The detection of DNA adducts, DNA base changes and chromosome damage for the assessment of exposure to genotoxic pollutants

Aquatic T~~~~~~~~s~, 22 ( 1992) 323-344 0 1992 Elsevier Science Publishers B.V. All rights reserved 0166-445X/92/$5.00

AQTOX

323

005 14

he dete~tiQ~ of and chromosome damage for the assessment of exposure to genotoxic Qll~ta~ts

(Received 2 September 1991; accepted 15 January 1992)

The exposure of aquatic species to a variety ofgenotoxic chemicals raises the question as to the potential effects of exposure upon the hcdlth slatus of both current and future aquatic populations. The induction of DNA lesions in exposed species may initiate the formation of both structural and point mutations in genes. the modifications of which may lead to abnormality in both somatic and germ cells. The assessment of exposure levels of genotoxic agents and the genetic consequences requires the application of a set of inter-related techniques capable of providing both qualitative and quantitative information. In this papr we describe some m~thodolo~ies available for the (a) assessment of genotoxin induced DNA adducts using the ‘“P-postlabelling procedure (b) measurement of point mutations using the restriction site mutation assay and (c) the measurement of the production of chromosome structural and numerical aberrations using the micronucleus assay. This package of technologies may be optimised to use with individual species present in the aquatic environment to provide a cost effective package, capable of monitoring

genotoxin exposure of ir~~viflual environments.

Key words: DNA adducts; “P-postlabcling; toxic pollutants

Restriction site mutation assay; Micronucleus assay; Geno-

INTRODUCTION

The contamination of the envjronment with bjologi~~lly active chemicals is becoming an issue of increasing concern and awareness. Such chemicals have always been present in the environment as a result of natural processes (Nagao and Sugimara, 1976; Lippmann and Schlesiner, 1979). However, with the advent of the industrial revolution the release of man made contaminants with toxic or harmful effects has inevitably increased (Kirsch-Voiders, 11984).In 1976 we demonstrated that the body Ci,rrc:~[~“rteil,tcv lo: Dr. N.J. Jones, Molecular University

Biology Research Group. School of Biological Sciences.

College of Swansea, Singleton Park, Swansca SA2 SW, U.K.

324

tissues of marine mussels collected from polluted sites contained genotoxic chemicals capable of producing positive responses in a variety of short-term in vivo test systems fParry et al,, 1976). Environmental pollutants that react in vivo with DNA, either directly or following cellular metabolism, are likely to have detrimental effects on any exposed populations. DNA adducts or lesions formed by reaction with genotoxic contaminants may ultimately give rise to genetic changes such as point mutations and structural and numerical chromosome aberrations. In view of the role of such genetic alterations in somatic and inherited disease, such pollutants should be regarded as potentially mutagenic, carcinogenic and teratogenic. Indeed, a number of studies have demonstrated the presence of liver and other neoplasms in fish collected from contaminated areas (Dawe et al., 1964; Brown et al., 1973; Couch et al., 1985). It is therefore of importance that we develop sensitive assays for the detection and measurement of DNA lesions as these may serve as early indicators of environmental contamination by genotoxic agents. In turn, complementary methods capable of monitoring induced genetic changes in exposed species are also required. In this paper we describe three sensitive techniques for detecting DNA adducts, DNA base changes and chromosome damage. The ASP-postlabelling assay (K. Randerath et al., 1981; Gupta et al., 1982; Reddy et al., 1984). the restriction site mutation (RSM) assay (Parry et al.. 1990) and the micronucleus assay (for review see Heddle, 1983) comprise a powerful combination of biochemical, molecular and cytological techniques which provide a comprehensive methodology for the assessment of the impact of aquatic germtoxin exposure. The three techniques can readily be applied to a wide array of organisms land tissues) including aquatic species and man, and can thus be utilised to monitor a variety of environments and situations. DETENTION OF DNA ADDUCTS BY ~~P”POSTLABF~LLING

