DNA alteration and repair

ECOTOXICOLOGY

AND

ENVIRONMENTAL

SAFETY

4, 85-95

DNA Alteration LEONARDO

DNA

SANTI,

alteration:

(1980)

and Repair

S. PARODI, M. TANINGHER, AND C. BOLOGNESI~’

A primary

C. F. CESARONE.

event in carcinogenesis

DNA alteration and repair is undoubtedly as essential stage in the mechanism of chemical carcinogenesis. Present knowledge has in fact shown, and in a rather convincing manner, that the majority of known chemical carcinogens, in addition to radiations and viruses, are also mutagens in the widest sense of the word and usually induce alteration of the genome. Proof that carcinogenic or procarcinogenic chemical agents display muta.genic action is based on the recent experiments by McCann and Ames (4), who demonstrated a close relationship betneen carcinogenicity and mutagenicity in 9OcTo of the tested products (157/ 173, whereas the percentage of non-carcinogenic substances which were also non-mutagenic was 8770 (94/108) (Fig. 1). Initial experimental data allso confirm that failure of DNA repair could result in the appearance of tumours: for example, the potentiation of the oncogenic action of ultraviolet radiations in patients suffering from Xeroderma Pigmentosum who show a defect in DNA repair, in the same way as Cleaver (1) has ascertained recently for Ataxia Telangiectasia and Cokayne’s syndrome. At this point it is perhaps necessary to try to understand why alterations in nuclear DNA should be considered as the basic mechanism of carcinogenesis induced by chemical agents, when interaction has certainly been observed between the latter and other molecules involved in gene expression, such as RNA or regulating proteins. The possibility of chemical interactions between damaging agents and other target molecules has in fact already been shown in the literature. Two examples can be quoted: Wunderlich et al. (9. 10) observed a mcthylation of the mitochondrial DNA by methylnitrosourea (MNU) or dimethylnitrosamine (DMN); and the experiments by Sarma et al. (5) on the relationship between carcinogens like 2-AAF, MNU and ethionine and some types of RNA. In our view. * Presented

by L. Santi.

85

0147~6513/80/010085-I1$01.00/0 Copyright ‘i 19X0 hy Academic Press, Inc. 411 nghta of reproduction in any form revzrved

86

SANTI ET AL. Carcinogen

-mutagen 60

6 4 Non-carcinogen

Fig. 1. Correlation

between carcinogens

and mutagens (from Sugimura

ef al. (7)).

this can be explained by the fact that a modification of a whole population of a certain regulating protein, following an alteration of its single codifying gene, is certainly much easier than a direct alteration of the whole population of the regulating protein in question.

MNU

URETHAN 7

I

7

BrMBA

H

A-

,-DMN,DEN.MNU.ENU.MNNG.SAFROLE

2AAF,MAW,aNOO~

,7BrMBA

DNA CHAIN

B

Fig. 2. Sites of interaction

I

tine,

DMS

7BrMBA

of chemical carcinogens with DNA in

viva

and

irz vitro.

DNA ALTERATION

Fig. 3. Mispairing

Molecular

during DNA replication

interaction:

Carcinogen

AND REPAIR

of guanine methylated

87

in the 06 position.

- DNA

Interactions between chemical agents and DNA can be divided into two main groups: covalent and non-covalent. Covalent interactions are possible due to the presence in the DNA molecule of nucleophilic sites specific for covalent bonds with the ultimate reactive forms of carcinogens. In the DNA bases the most reactive points of attack are, in the purines. the nitrogen atoms (for example. in increasing order of reactivity: N7 of guanine. N7 and N3 of adenine, and N3 of guanine): on the purine bases, 06 and C8 of guanine are also subject to attack. As regards the positions on the pyrimidine bases, Nl, N3, CS for cytosine, CL! and C6 for th:mine can be mentioned. Fig. 2 shows the nuclcophilic sites of interaction and alkylation by the ultimate reactive products of the chemical agents. A typical example suggestin g a relationship between carcinogens and mutagenesis through transition is the one derived from the effects of methylation in 06 of guanine. As can be seen in Fig. 3, 06methylguanine does not pair with cytosine (normal situation shown in the formula on the right) but with thymine (left-hand section). A stable mutation results and thus incorrect genetic information in the daughter cells. Lastly. I would also mention that the phosphor groups can produce covalent bonds. The chemical agents that can induce covalent bonds and that are abIe to react directly or indirectly with DNA are of two types: the first type includes the classes of compounds such as nitrosamines, diazoalkanes, alkylmenthanesulphonates, epoxides, nitrogen mustard and mustard gas; examples of the second type include the nitrosamines, which appear to become alkylating agents after a probably enzymatic oxidative dealkylation (Fig. 4). Other substances which can interact covalently with DNA after metabolic activation are: 4-nitroquinoline-N-oxide, safrole and compounds belonging to the classes of aromatic amines and amides of polycylic hydrocarbons, of nitrofurans and of aflatoxins (Figs. 5-6). A defective DNA codification and in turn an erroneous replication of the genome can also arise from a non-covalent interaction: an example of an intercalating compound between the bases is given by the planar molecule of acridine.

