation detecting method which can be applied in anirnals or in cell cultures exposed to chemical or physical mutagcns13_ Most currently available rnutation sys- tems for the analysis of rnutations depend upon the isolation of a few mutated cells with a selectable mutant phenotype. Thus, these systems are limited to detecting mutations in a few genes only. The RSM assay can theoretically be applied to the de- tection of mutations in any gene for which the DNA sequer,:.ce is knovvn without the selection of a mu-
tanı phenotype. This assay is based upon the iso- lation of resistant restriction enzyme recognition se- quences by using restriction enzymes and arnplifica- tion of surrounding sequences With PCR. The prin- cipal steps of the RSM assay are outlined in Figurel.
The RFLP /PCR was used to measure mutations in the Pvu!I and Mspl restriction enzyme recognition
sequences in the hurnan c-Ha-ras 1 genel4_ in 1992, Sandy et al., applied the technique to analyze single base pair mutations in the Taql endonuclease rec- ognition sequence of the human c-H-ras 1 gene15_
RFLP /PCR was alsa used to detect oxy-radica[16 and ultraviolet B light induced rnutations in the p53 gene hurnan fibroblasts17_
Laboratory animal (Rat)
l
DMBA treatrnent ( 4 and 16 µg/kg of DMBA, i.p.)Isolation of genomic DNA frorn a variety of tissues
l
Restriction endo- nuclease (RE) digestionPCR amplification of mutant sequences
l
RE digestion RSM product analysis by polyacrylarnide gel electrophoresisı
Direct DNA sequence analysis of resistant RSM products
Figure 1. Basic steps of the RSM assay analysis in the DMBA treated rats.
The RSM assay was applied in vivo to the detection of induced mutations in BsiLI site of the al-globin gene of Xenopus leavis18. The other published in vivo application of the assay was the detection of N- ethyl-N-nitrosourea (ENU) induced rnutation in var- · ious restriction enzyme sequences in the p53 and a- haemoglobin genes of the rnouse19_
Our previous results established the RSM assay fea- sibility for direct acting genotoxins20_ in this study, DMBA was chosen as a model compound for me- tabolically activated genotoxin to investigate the po- tential of the method for use in the rat. The RSM technique was performed on two important classes of gene mutations which can cause transformation.
These were H-ras and K-ras oncogenes and p53 hı
mour suppressor gene. The induced DNA lesions by DMBA and genotoxic effects of this compound were analyzed by the RSM assay.
FABAD J. Pharnı. Sci., 24, 67-73, 1999
MATERIALS and METHODS Experimental animals
The RSM assay was performed on rats. The low dose21 DMBA treatment experiment was carried out in the Laboratory for Carcinogenesis and Mutagene- sis, National lnstitute of Public Health and Environ- mental Protection, Bilthoven, The Netherlands.
Groups of 2 rats were exposed to 4 µg (2 rats) and 16 µg (2 rats) of DMBA in 5% ethanol in tricapryline (ETC) per kg body weight, by i.p. injection. The con- trol animal was given ETC. All animals were sac- rificied 7 days later, and the rats were dissected, the brain, kidney, liver, pancreas, spleen, testis and gra- n11lorna pouch tissue were rernoved and stored at -70°C until DNA extraction.
DNA extraction
Genomic DNA was extracted from control and treated animals by a rnodified high salt method22 using a DNA extraction kit (Strategene). The con- centration of each DNA sample was quantitated by UV spectroscopy. The extracted DNA purity was deterrnined from the absorbance ratio at 260 and 280 nm.
Restriction enzyme digestion
Af ter obtaining the absorbance ratio (A260 / A280) between 1.7 and 2.0 far each sarnple, resh·iction en- donuclease digestion was carried out on the DMBA treated and control animals.
1 µg of aliquots of genomic DNA were digested in a reaction mixture containing 1 X amplification buffer (10 mM Tris-HCI pH 8.5, 50 rnM KCI, 1.5 mM MgCl2, 0.1 % Triton X-100) and 10 or more units of restriction endonuclease at the appropriate tem- perature overnight (16 hours). The final volume was adjusted to 20 µl with sterile water. Restriction en- donuclease digestion was stopped by heating the re- action mixture at 90°C for 5 min.
PCR amplification
The digested mixtures were amplified in 50 µ! re- action using 1.25 U Taq polymerase, 1 X amplifica- tion buffer, 20 pmol of each primer, and 200 µM of
each dNTP. PCR was carried out using the fol- lowing thermocyle protocols: initial denaturation far 2 minutes, followed by 25 or 35 cycles at 94°C far 1 minute, annealing for 20 seconds, and extension at 72°C for 20 seconds. Af ter amplification, 17 µl of the PCR products were subjected to a second round of restriction endonuclease digestion in 20 µl reaction
mixhıres containing 1 X restriction enzyme buffer at the appropriate temperature overnight. 10 µl of the RSM products were mixed with 2 µl loading buffer (50% v/v glycerol, 0.1 rnM Na2EDTA pH 8.0, 1% w/
v SDS, 1 % brornophenol blue) and electrophoresed in 1 X TBE buffer (0,089 mM Tris-HCl, 0.089 M Boric acid, 0.002 M EDTA, pH 8.0).