“P-postlabelling is a technique which has been developed for the detection of DNA constituents which have been chemically altered by reaction with genotoxins. The original procedure was developed by K. Randerath and co-workers (K. Randerath et al., 198 1) and is analogous to the methods developed by them for the analysis of RNA and DNA composition (E. Randerath et al., 1972; Randerath et al.. 1980; Reddy et al., 1981). With “P-postlabelling it is possible to detect DNA damage induced by a wide variety of genotoxins, including polycyclic aromatic hydrocarbons, heterocyclic polyaromatics, alkenyl benzenes, arylamines and amides, pyrolysis products, mycotoxins, alkylating agents and free radicals (for review see Gupta and Randerath 1988: Watson 1987). The “‘P-postlabelling assay may be performed on virtually any biological sample from which DNA can be extracted and involves the enzymatic radiolabelling of axon-radioactive nuclcotides. The basic protocol (Fig. I) entails the enzymatic hydrolysis of DNA to deoxyribonucleoside 3’-monophosphates (dNps) with micrococcal nuclease and calf spleen phosphodiesterase. The dNps are then radioactively labelled via T4 polynucleotide

325

NNNXNN

Adducted DNA

Mtcrococcal

Enzymattc Digestion

Cal! spleen

13kadenoslne

nuclease phosphodlesterase

tttphosphate

(100-300

Cl/mmol)

5’ phosphorylation T4 polynucleotide

klnase

JI N

X 31

31 p

Lk

3~-~a~l~~sdp~~so;~~~eonucleoslde ‘- ’ (dpNps + dpXPs)

P

Thln layer chromatographtc purtllcatlon and resolution of adducted blsphorphates (dpXps) on PEI-cellulose plates

Autoradiography Fig. 1. Standard procedure for the “P-postlabclling

of DNA

adducts developed by K. Randcrath

and

co-workers (K. Randerath CI al.. 1981; Gupta ct al.. 1982).

kinase catalysed [-“PI-phosphate transfer from [y?‘P]-adenosine triphosphate to yield deoxyribonucleoside-3’-5’-bisphosphates (dpNps). Any damaged or adducted deoxyribonucleoside-3’-S-bisphosphates (dpXps) present in the sample are then purified and separated from their normal (undamaged) counterparts (dpNps). This is generally achieved using multidimensional anion exchange thin layer chromatography (TLC) on polyethyleneimine (PEI)-cellulose plates. Fig. 2 shows the typical solvent

326

/

ftltsr aper uttache 8 with

wtck

staples

DI

2Ox2Ocm PEI-cellulose T.L.C. plate

1

OR16lN

D4

7----+

-_a

D2

1 D3

Development

Ditectlon and Solvent - -.._ - ---~__.__~__~______~~~ Dl,

i.CM

sodium

phosphate,

D2, 2.5M ammonium

D3, 3.5M lithium 8.5M urea

pH 6.8

rormate,

iormate.

pH 3.5

pH 3.5

---

--.--

phosphate

---

Overnight onto wick; Wick and top 3cm of plate removed; Plate washed ln water. 6h onto wick; Wick and top 3cm of plate removed; Plate washed ln water. To top o! plate (Ibh); Plate washed once tn 13mM Trls base and once in water. To top ot plate (5.5h); Plate washed twice tn water.

D4, 0.8M lithium chloride 0.5M TrlP-HCl pH 8.0 8.5M urea D5. 1.7M sodium

and washes

pH 6.0

_

Overnlght onto wick. Wick and orlgln removed; Plate dried and autoradlography performed.

Fig. 2. Typical solvent conditions for the chromatographic

purification

and resolution of DNA

adducts

containing aromatic moieties (Jones et al., 199 I ). Solvents Dl and D2 remove normal nucleotides, residual “P-ATP

and inorganic phosphate onto the filter paper wick attached to top of plate. Solvents D3 and DJ

move DNA

adducts tdpXpj

in the direction

indicated.

D5 removes residual non-adduct

radioactivity

reducing background.

conditions for the chromatographic purification and resolution of DNA adducts containing aromatic moieties (Jones et al., 1991). The labelled adduct digest (0.2-20 pug DNA) is applied to a 20 x 20 cm plastic backed, PEI-cellulose TLC plate and nonadduct material (normal nucleotides, residual ATP and inorganic phosphate) is removed from the plates onto filter paper wicks using the aqueous solvents DI and D2.