88

SANTI ET AL. ALKYLATING

AGENTS

CH2-4H2 ..:. ( 6-c

CH3?\ ctijo

‘0

f~ - PROPIOLACTONE

0

,o S \o

DIMETHYL

SULFATE

CH \2 CH/2 CH2 ..\. i o-s-+0

‘,CH2

CH3(CH2),SC-N,;H

2 N-STEAROYLETHYLENE IMINE

0 PROPANESULTONE

H2C\3CH--CQ,y42 DIEPOXYBUTANE

ACYLATING

Ii UAACIL

MUSTARD

AGENTS

Y=CH ;N + C-W3 iH=CH ’ 6 I-ACETYL-IMIDAZOLE

(CH~)ZN-C;‘CI 0 DIMETHYLCARBAMYL CHLORIDE

Fig. 4. Chemical carcinogens reactive

DNA

per

se.

repair

There are enzymatic systems which are capable of counteracting the aggression against DNA by chemical agents. A basic pattern of such repair mechanism depends essentially on models obtained experimentallv with bacteria. From data now coming in, it seems that the validity of such models can be extended to mammals. There

CH3 BENZ(a)PYRENE

f+&% “2 2-ACETYLAMINOFLUORENE

7,l2-DIhiETHYLBENZbb ANTHRACENE

Q$@N~ N-METHYL-4-AMINOAZOBENZENE

URETHAN 4-NITROQUINOLINE-I-OXIDE ‘2”s

:N-NO

C,H,-S-CH,CH,-iI-COOH

c2H5 DIETHYLNITROSAMINE

2 ETHIONINE

Fig. 5. Synthetic chemical carcinogens non-reactive

per

se.

DNA ALTERATION

89

AND REPAIR

PRECARCINOGEN b PROXh-JCARCINOGEN(S) I CARCINOGEN(S1

ULTIMATE

electrophilic(electron-deficient),

INACTIVE METABOUTES ,p

mutogenlc I nucleophilic m cellular

CARCINOGEN INFORMATIONAL ~TIATlOt+

SPECIFIC

RESIDUES BOUND MACROMOLECULES \1 ALTERATIONS

mechanisms:

(electron-rich) mocrom0lecules

IN CRITICAL

MACROMOLECULES

1

& PROMOTIQCI’

EXPRESSION

atoms

COVALENTLY TO (DNA’s.RHA’~.PROTEINSI

genek?, epigenetlc?, wrol-genetlc?,wrol-eplgenehc?

[

O.N.S,C

OF ALTERED

INFORMATION

c GROWTH

OF CLONES \1

GROSS

TUMORS

Fig. 6. Scheme for the metabolism of organic

chemical

carcinogens.

are three main types of known repair processes: photoreactivation, repair by removal and restoration of the damaged site (dark repair) and post-replicative repair. Photoreactivation

This is a DNA repair process which depends on a single enzyme (photoreactivating enzyme) that, after binding specifically with the pyrimidine dimers induced in DNA by U.V. radiations, is activated by wave-lengths of visible light, between 300 and 500 nm and then splits the dimers into monomers. Although previous studies suggested that the enzyme was lacking in placentated mammals, it has recently been identified in human leukocytes and in other mammalian cells. The r61e or r6les of this repair process in placentated mammals has still to be elucidated, even though it has been demonstrated that photoreactivation promotes the capacity of forming colonies and of reducing the frequency of mutations. It is interesting to observe how RNA damaged by U.V. radiations is not attacked by the photoreactivating enzyme which, however, is capable of monomerizing the dimers in DNA even if monohelical, for example phage @x 174 (2,6). Dark repair (or excision repair)

This is a pre-replicative repair, presumably error-free, and it is the DNA repair process that is best characterized both in bacterial and mammalian cells. It has been shown with defective strains of Escherichia Coli that this type of repair is controlled by U.V. radiationspecific genes. Patients with Xeroderma pigmentosum are an example of a defect in one of the stages of dark repair. In this repair process,

90

SANTI

ET AL.

even in the case of damage by U.V. radiations, the damaged portion is completely removed from DNA; this fact distinguishes dark repair from photoreactivation where the pyridimine dimers are directly monomerized on the DNA molecule. Dark repair takes place through the following stages: 1. Incision of the DNA chain at the damaged site (endonuclease); 2. Removal of the damaged part (exonuclease): 3. Insertion of nucleotides in the free space, using the undamaged complementary DNA chain as a pattern (polymerase reparative replication); 4. Formation of phosphodiester bonds on the repaired DNA chain (ligase) (Fig. 7). Post-repiicativc

repair

This type of repair is considered error-prone; however, its molecular bases are not yet fully understood. It involves examples of recombination certainly in bacteria and probably also in mammalian cells. When the unremoved lesions come into contact with the replication apparatus during the semi-conservative synthesis of DNA, they act in such a way that the replication apparatus bypasses the damaged bases. DNA synthesis is resumed beyond the damaged site, with the resulting formation of gaps or empty zones on the chain which can even be as long as the space occupied by 1000 bases.

