DNA alkylation damage: consequences and relevance to tumour production

Mutation Research, 233 (1990) 247-252 247 Elsevier

MUT 02873

DNA alkylation damage: consequences and relevance to tumour production

Janet Hall and Ruggero Montesano Unit of Mechanisms of Carcinogenesis, International Agency for Research on Cancer, F-69372 Lyon Cedex 08 (France)

(Received 8 May 1990) (Accepted 6 June 1990)

Keywords: Alkylation damage; DNA repair; Tumour formation

Considerable progress has been made in the understanding of the cellular and molecular changes leading to the initiation of the cancer process and its progression. Much of this informa- tion was derived f rom studies in cultured cells or cell-free systems and to a lesser extent from studies in animals. The induction of cancer in experimental animals by the alkylating N-nitroso compounds has provided an informative model to examine the temporal sequence of such changes and to assess their biological relevance to the cancer process (see Lawley, 1989; Magee, 1989). In the understanding of cancer induction by such carcinogens, a relationship between metabolism, formation of specific D N A adducts, and in par- ticular the promutagenic D N A lesion O6-methyl - guanine (O6-meG), D N A repair and molecular changes which occur in critical sequences of the cellular genome, has been shown in various experimental systems (Samson and Cairns, 1977; Zarbl et al., 1985; Van Zeeland, 1988; Ellison et al., 1989). Such experiments provide evidence that carcinogen-specific mutations are a critical step in n i t rosamine- induced tumour fo rmat ion (see Bartsch and Montesano, 1984; Barbacid, 1987; Mitra et al., 1989; Magee, 1989). The studies that are presently under way in our laboratory to examine the relevance of specific D N A lesions and

Correspondence: Dr. R. Montesano, Unit of Mechanisms of Carcinogenesis, International Agency for Research on Cancer, 150 cours Albert Thomas, F-69372 Lyon Cedex 08 (France).

their repair in cancer induction by nitrosamines are briefly discussed here in the context of other contemporary work.

DNA alkylation and the modulation of repair activity in rodents

In many species, metabolism of nitrosamines, such as dimethylnitrosamine (DMN), is largely confined to the liver, kidney and lung and it is only in these organs that significant alkylation of nucleic acids takes place (Swann and Magee, 1968), suggesting that the distribution of activating systems in different organs may have an important influence on the organ specificity of the compounds (Druckrey et al., 1967). Table 1 shows a comparison of the in vitro metabolism of D M N in

TABLE 1

COMPARISON OF IN VITRO METABOLIC AND RE- PAIR ACTIVITY IN LIVER FROM VARIOUS SPECIES

Species 7-Methyl- O6-methylguanine, guanine " DNA-methyltrarts-

ferase b

Trout (rainbow) 0.0004 23 c Hamster (Syrian golden) 0.29 199 (88-298) Rat (Wistar, BDIV) 0.19 130 (112-163) Monkey (Macacus

cynomolgus ) 0.018 318 Man 0.13 1443 (710-2790)

a mole/100 mole of guanine, data from Montesano et al. (1982).

b fmole/mg protein (range of values). c Data from Nakatsuru et al. (1987).

002%5107/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

248

liver slices and the O6-meG-DNA methyltransferase activity in the liver of various species. The liver of Syrian golden hamsters showed the highest rate of metabolism and an intermediate level of repair. A single dose of 25 mg /kg DMN induces a 30% incidence of liver tumours in hamsters, but, in contrast, no liver tumours in rats (except when the nitrosamine is given after partial hepatectomy) (Tomatis and Ceils, 1967). The critical role of DNA repair became apparent from studies such as those of Goth and Rajewsky (1974) who clearly demonstrated the repair of O6-alkylguanine from non-target tissues in the rat after ENU exposure and the lack of removal of this modified base and subsequent tumour formation in the target organ (brain). Subsequently, the formation and persistence of this DNA adduct in mammalian cells and tissues was studied in many laboratories, by following the amounts of alkylated bases present in DNA in vivo at various times after a dose of radiolabelled alkylating agent. Using such techniques, it was demonstrated that the repair process for O6-meG was much more efficient after low doses of carcinogen (Nicoll et al., 1975; Klei- hues and Margison, 1976; Pegg, 1977) in both kidney and fiver in many rodent species. Chronic administration of the alkylating agent DMN was found to increase the excision of O6-meG from rat fiver (Montesano et al., 1979) in a manner which appeared analogous to the adaptive response earlier reported in E. coil (Samson and Cairns, 1977; Karran et al., 1979). This increase was subsequently shown to be due to a specific induction in the O6-meG repair enzyme level in the rat liver (Montesano et al., 1980) and has been shown to be dependent upon the dose and length of DMN pretreatment and to persist for several weeks after the end of the chronic exposure (Montesano et al., 1983). Most of these early studies have measured the level of alkylation in total liver DNA; however, Lewis and Swenberg (1980) demonstrated that differential repair of O6-meG from DNA occurred in rat fiver hepatocytes and non- parenchymal cells. Their study showed that al- though the initial level of alkylation after 1,2-di- methylhydrazine (DMH) exposure was slightly less in non-parenchymal cells than in hepatocytes, after 24 h only 10% of the initial amount of O6-meG remained in the hepatocytes compared to 53%