327

Any aromatic carcinogen-adducted nucleoside-3’“5’-bisphosphates (dpXp’s) are retained at the origin due to their greater affinity for PEI-cellulose. Solvents containing high molarity urea (D3 and D4) are then utilised to move and s‘parate structurally distinct dpXp’s from one another. Resolution of the adducts with these solvents is determined by pH, urea concentration, overall ionic strength and the nature of the anions (Gupta and Randerath, 1988). Adducted nucleotides are visualized as a twodimensional pattern of discrete spots following autoradiography of the chromatograms. The radioactivi~ in adduct spots can then be quantified by excising the spots from TLC plates, followed by liquid scintillation counting. The amount of radioactivity in normal nucleotides (dpNp) is determined in a similar way by performing chromatography on a diluted aliquot of the labelled digest (0.01-10 ng DNA), using solvents which separate the normal nucleotides from each other in either one or two dimensions (Figs. 3 and 4). If one assumes that normal and adducted nucleotides are labelled to an equal extent then the relative adduct labelling (RAL) may be calculated (Gupta, 1985): RAL =

dpm in adduct dpm in total (normal) nucleotides x dilution factor

Alternatively RAL may be calculated as described by Reddy and Randerath (1986) based on the specific activity of the [y”-PjATP (expressed as dpm/pmol) and the amount of DNA used (1 pug DNA = 3240 pmol dNp): RAP. *=

dpm in adduct Sr;. ,c:. ATP x pmol dNp used for analysis

RAL values can then be translated into adduct frequencies (l/RAL) or into attomol adduct per ,ug DNA by assuming that 1 yg DNA is equal to 3.24 x 10” attomol nucleotides. It has been estimated that with the original standard “P-postlabelling assay (using m 0.2 pug DNA) it is possible to detect a single DNA adduct in IO’--IO’ normal nucleotides {Gupta et al., 1982 j. However, in recent years various enhancemeilt methods have been developed which increase the assay’s sensitivity to I adduct in IO”’ nucleotides. These enhancement techniquc; (Fig. 5) involve an additional step in which the vast bulk of normal nucleotidl:s are removed from, or prior to, the “P-1abelling reaction and thereby permit the use of larger amounts of DNA (5-20 pg) and very high specific activity [y”PjATP (5000-9000 Ci/mmol). In the nuclease PI procedure (Reddy and Randenlth. 1986) the deoxyribonucteoside-3’-monophosphates of normal nucleotides are cleaved to deoxyribonucleosides which do not then serve as substrates for the T4 polynucleotide kinase in the subsequent labelling reaction. However, most aromatic adducted nucleotides are totally or partially resistant to the 3’-dephosphorylating action of nuclease Pl and remain availrable for “P-1abelling. In the HPLC-based procedure (Dunn et al., 1987;

Fig. 3. One-dimensional

separation

of normal “P-labelled

nucleoside 3’-5’-bisphosphates

sodium phosphate pH 6.8. The equivalent of 0.0 I2 ng of X~opus TLC plate and autoradiography

larvae DNA l~w~vt~

using 0. I2 M

was spotted onto the

performed for I h. The bisphosphates of adenosine. cytidinc. guanosine

and thymidine arc indicated. Pi is inorganic phosphate and residual “P-ATP

was destroyed with potato

apyrasc.

Dunn and San, 1988) hydrophobic DNA adducts are isolated prior to “‘P-postlabelling using methanol gradients. Enrichment with the butanol extraction procedure (Gupta, 1985) involves the organic extraction and concentration of adducted deoxyribonucleoside-3’-monophosphates in the presence of phase transfer agent tetrabutyl ammonium chloride. Residual normal nucleotides are removed from the butanol phase by back extracting with H,O. As each of the enhancement procedures relies on the removal of normal nucleotides from the labelling reactian it is important to monitor whether they arc indeed removed effectively. The efficiency of 3’-dcphosphorylation by nuclcase PI for

329

Fig. 4. Two-dimensional separation of normaf “‘P-labelled nucleoside 3’-S-b~sphosphates. DI : I .5 M ammonium formate pH 3.5, D2: 0.2 M ammonium sulphate. The position of the bisphospksles of adenosine, c~t;di~~. g~anosi~e and thymidine are indicated. Pi is inor~dnic phosphate and residual “P-ATP was destroyed with potato apyrase. Autoradiography was for I h.

example or the removal of normal nuclleotides by butanol extraction can easily be assessed by examining a small aliquot of the subsequent labelled adduct digest using the chromatographi~ conditions utilised for the separation of the normal nucleotides. This control and others used to monitor the digestion of DNA to dNps, the availability of excess [“‘PJATP in labelling reactions. adduct enrichment and RNA contamination should be i~~corpo~ted into all ~~P-pos~l~~be~~ing protocols (Jones et al.,

330 NNNXNN

Enzymattc

digestion

1

4

MiCtOCOCCCfl

Cal! spleen

nIXleaSe

phosphodiesterase

Deoryrtbonucleoslde p 3’ monophosphates (dNps

+

dXps)

I \?