-a-

/I;,“,:

ENDONUCLEASE.

' 4

P~~~SPHATASE

_/h

5' EXONUCLEASE

977777

~'CXONUCLEASE

4d

Fig.

7. Excision

repair

(dark

repair).

NIP

Basic

steps

in the repair

process.

DNA

ALTERATION

AND

91

REPAIR

In the last few years, experimental data have accumulated, essentially with E. Coli, that suggest the existence of repair mechanisms that do not have the primary aim of re-establishing the correct nucleotide sequence, but the normal continuity of the DNA strand. A DNA chain that is interrupted or that has chemical alterations therefore constitutes a steric obstacle in the path of the replicative machinery with consequent lethal effect for the cell. Such an error-prone repair mechanism would save the cell but not protect it from mutations. Short-term predictive tests for the evaluation genie activity of chemical compounds

of the potential

onco-

The possibility of demostrating a DNA aIteration and/or repair is a reliable approach in evaluating the alterations induced by chemical agents. The techniques for detecting DNA. repair or unscheduled synthesis of DNA correlated with dark repair could therefore be an excellent means of demonstrating an alteration in cellular DNA. Of these techniques, prefer’ence should be given to those which reveal unscheduled synthesis by determination of the radioactivity incorporated into DNA as thymidine during the repair process. Radioactivity can be measured essentially by two techniques: directly, determining the incorporated thymidine by liquid scintillation counting or by autoradiography. Arttoradiographic

technique

With the autoradiographic technique it is possible to distinguish the tritiated thymidine incorporated into cells in S phase and that incorporated into cells out of S phase. By measuring the radioisotope uptake according to the number of grains detected with photographic emulsion. it can be observed that the cell population not in S phase is around a mean value of 0 grains per nucleus in the case of control cells, and generallv around a mean value of a few dozen grains in the case of treated cells. The cell population in S phase shows the majority of nuclei as compietely black and in practice no nuclei with less than one hundred grains/nucleus are observed. The cell population during DNA repair synthesis therefore appears quite distinct from the cell population in S phase. Evaluation

of DNA

repair sjwthesis in r’ivo in mouse germ cells

Unscheduled synthesis is induced by in viva administration of chemical agents followed by intraperitoneal injection of (3H)TdR and is then evaluated on the spermatids taken from the caudal portion of the epididymides. Unscheduled incorporation into DNA is determined by liquid scintillation counting (LSC) after purification of the sperm nuclei and identification of the s!Jnthesis out of the S phase (Figs. 8-9).

92

SANTI

CHEMICAL TREAThENTOF THE ANIt!ALS

2 HRS,

ET AL.

[~H]T~IR~ICE

)

2-4 CAUDAEEPIDIDWIDES HOMOGENIZATION

FILTRATION 8 SONICATION

c

CENTRIFUGATION THROUGH

5%BSA ( 3000 G )

PELLET SUSPENDED ----) -7

CENTRIFUGATION (30ooG)

RESUSPENSIONIN DNA MICROFLUORIRETRIC DETERMINATION ’

o’154i

NACL

HMOCYTOI(ETER OBSERVATION 8 COUNTING OF SPERR HEADS

1

FILTRATION THROUGHWHATMN GF/C 6 WASHINGS 1 LIUIID SCINWATION

CCUHTIC (LSD

Fig. 8. Basic scheme of the technique for the evaluation in mouse germ cells of DNA repair synthesis in viva

-

I 10 DAYS A-.

US-DNA

I 12

I 14 AFTER

CONTROL

-

I I 16 IP TREATMENT

I 20

o.--..-oTREATED

Fig. 9. Unscheduled uptake of (‘H) TdR during post-meiotic phase of male mouse germ cells. The animals were treated with a single dose of MNU (N-Methyl-N-Nitrosurea) 75 m/kg. US - DNA: unschedu!ed synthesis of DNA. M : DNA synthesis during meiosis.

DNA

ALTERATION

AND

93

REPAIR

--

-I

TOP

BOTTOM FRACTION

A i

DGA

C I

DGA 062

IOmM;

N.