Polyacrylamide gel electrophoresis (P AGE) pur- ification of resistant bands
The rernaining RSM products (10 µl) containing re- sistant restriction endor.uclease r·ecognitiol'. se- quences were resolved by 6% polyacrylamide gel and stained with ethidium bromide (final concentra- tion 0.5 µg/ml) in 200 ml 1 X TBE buffer for 30 min- utes.
After staining, the gels were washed with distilled water and placed on a UV transilluminator. The re- sistant bands were cut out of the gel and slices of the gel were inserted into a Spin-X hıbe (Costar). The re- sistant sequences were separated from the gel slice by centrifugation after placing the tube in a freezer at -20°C for 30 minutes.
Amplification of resistant sequences and direct sol- id-phase purification
The PAGE purified RSM assay products were di- luted with sterilised water. 10 µl of diluted products were then amplified in 100 µl PCR reactions using 2.5 U Taq polymerase, 1 X amplification buffer, 200 M each dNTP and 20 pmol of each primer of which the antisense primer had been biotinylated at the 5' ter- minus. PCR was performed initial with de- naturation at 94°C for 2 minutes, followed by 20 cy- cles of 94°C 1 minutes, at the approp!iate annealing temperature for 20 seconds and at 72°C lor 20 sec- onds. After amplification, 40 µl of the biotinylated products were purified using Dynabeads M-280 Streptavidin (Dynal, Norway) to separate the bio- tinylated strands from the non-biotinylated strands
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and excessive primers, nucleotides and other PCR reaction components. The immobilised strands were resuspended in 7 µl of sterilised water.
DNA sequencing
The single-stranded templates were sequenced with the Sequenase 2.0 DNA Sequencing Kit (United State Biochemicals, USA) according to the did- eoxynucleotide chain termination method23 using nucleotides labelled with 355. Autoradiographic de- tection of the sequences was performed using Hy- perpaper355 sequencing film (Amersham). Gels were exposed to film in X-ray casettes lor 24-48 hours at room temperature.
RESULTS
The RSM analyses were performed on the genomic DNA extracted from brain, liver, kidney, pancreas, spleen, testis, and granuloma pouch tissue of the control and 4 DMBA treated animals. The assay was carried out using 13 restriction endonucleases locat- ed in the exon 5 (AluI, Dde!, Ncol, Nla!V), 6 (Mspl, Ddel), 7 (Esli), and 8 (Esli) of the rat p53 gene; and exon 1 ( Cfol, Hindili) and 2 (Hinfl, Cfol) of the rat H-ras gene; and exon 2 (NJa!V) of the K-ras gene.
A total 455 RSM assays with 91 assays from the con- trol and 364 assays from treated animals were per- formed using 13 restriction endonucleases in the p53 and ras genes. No resistant restriction endo- nuclease recognition sequences were detected in the untreated control tissue samples. Of 13 restriction endonuclease recognition sequences analyzed, DMBA induced mutations were detected only at the Bs.II. restriction endonuclease recognition site of the p53 gene. 5 resistant restriction endonuclease rec- ognition sequences were observed in the exon 8 of this gene. 2 granuloma pouch tissues, 2 spleen and 1 pancreas gave mutated RSM products in the exon 8 segment. None of the these resistant products were in the low dose DMBA (4 µg/kg) group. All re- sistant restriction endonuclease sequences were de- tected at the 16 µg/kg dose of the DMBA group.
DNA sequencing analysis revealed that the resistant restriction endonuclease sequences detected at the Esli recognition site were G to A transition. These mutations were detected in the DNA extracted from
pancreas, spleen, and granuloma pouch tissue. The base changes detected in the resistant restriction en-
zyme site are given in Table 1.
Table 1. DNA sequence analysis of resistant re- striction endonuclease recognition site in the p53 gene.
Exon Tissue Restriction endonucleases and Mutation Their recognition sequence
8 Spleen Bsll CCN7GG G to A 8 Spleen Bsll CCN7GG G to A
8 Pancreas Bsll CCN7GG GtoA
8 G. pouch Bsll CCN7GG GtoA
8 G. pouch Bsll CCN7GG GtoA
DISCUSSION
The aim of the study was to detect mutations in the restriction endonuclease recognition sequences us- ing RSM assay. The assay was performed on the rat genomic DNA extracted from DMBA treated an- imals. The RSM analysis was conducted using 13 re- striction endonucleases in the p53 and ras genes.
Resistant restriction endonuclease recognition se- quences were detected in the one restriction enzyme recognition site out of 13 restriction endonucleases.