ADDUCT ENRICHMENT

Bu tan01 Extraction

HPLC Separatlon

Nuclease

‘+’ 32P-ATP (5000-9000 8 T4 PNlC

5’ phosphorylatlon dpXps

Pl Digestton

Ci/mmol)

at

P

P

Q TLC and AUTORADIOGRAPHY Fig. 5. “P-postlabelling tanol extraction

(Gupta,

enhancement proccdurcs. In the nuclcasc PI (Rcddy and Randcrath, 1085) and

I%%), bu-

HPLC (Dunn and San, 198X) procedures, the bulk ol’ normal nu-

cleotides are rcmovcd from the labclling reaction. allowing the use of high specific activity “P-ATP.

1991, Reddy and Randerath, 1987). In this way the efficacy of the “P-postlabelling analysis may be ensured for each sample (Jones et al., 1991). It is also important to be aware that although the various enrichment techniques enhance the detection of broadly similar classes of DNA adducts the overlap is not an exact one. A numbr:r of studies have dcmonstratcd that certain adducts arc detected

331

more efficiently with one method than another and vice versa (Gupta and Early, 1988; Gallagher et al., 1989; Jones and Waters, unpublished data). For example, Gupta and Early (1988) found that most deoxyguanosine-C8-arylamine adducts examined were almost completely lost in the nuclease Pl-mediated procedure unless a polar group was present in the aromatic amine moiety. The butanol extraction method, on the other hand, was very efficient in enhancing the detection of both types of adducts. Consequently we therefore strongly recommend that when a study is initiated a number of the enhancement versions be utilised until it is determined which one(s) are the most appropriate. In addition, the differential enhancement of adducts with these methods may provide some insight into the nature of the adducts themselves. As well as the extreme sensitivity achieved by the use of the various enhancement techniques the 3”P-postlabelling assay has a number of features which make it a powerful tool for assessing exposure to genotoxic chemicals. One of the most important aspects is its versatility. The requirement for only microgram quantities of DNA enables the assay to be performed on small amounts of samples from a variety of sources including human and animal tissues, lymphocytes and cultured cells (including cell lines derived from mammals, amphibians and fish). In addition, it can be used to detect a vast array of DNA adducts, including unidentified damage, as prior knowledge of the nature and structure of the adduct formed is not a pre-requisite for its detection. This is primariiy because the method is not dependent on the availability of genotoxic chemicals in an isotopically labelled form as the adducted nucleotides are radiolabelled after their formation. This makes “P-postlabelling particularly well suited to detecting DNA damage induced by complex mixtures such as those associated with petrochemicals, heavy industry, exhaust emissions and tobacco smoke. These advantages of the “‘P-postlabelling assay (i.e. its extreme sensitivity and applicability to a pkiher a ofgcnotoxic substancesj nrahe it an Ideal analytical method for both the assessment of environmental contamination in indicator organisms and for studies in humans. The studies in humans are now quite extensive and serve to illustrate the potential use of the technique in other species. For example, “P-postlabelling analysis has been utilised successfully to identify tobacco smoking associated DNA adducts in a variety of tissues including lung, heart, placenta and monocytes (Phillips et al., 1988a; Everson et al., 1986; E. Randerath et al., 1989; HoJz et al., 1990). In these experiments DNA damage is generally visualized as diagonal radioactive zones (DRZ) on autoradiographs indicating the presence of diverse types of adducts. These DRZ are typical following exposure to complex mixtures such as tobacco smoke, creosote or diesel exhaust (E. Randerath et al.. 1988: Schoket et al.. 1988; Schoket et al., 1989) and similar DRZ have been demonstrated in aquatic species (see below). An example of such a DRZ is shown in Fig. 6 and this was obtained in our laboratory following the 3’P-postlabe!ling analysis of DNA extracted from the oral tissue of a heavy cigarette smoker (Hoskins et al.. 1991). Riomonitoring in the work place has demonstrated the presence of aromatic carcinogen-DNA ad-

Fig. 6. Example of a diagonal radioactive zone (DRZ) indicating the presence of diverse types of DNA adducts typically found following exposure to complex mixtures of genotoxi~ s~lbstan~es. Such DRZ have been found in various human tissues of tobacco smoking individuals (see text) and in the livers of fish from polluted waters (Dunn et al., 1987; Varanasi et al.. 1989). in this instance DNA was extracted from the oral squamous ccl1 cpithelia of a heavy cigarette smoker (-3Wday) and analyzed using the butanol extraction procedure. Chromatography was performed as given in Fig. 2 and autoradiography was for 96 h.