remaining in the non-parenchymal cells (the target cell population in DMH-induced carcinogenesis). Cell type-specific differences were also found in the induction of O6-meG repair by alkylating agents. Planche-Martel et al. (1985) showed that the specific increase in O6-meG repair observed after DMN pretreatment was confined to the parenchymal cells of the liver and a much lower repair activity has been observed in non- parenchymal cells.

In both bacteria and mammalian cells (see Lin- dahl, 1982; Pegg, 1984) the repair mechanism for O6-meG involves the transfer of the methyl group from the 0 6 position of guanine to a cysteine residue within the receptor protein. This is a stoichiometric reaction and results in the inactiva- tion of the repair protein and restoration of the guanine in the DNA. No mechanism for reactiva- tion of the methylated protein has been found and for the repair reaction to continue new protein is required. This peculiar mechanism of DNA repair suggests that the reconstitution of active O6-meG - DNA methyltransferase (MT) in a tissue will be an important factor in determining the probability of the occurrence of mutations (and probably tumours) resulting from unrepaired DNA lesions, particularly during chronic exposure. A depletion in the level of active enzyme is observed im- mediately after DMN treatment in both rat and hamster liver (Stumpf et al., 1979) and in cells in culture after treatment with alkylating carcinogens (Domoradzki et al., 1985). Distinct species differences in the time course of recovery of active MT to control levels are however observed (Hall et al., 1990). In the rat liver the levels of active enzyme have returned to control levels 72 h after treatment with DMN (20 mg/kg) and a subsequent increase in MT levels relative to control levels was observed. In the hamster liver, which has a similar constitutive level of expression of this gene, and after DMN treatment, which pro- duces a similar initial level of DNA modification (25 mg/kg), no recovery of active enzyme was detected up to 96 h after treatment. Only in the lowest treatment group examined (2.5 m g / k g DMN) was active enzyme measurable and recovery to control levels reached at 264 h. How- ever, in contrast to the rat liver, no substantial induction in MT level above the initial constitu-

T >,

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.g E <

g c~

%

300

200

1001

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O ~ < 0 / 0 ,'"'"El

, [~] . / "

, , t , 1 2 3 4

Doys o f t e r DMN t r e o t m e n t

I 800 ~'~

600

-5 400 E

200 "i

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Fig. 1. O6-methyldeoxyguanosine (#mole/mole dG) in fiver DNA and O6-meG-DNA methyltransferase activity in fiver protein extracts of BDIV rats and Syrian golden hamsters after administration of a single dose of DMN (20 mg/kg and 25 mg/kg) respectively. Each data point represents the mean level found in each treatment group (3-9 animals). 1005 Or-meG - DNA methyltransferase activity represents 0.19 units/mg protein and 0.17 units/mg protein in rat and hamster fiver respec-

tively, n, rat; o, hamster.

tively expressed level was observed. Fig. 1 shows the depletion and recovery of the MT in the liver of these two species, as well as the time course of disappearance of O6-medG from the liver DNA. Treatment of other species with various DNA- damaging agents has also failed to demonstrate an increase in MT activity (see Saffhill et al., 1985). The molecular events that control this recovery and the mechanism for the enhancement in the repair activity specifically seen in rat tissues re- main obscure. The recent cloning and sequencing of the human gene (Tano et al., 1990; Rydberg et al., 1990) for the MT will allow these differences to be studied at the molecular level.