The RSM analysis of the p53 gene sequences de- tected DMBA induced mutations at the Esli re- striction endonuclease recognition site of the exon 8 segment. 5 resistant RSM products were observed in the total of 364 assay products. No resistant re- striction endonuclease recognition sequence was ob- served in the DNA from the untreated control an- imal tissues in a total 91 RSM assay products. DNA sequence analyses revealed !hat the induction of mutations at the Esli restriction endonuclease rec- ognition site were G to A transitions. These muta- tions were detected in the DNA from granuloma pouch tissue, pancreas, and spleen.
P AHs are metabolically activated to e]ectrophilic species that bind covalently to nucleophilic sites in DNA and formed adducts whlch may occur at a number of sites within the DNA molecule. However, adduct formation involves specific electronic and stereochemical factors24. It is reported that guanine bases in DNA are the predominant sites lor attack by chemical carcinogens and P AHs prefer the N2
FABAD J. Pharm. Sci., 24, 67-73, 1999
position of guanine25. Diple et al. have demonstrat- ed that experiments involving prelabelling of cel- lular DNA with carbon 14-labelled bases, suggest that the DMBA reactive metabolites bind extensive- ly to both guanine and adenine residues in DNA26.
in this study the G to A transitions faund in the . DMBA induced mutations are consistent with the ability of DMBA and other PAH to interact ex- tensively with guanine residues27. The other ex- planation of this type of mutation is that the rec- ognition sequences of the Bsl! (CCTGTCCTGGG) has no A residue and the other restriction endo- nucleases employed in the RSM assay are G:C rich (68.83%) and thus limited the ability of the assay to detect A to T mutations. Fong et al, have also shown that K-ras gene mutations in hepatic tumours from rainbow trout exposed to DMBA contain G to A transitions at codon 1228. The identified mutations were observed only in the Bsl! restriction endo- nuclease recognition sequence in exon 8 out of a to- tal of 8 restriction endonucleases in the p53 gene.
This indicates that this sequence may be a highly se- lective target far DMBA. No resistant RSM products were observed within the other restriction endo- nuclease recognition sequences. This could be due
to the factors that affect the mutability, such as loca!
chromatin structure and sequence context, its rep- lication schedule and the repairability of the mut- agen-induced lesions. The fact that the induced base substitutions are G to A transitions in DMBA treat- ed animals suggest that N2-7,12-dimethylbenz [ a ]anthracenediol-epoxide-deoxyguanosine adducts are responsible for these mutations.
,
Although DMBA induced mutations were detected in pancreas, spleen and granuloma pouch tissue in this study, the techniqiıe failed to detect mutations in the other tissues. This may be a consequence of the capacity far repair of the other tissues, which may efficiently remove the,- DMBA-induced DNA damage. This tissue difference may alsa be a func- tion of the celi division rates of the pancreas, spleen and granuloma pouch tissues, which are higher than those of the other tissues and may fix DNA damage as rnutations before repair can occur.
A problem which has been associated with the RSM assay is the possibility !hat restriction endonuclease recognition sequences resist;ınt to digestion may
arise in vib:o due to misincorporation by Taq poly- merase over adducted site~ in the extracted genomic DNA. There is evidence that DNA synthesis can be terminated when DNA polymerase encounters such DNA modifications by either 1) inability of the poly- merase to insert a nucleotide damaged base through a change in the DNA confirmation at the lesion site29,30 or 2) the removal of an inserted nucleotide due to the 3'-5' exonuclease activity of the poly- merase31. The inhibition of the polymerases 3' S proofreading activity could therefore enable !he en- zyme to bypass certain lesions, thereby inducing er- rors in the synthesis leading to mutations. The use of a non-proofreading enzyme (Taq polymerase) throughout the RSM assay could therefare have led to the production of in vitro induced mutations. The levels of these in vitro induced rnutations are how- ever difficult to estimate. Meastıres involving the optimisation of the PCR conditions were laken to ensure high fidelity. in addition, the RSM results were obtained from animals which had been sac- rificed after 7 days when the animals would have been expected to have repaired a considerable pro- portion of the lesions. It is likely therefore that the identified DMBA induced mutations were due to İlı vivo base substitutions.
Most of the RFLP /PCR studies have been con- ducted in vitro/ using cultured mammalian cells as targets far mutagenesis14, ıs. The RSM has been ap- plied to measuring mutations in Xenopus and mouse18, 19. Although different target genes and re- striction enzymes have been used in these studies, the feasibility of the assay combining with our re- sults has proves that the RSM assay is a potential method far identifying mutations in any species, any tissue and any gene.
in conclusion, these results of the RSM assay sug- gest that the method could be used far detection of base changes in DNA from several rat issues that were exposed to metabolically activated genotoxin, DMBA. The RSM assay has the potential far use in the regulation and assessment of the mutagenic po- tential of unknown chemicals and biomonitoring of mutagens in·the environment as a short term mu- tagenicity test in different species.
Süzen/ Parıy
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