ducts in the white blood cells of iron foundry and aluminium production plant workers (Phillips et al., 1988b; Savcla ct al., 1989; Schoket et al.. 1991). ASP-postl~belling analyses in aquatic species have been much less extensive than thosr in humans or laboratory rodents but the limited number performed to date give some indication of the promise of the technique. Dunn et al. (1937), using both the nuclcase Pi and HPLC adduct enrichment procedures demonstrated the prcscnce of

333

DNA adducts in the livers of fish (Ictulurus nebulasus) from polluted areas. These adducts, detected in the form of DRZ, were not present in the liver DNA of fish raised in aquariums. The DNA from fish collected from the polluted Buffalo River contained 227.1 + 94.0 attomol adduct/~g DNA whilst the figure for aquarium raised fish was 47.6 ,+ 29.8 attomol&g. Varanasi et al. (1989) demonstrated an excellent correlation between the level of hepatic DNA adducts in the marine flatfish English sole (f~rophrqts vetuh), levels of fluorescent aromatic compounds in bile and the levels of contamination by sediment associated polycyclic aromatic hydrocarbons at three sites with low, intermediate and high levels of pollution. In contrast, however, KUrelet et al. (1989a) observed no statistically significant differences in DNA adduct levels in both freshwater (e.g. Cyprirm curpiu) and marine (Mugil uurutus) fish from polluted and unpolluted waters. Clearly, further studies are required to fully assess the utility of ASP-postlabelling for such studies and rigorous determinants of what constitutes an unpolluted site are also essential. Kurelec and collaborators (Kurelec et al., 1988, 1989b) have also demonstrated the use of the mussel, Myth gullopnwimkl, as a potential indicator organism for risk assessment of genotoxic pollution in the marine environment. However, in both the fish and mussel studies published to date only a single tissue has been examined for the presence of DNA adducts (liver and digestive gland, respectively). A possible confounding factor with these tissues may be occurrence of adducts which are apparently unrelated to any known exposure to genotoxic agents (Kurelzc et al., 1989a.b). These so-called ‘natural’ or background adducts may interfere with the detection of pollutant-mediated DNA damage. It is therefore important to examine other tissues which may have a lower background level of adducts and consequently prove to be valuable for environmental monitoring. Variation in the levels of background adducts has been demonstrated in a number of rodent studies with liver usually exhibiting the highest levels (e.g. Gairola and Gupta. I991 ). The USCof the gill and mantle of mussels as alternative tissues to the digestive gland arc presently in progress in our laboratory. Wc are also exan~ining the larvae of the amphibi~~n ,~c~~~~~~?i~.s kresh as a possible tool for monitoring freshwater contaminations We have demonstrated a clear dose response in the level of DNA adducts in larvae treated with the polycyclic aromatic hydrocarbon benzo[a]pyrene at doses between 0.02 and O.S~glml (Morse, Jones and Waters, unpublished) indicati~lg the extreme se!~sitivity of the assay. Fig. 7 shows an a~ltoradiograph obt~~i;led following ASP-~tostlabell~ngof DNA extracted Iii-om larvae treated with 0.5 pg/ml benzo[a]pyrene for 24 11.A number of distinct adduct spots are visible consistent with those obtained in other studies (Reddy et al.. 1984 Canella et al,, 1991). ‘UP-postlabelling therefore has great potential for n~onitoring aquatic enviro~llnelits for the effects of contamination by genotoxins. With specific reference to aquatic pollutants the technique will detect DNA damage dw to oils or otl?er petrochemical associated compounds, or indeed any other contammant capable of reacting with

334

Fig. 7. Autoradiograph

of DNA

adducts detected by the nuclease PI version of “P-postlabelling

r~p~.sItwis larvae cxposcd to 0.5 pg/ml benzo[a]pyrene for 24 h. Chromatography

in XUI-

was as described in Fig.

2 except for D3: 4.0 M lithium formate, 8.5 M urea pH 3.2 and D4; 0.8 M lithium chloride, 0.5 M Tris-HCI, pH 9.0. 8.5 M urea. Autoradiography

was for 66 h (autoradiograph

kindly provided by Ruth Morse).