P e r s p e c t i v e s

Distinct cell-, tissue- and species-specific differences exist in both the levels and distribution of DNA modifications produced by alkylating carcinogens and in the ability to repair such lesions (see Table 1). Many studies of the relationship between the formation of DNA damage and its repair, and biological end-points, such as

249

survival, mutagenesis and tumour formation, have assumed a degree of uniformity in these processes throughout the genome. Experimental data have shown that this is not always the case (see Hana- walt, 1987). Many of the methods used to study the levels of DNA modification, the efficiency of repair and mutation induction in DNA isolated from whole tissues cannot be easily applied to the study of intragenomic heterogeneity.

Various immunological and molecular probes are becoming available which will allow such processes to be analysed at the individual cell level and eventually at the nucleotide level. Immunocy- tological assays using monoclonal antibodies in conjugation with electronically intensified im- munofluorescence techniques have been developed that allow the detection of modified DNA compo- nents in the nuclei of individual cells (see Adam- kiewicz et al., 1985; Wild, 1990). Monoclonal and polyclonal antibodies available against 7-medG (imidazole ring-open) and O6-medG have been applied to examine by immunohistochemistry the capacity of the various cell types in rat and hamster liver after treatment with dimethylnitrosamine; the results are in good agreement with the findings based on the measurement of these adducts in DNA extracted from these different cell populations of the liver (Asamoto et al., unpub- lished data).

In bacteria, Saccharomyces cerevisiae and mammalian cells the correlation of specific adduct formation and mutation induction has been measured by the phenotypic selection of specific gene mutations, e.g., nalidixic acid resistance (E. coil), cdc mutants (S. cereoisiae), HPRT locus (V79, CHO, human lymphocytes) (see Van Zeeland, 1988). Somatic cell mutations occurring in vivo in humans have been measured by determining the frequency of the 6-thioguanine resistant lymphocytes in peripheral blood by autoradiographic techniques or direct cloning of the mutants, allow- ing the characterization of the hprt gene alterations (Albertini et al., 1985; Tates et al., 1989; Cole et al., 1989). With the recent technological advances in molecular biological techniques such as the polymerase chain reaction and denaturing gradient gel electrophoresis (Myers et al., 1987), it now appears feasible to detect mutant cells at the single-cell level (Kumar and Barbacid, 1988) and

250

to detect mutations within a section of a coding sequence.

Loci have been identified within the genome that may be more biologically relevant targets of DNA-damaging carcinogens than others. The demonstration by Barbacid and coworkers of the activation of an oncogene (H-ras) by base sub- stitution mutation (Reddy et al., 1982) has led many groups to investigate the role of the activation of this and other oncogenes in human cancers. It is apparent from the data so far accumulated that in human cancers the prevalence of activation of ras oncogenes varies from one type of human cancer to another and that the specific mutations that seem responsible for their activation are not occurring randomly. The prevalence of ras activation ranges from more than 80% in colon cancer to 0% in oesophageal cancer (Bos, 1989; Hollstein et al., 1988) or glioblastoma or neuroblastoma (Ballas et al., 1988; Bos, 1988). In colon tumours the majority of the mutations in K-ras are G-A transitions at the second G of a G G pair at codon 12, whereas in adenocarcinoma of the lung the most frequent mutations are G-T transversions (see Bos, 1989). This pattern of oncogene activation and mutation spectrum probably indicates a different aetiology. It is well known that in experimentally induced mammary tumours in rats and in other experimentally induced tumours the mutation spectrum appears specific for the carcinogen used and correlates with its known chemistry of interaction with DNA (Zarbl et al., 1985; Quintanilla et al., 1986; Mitra et al., 1989). Re- cently, Fuchs and coworkers (Burnouf et al., 1989; Freund et al., 1989) showed that hot spots for spontaneous deletions occur in DNA sequences that acquired the Z-conformation following treatment with the carcinogen 2-acetylaminofluorene. These data indicate that the occurrence of multiple genetic alterations, known to occur in the natural history of certain cancers like colon or brain (Vogelstein et al., 1988; James et al., 1988), are greatly determined by exposure to environ- mental carcinogens and by the processing of the DNA damage in a given cell or tissue.

Acknowledgements

The authors thank Mrs A.-M. Maillol for the preparation of the manuscript and Dr C.P. Wild

for helpful discussions. These studies were par- tially supported by U.S. NIEHS Grant No. 5 U01 ES04281-02 and CEC Contract No. EV4V 0040-F (CD).

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DNA alkylation damage: consequences and relevance to tumour production