B:

OGA

D :

control

ZSmM,

Fig. 10. Sedimentation pattern of DNA in alkaline Cells were treated with different doses of DGA

sucrose gradient. (N-Diazoacetyl-glycine

amide).

A method widely used to demonstrate that a chemical compound has interacted with DNA is to measure the breakages in the individual DNA in alkalis using gradients or the elution techniques. Alkaline gradients By centrifugation, the individual DNA chains migrate in bands at different distances from the top according to their sedimentation coefficient. The undamaged DNA chains sediment more rapidly, while the lighter chains, derived from the breakage of damaged DNA, are retained in the higher zones of the gradient (Fig. IO). Alkaline elittion This technique measures the same parameters as the alkaline gradient (breakages in the individual .DNA chain), but it is a more simple, rapid and independent technique than the alkaline gradient as far as its working principle is concerned. Essentially, it measures the velocity of DNA elution through a filter. Elution velocity is a complex function of the molecular weight of DNA under study (Fig. 11). This method has the advantage of being applicable not only to mammalian cells treated in vitro but also to preparations obtained from animals treated in vi\,0 (Fig. 12). The alkaline elution technique, set up for the first time in 1973 by Kohn (3), has already been applied with success by various Authors, and Swenberg et al. (8) have assayed about 40 compounds positive in the Ames test, and have obtained a very good correlation.

94

SANTI ET AL. CHEMICAL TREATMENT OF THE ANIMALS

)

SACRIFICE

)

LYSIS OF NUCLEI DN MLLIPORE FILTER

WlOGENIZATION-CEMRIFUGATION

CRUDE NUCLEAR SUSPENSION

c DNA ELUTION 8 FRACTIONS COLLECTION 1 tlICRWlJORIHETRIC DEIEWIIN4TION OF THE ELUTED DM

Fig. 11. Basic scheme of the alkaline elution technique.

These Authors are still using this technique, the presentation given at the recent Congress Due to the fact that this method is so pratical it will be possible to obtain further evaluation

as can be seen from in Edinburgh (1977). and simple to perfom, data in a short time.

A test which, from a systematic point of view, could be placed in an intermediate position between the methodologies evaluating chromosome damage and those analyzing DNA repair synthesis is sister chromatid exchange (SCE). The biological significance of SCE is still largely unknown; undoubtedly a recombination process is involved and by means of exchanges between chromatids just replicated, allows removal of the damage persisting even after passage of the replication apparatus. The appearance of SCE is probably an expres-

I

0

5 FRACTION

10 No.

Fig. 12. Elution pattern of mouse liver DNA, damaged with cycasin.

DNA

ALTERATION

AND

REPAIR

sion of the fact that the cells, through recombination, eliminate the damage not already removed by other mechanisms. Even though the details of this process have not yet been elucidated, it seems nonetheless that it may be considered as distinct from other types of repair. Therefore we think that SCE. possesses such features as to recommend its inclusion in a battery of short-term tests. In fact a number of chemical and physical mutagens at very low doses increase the incidence of SCE, resulting in a high sensitivity plus good statistical correlatability and relative methodological simplicity. As far as we know (up to date only 50 substances have been tested), predictivity seems very promising. Several in vi\lo/in vitro methods have already been developed which should make it possible to overcome the dithculties involved in in \sitro metabolic activation that give false negative results. For the purposes of evaluation of the test, a positive result may be considered strong evidence of persistence of DNA damage, while a negative result would leave the possibility of the damage being removed by other means. REFERENCES 1. CLEAVER J.E. In: Genetics of Human Cancer, Eds. J.J. Mulvihill, R.W. Miller and J.F. Fraumeni Jr., Raven Press, New York, 1977, pp. 355-363. 2. HANWALT P.C. Endeavour. 31: 83, 1972. 3. KOHN K.W., GRIMER-EWIG R.A. Cancer Res., 33: 1849, 1973. 4. MCCANN J., AMES B.N. Proc. Nat. Acad. Sci., USA, 73: 950, 1976. 5. SARMA D.S.R., RAJALAKSHMI S., FARBER E. In: Cancer, Vol. I. Ed. F.F. Becket, Plenum Press, New York, p. 235, 1975. 6. SETLOW R.B. Prog. Nucl. Acid Res. Mol. Biol., 8: 257, 1968. 7. SUGIMURA T. et al. In: Screening Tests in Chemical Carcinogenesis, IARC H. Bartsch, L. Tomatis, Scientific Publication No. 12, p. 81, Eds. R. Montesano, 1976. 8. SWENBERG J.A., PETZOLD G.L., HARBACH P.R. Biochem. Biophys. Res. Comm., 72: 732, 1976. 9. WUNDERLICH V. et al. Biochern. J., 118: 99, 1970. 10. WUNDERLICH V. et al. Chem. Biol. Interact.. 4: Sl, 1971/1972.

95