DNA. The ability to store tissue samples or DNA extracts at -70°C, almost indefinitely. provides the opportunity to monitor a site of interest over a period of time. For example, it should be feasible to assess and follow the effects of genotoxin contamination at a particular site or sites after an event such as an oil spill. When using “P-postlabelling it is not essential to suspect a specific contaminant, as the method can detect uncharacterised damage by virtue of additional spots on autoradiographs DNA damage due to exposure to environmental pollution can be determined by comparing autoradiographs from captured individuals with those reared in the laboratory although possible artefacts arising from factors such as diet should always be borne in mind. Subsequent identification of pollutant-mediated DNA adducts is a potentially problematic area of “‘P-postlabelling analysis. Although no information on the chemical structure of the adduct is required for detection characterization of the adduct may be troublesome. Potentially, specific DNA lesions may be identified by reference to autoradiographs generated from cultured cells or laboratory raised

335

individuals which have been exposed to a single specific genotoxin. Additionally the availability of synthesized adduct standards (e.g. (-t)-(?R.8S.9R-trihydroxy-7,8,9,10tetrahydrobenzo[a]pyrene-lOs-y1)2’-deoxyguanosine) will considerably aid the identi~cation of induced adducts. Unfo~unately, as yet those standards are somewhat limited in number but more should become available with time. One important aspect of 32P-postlabelling procedures that merits particular mention is safety due to the use of high specific activity [y3*P]ATP. All manipulations must be performed behind 1 cm acrylic screens whilst wearing protective aprons and eyeware. Tubes containing 32P must be handled with forceps and several layers of disposable gloves should be worn at all times. A suitable radiation monitor is essential, indeed mandatory, for work involving high specific activity ‘*P solutions. Frequent monitoring of gloved hands, equipment and work areas is imperative to detect contamination and to avoid possible cross-contamination. The -“P-postlabelling methods described in this paper are primarily suited to the detection of aromatic and/or hydrophobic DNA adducts. They are inherently unable to detect smaller DNA lesions caused by small alkylations or free radical damage. Adducts such as ~~-methyldeoxyguanosine, ~‘-methyldeoxyguanosine, thymidine glycol and 8-hydroxydeoxyguanosine are too similar in structure to normal nucleotides for the nuclease PI and butanol extraction enhancement methods to be effective and too similar in size to be separated using the chromatographic conditions described in Fig. 2. However, methods incorporating a combination of techniques such as immunoaffinity or high performance liquid chromatography and “P-postlabelling are becoming available for these adducts (Wilson et al., 1988; Shields et al., 1990; Watson and Crane, 1989; Hegi et al., 1989; Povey et al., 1989). In our laboratory we are currently using and developing methods to detect an array of DNA damages including those induced by tobacco smoke; complex mixtures associated with petrochemicals; various genotoxins associated with industrial processes such as 1,3-butadiene, styrene and ethylene oxide: the mycotoxins sterigmatocystin and allatoxin and other environmentally important genotoxins. THEMEASUREMENTOFTHEINDUCTIONOFPOINTANDCHROMOSOMALMUTATIONS

The measurement of the induction of DNA adducts described above, provides a methodology for the assessment of exposure to DNA damaging agents. However, DNA lesions may have a number of consequences in exposed species. Lesions may undergo error fret: repair leading to the reconstitution of the original DNA sequence, lesions may lead tc cell death and thus make no contribution to the genotype of progeny cells or alternatively lesions may lead to modification of DNA sequence and structure such that they lead to mlltations in progeny cells. mutational change may occur at three levels of organisation: by undergoing point mutation, chromosome structure and chromosome numerical changes. Assessment of the consequences of DNA lesion induction, requires the development and application of techniques

336

capable of measuring all three types of mutatiors. In this section we will describe some methodologies which have been developed for the measurement of induced mutations in aquatic species. THE

INDUCTION

OF MICRONUCLEI

Following the exposure of living cells to clastogenic (chromosome damaging) agents, the cytoplasm of dividing cell populations can be seen to contain micronuclei (Evans et al., 1959). These micronuclei are small membrane bound masses of chromatin which may contain acentric fragments of chromosome material, centric fragments and whole chromosomes. It has generally been assumed that acentric fragments are the product of chromosome breakage (structural damage) whereas centromere containing micronuclei are the product of the failure of the fidelity of cell division leading to the production of numerical chromosome changes. In both cases the micronuclei represent chromatin not included in the main nucleus, presumably because of its failure to segregate to the progeny nuclei produced as a result of mitotic cell division. The induction of micronuclei in the polychromatic erythrocytes of rodents has become the most widely used in vivo assay for the detection of genotoxins (Heddle et al., 1983). A particular advantage of the mammalian erythrocyte is that it is annucleate and thus micronuclei are readily observed in such cells. The micronucleus test has been modified by Jaylet and his colleagues for use in a range of amphibian species including the clawed toad XCWO~US kr~vis (for review see Jaylet et al., 1986). There is now an exte.:sive literature published by Jaylet and his colleagues which demonstrates the suitability of the amphibian micronucleus assays in the detection of genotoxins present in fresh water samples. The micronucleus assay has also been successfully adapted for use in the gill tissue of species of the mussel Mytilzrs as a monitor of the genotoxic effects of marine waters (Majone et al., 1987). In our laboratory we have been making use of the amphibian X~~zopusIueris as an experimental species for the assessment of the chromosome damaging effects of aquatic genotoxins. The potential of X~~opus for the monitoring of water quality has TABLE

1

The induction of micronuclei in the erythrocytes of larvae of Xmops ethyl methane sulphonate. Data derived from van Hummclen Developmental

stage

Concentration (Median

of ethyl

hiv

following

et al. (1989).

methane sulphate

frequency of micronuclei/ 1000 erythrocytcs)

49

I

8

45

51

0

3

I6

0

4

56

___”

14 -_i___l_l

14 days exposure to

Plate I. The induction of micronuclei in the red blood cells of stage 50 larvae of Xerqws kwrisproduced by exposure to 50 fig/ml of nitroso-guanidine (plate kindly provided by Colette Whams).

been outlined by Van Hummelen et al. (1989) who described the use of Xenopus larval stages to detect the induction of micronuclei after exposure to the alkylating agent ethyl methane sulphonate and the polycyclic aromatic hydrocarbon benzo[a]pyrene. In the Xmopus micronucleus assay larvae at development stages 49 to 56 are exposed to water borne genotoxins for periods of up to 14 days. The animals are then anaesthetised and blood smears prepared from cardiac punctures (Van Hummelen et al., 1989). An example of micronuclei induced by nitroso-guanidine exposure in the erythrocytes of Xenopus stage 50 is shown in Plate 1. It should be noted that the peripheral blood erythrocytes of Xenopus Zaevisare nucleated and thus the scoring of micronuclei is rather more difficult than in the equivalent rodent cell type. An example of the data obtained by Van Hummelen et al. (1989) is shown in Table 1. These data demonstrate that following exposure to ethyl methane sulphonate at concentrations of 50 and 100 pug/ml there were dose dependent increases in the frequency of micronuclei in Xrnopus larvae treated at stages 49, 51 and 56 with stage 49 larvae showing the greatest sensitivity. Current experience indicates the high sensitivity of the Xenopus assay. For example Jaylet and his collaborators (Jaylet et al., 1990) have demonstrated that benzo[a]pyrene produces a positive result in the micronucleus assay at 0.06 pug/ml and methyl mercury at 0.0025 pg/ml. THE INDUCTION OF DNA BASE CHANGES

Methods for the measurement of induced point mutation after chemical exposure are generally based upon the quantification of the frequencies of specific genotypes detectable in the presence of various selective agents (for examples, see Venitt and Parry, 1984). These selective systems include resistance to the effects of toxic drugs. as is widely used in mammalian cell culture systems (Cole and Arlett, 1984) and the growth of prototrophic cells in auxotrophic cell populations, such as are used in the Salmonella/mammalian microsome assay (Venitt et al., 1984). These selective systems have provided us with methods for the measurement of mutant frequencies in bacteria and cultured cells. In vivo, the availability of point mutational assays is more limited and is at present confined to methods such as the mouse spot test (see Fahrig, 1975) and more recently assays based upon the use of transgenic animals carrying selectable genetic markers particularly those of bacterial origin (Gossen and Vijg, 1990). In terms of environmental monitoring, the fundamental problem with all the above assays is that ihey are based upon the use of specialised laboratory methodologies that are not applicable to species present in the natural environment. We have been developing a methodology based upon the use of the polymerase chain reaction (PCM.) (for review see White et al., 1989) which may theoretically be applied to the study of DNA base changes in any gene of any species for which DNA sequence information is available. The methodology is based upon the measurement of base changes which occur in the DNA sequences which code for recognition sites for bacterial restriction enzymes (Parry et al., 1990; Zijlstra et al., 1990). As part of

339

our work on the development of methodologies for detecting genetic damage and genetic changes in aquatic species we have been utilizing the methodology which we term the restriction site mutation assay (RSM) to study the effects of genotoxin exposure to the olglobin gene XEL HBAl of Xenopus laevis (Partington and Baralle, 1981). The polymerase chain reaction (PCR) by alternating cycles of polymerisation and denaturation allows the amplification of DNA sequences between two unique reaction primers (amplimers). For the XEL HBAI gene we can utilize the region between base 20 and base 203 to amplify a DNA fragment suitable for mutational analysis. Using matched base primers 5’-ATATTGTCTGAATGAATGAATG-3’ and YAGATGTCCTAGAAGTATCAGT 5’ we can amplify a region of 204 bases by 30-35 cycles of incubation with substrates and Taq polymerase in a temperature cycler. The resulting amplified product can then be run on a polyacrylamide gel and can readily be identified as a characteristic band. Within the DNA sequence of XEL HBAl amplified by the primers there are unique enzyme recognition sites for the enzymes shown below:

Enzyme

Recognition

Hpa1 Hae 1 Dpn 1

GTTAAC

Mb0 1 Sau3A Xl102 Bait 1 N/a4 Mae 1 Rw 1

site

GGCC

GATC GATC GATC ;GATC;: GG’*CC GG%CC

CTAG GTAC

Treatment of Xenopzrs DNA with any of these enzymes results in breakage in the region 23 to 203 of XEL HBA 1. The resultant degraded DNA fails to amplify in the polymerase chain reaction when the reaction mixture contains amplimers specific for the region. Conversely, if the region 20 to 203 is amplified and then treated with the individual restriction enzyme the 204 base pair product can be cut to produce unique size fragments characteristic for each restriction enzyme. The basic principle of the RSM methodology is that DNA extracted from an organism exposed to a genotoxin may contain a sequence of interest which contains a wild type restriction site or may contain a sequence change leading to a mutant restriction site. DNA containing wild-type sequences, For example the sequence 5’-

340

CTAG-3’ is cut by the restriction enzyme Mael, whereas DNA containing a mutant site such as a C to T transition at the first base of the sequence, i.e. to S-TTAG-3’ will be resistant to the cutting action of Mael. Thus after treatment of DNA with restriction enzyme under optimal conditions all wild type sequences are cut whereas the 204 base region under study containing a restriction enzyme resistant site will remain intact. The restriction enzyme resistant sequence can then be amplified by PCR treatment to produce a sample of mutant DNA. Thus the Sasis of the assay is the selective amplification of mutant sequences. The procedure described above can be applied for each of the restriction enzyme sites present in the 204 base region of XEL HBA 1 thus allowing the identification of individual base sequence changes in each of the restriction enzyme sites. The extraction of DNA from a variety of tissues of an exposed species and subsequent restriction enzyme analysis will thus allow the analysis of induced base sequence changes in different tissues of the test organism. In principle, the methodology may be applied to any species, subject only to the availability of sequence information. The major potential problem with the RSM methodology is the degree of certainty we have over the complete restriction of wild type DNA prior to PCR incubation. As we have discussed elsewhere (see Parry et al., 1990) the presence of unrestricted wild type DNA could lead to artefacts in the method and it is important to use protocols which give maximum restriction of the genomic DNA samples. However, in our experience apparent artefacts can readily be eliminated by the routine restriction of the amplified product (Parry et al., 1990). At the present time the sensitivity of the RSM methodology to a range of mutagenic chemicals has yet to be determined. However, within the sequences we have selected for PCR amplification bases involved in restriction sites make up at least 10% of the amplified region and represent targets for mutagenic changes. The combination of the three technologies described here provide a methodology suitable for the assessment of genotoxin exposure from the starting point of lesion induction (“‘P-postlabelling) to the induction of point mutation (restriction site mutation analyses), to the formation of chromosome damage and numerical chromosome changes (micronucleus assay). Such analyses allow the assessment of the significance of lesion induction in the production of genetic changes which may be involved in the induction of deleterious mutations leading to tumour formation and inherited genetic changes. ACKNOWLEDGEMENTS

The work of the authors was supported by grants from the EEC Programme, the Health and Safety Executive, the Tobacco Products and Natural Environment Research Council. We are grateful to Dr. his comments during the preparation of the manuscript and to Dr. producing Figs. 1, 2 and 5.

Environmental Research Trust Ray Waters for Sian Ellard for

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