Xjenza Vol. 1 Issue 1 - 1996

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Editorial Xjenza: The First Issue Thc n d has long been felt among scientists in Malta for a local oullet for their work. parlicularIy for work dealing with local issucs that might not readiIy find acceptance by an internarional journal. It was from this need that thc idea for the journaI ,Yjcnza was conceived about cightccn months ago. an idea pul fonvard by a member of The Malta Chambcr of Scientists. This proposaI was later approved by the Council of thc same Chamber. Since thcrc was an encouraging response to a first call for subnlission of papcrs. it was decided to go ahcad with the ambitious vcnture. What you see in front of you today is the fruit of six mouths hard work which led to tirc publication of this first issuc 0fA7~nza.

All thc research articles in this jonrnal havc been subjected to pecr review. It is editorial policy to set high stsndards of peer revicw which should lead to cqually high standards of scientific research. This docs not meau that editorial policy will be soIeIy about publishing the best work of established scientists; it will also be our policy to cncouragc young scicntisls with promising new lines of r e s ~ ~ rtoc lput ~ pen to paper. In this way, .v:jenzn will provide a suitablc training ground for young scicritists secking thcir first publication. Aparl from original research articIcs, revicw articIcs on topical issues will also be fcatured.

A+ienzn wiII be fcnturing a section devoted to current research profiles which will serve to incrcase awareness among members of Ihe Chamber and the public, of the scientific rescarch being donc in MaIta in thc various disciplines of scicnce. This scction may include abstracts of papcrs aIrcady publishcd iu international journals.

The Editorial Board welcomes submissions from scientists in Malta specibing their research inleresl and activitics to ensure that the jor~rnal presents an opporlunity to all rcsearchcrs across the many scientific discipIines to featurc on a rcgular basis. It is hoped that ovcr timc A7enza would serve as a record of MaItesc scientific dcvelopxncnt and achevemcnt. . Y j c t m will also serve as a forum for the initiation and

dcvclopmcnt of debatc among thc Maltese scientific cornmnni~yon the ccntral scientific issues of the day. It wiII be a xnattcr for the Editorial Board to solicit contributions in this regard. Thc issues of debatc will tcnd to be thosc identified within and rcflccting the agenda of the MaIta Chamber of Scientists, but not esclusivcIy so. as thc range of issues might well include a debatc on the rolc of the Chamber itself. The journal will also carry a section on the activities of thc Malta Charnber of Scientists. In subsequent issues. a correspoildcncc colunm wi11 be included in which wc

hope to publish lettcrs from readers rclating to arliclcs featured in previous issues or on any topic of interest to our rcaders. We also welcome feed-back from readers on how, perhaps. we could improve the journal (assurniug that such a thing is possible!). Any readers with idcas for a new section should let us know. So plcase write in with all your comments. Wc assure you that we are a11 grownup women and men and mn takc criticism and ideas for change very wcll.

Another objective of .X]enza is to forge lirks between local scienlists and Maltcsc scicntists working abroad. A register of Maltese scientists working abroad will bc compiled with a vicw to famiIiarising local colleagues with their work and ericouraging cross-border Maltese collaboration. Attracting papers from such pcrsons abroad wouId help in this familiarisatiori process but more importantIy these scientists could be asked to act as rcferces for papers snbmniltd to the jounial. Such a register is being compi1cd. Rcaders who know of Maltese scientists working abroad and of their rcscarch interests. arc asked to write in with these details. Who knows what projccts and other benefits lnight be spawned in this wag'?

Since somc of the articles publishcd in this issue had been pending for some time bccause of a few teething problems, it was not possible this time round to includc dates for subn~issioriand acceptance of these arlicles. It will become standard practice from the next issuc of ,Yjenza which is planned for early 1997, by which tinlc wc also anticipate bcing on the Internet.

The first issue of &Jema has come into being as a rcsult of the supporl and dedication offered by my colleagues: Dr. Martin Ebejer. Dr. Richard Muscat, Dr. Emmanucl Sinagra and Dr. Christian A. Scerri. When spirits were low, they were always there with words of encouragcnicnt spurring me on. For this, and especiaIIy for their hard work. I do thank them.

I would also like to thank the many sponsors who havc made this jourual something akin to a viable proposition.

Finally I would likc to call on all Maltese scientists.

XJenza is really your journal! Make it come to life!

Wc look forward to receiving the best work from you.

ANGELA XUEREB EDITOR

Report

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The Malta Chamber of Scientists - The Second Anniversary Alex E. Felice and Richard Muscat Tiw Maialm I7mnher ofScientists, P.0. Box 45 I~bllcftn13.P.(3 In the first issuc o f .yj:icn:a. l'he Journal of the Malta Charnbcr of Scicntists. it is our privilege a s Foundation President a n d For~ndationmcmbcr of Council. to outline thc activities of the first two ycars of thc Chamber's life. This also gives us a n opporlunilg to reflect on what we scc as thc futurc of' the Chamber.

Thc MaIta Chanlbcr of Scicntists has bccorne thc national representative organisalion of practising scientists in Malta. Thcre is now an organisation which is adminis:ratively sound, with a hcalthy budgct. ready to facc the futurc with conficicnce.

The introduction of Ajenza roughly coincides with thc second anniversary of thc inaugural cvcnt with which l h c new Malta Charnbcr of Scicntists was publiclv launched. It completcs a packagc of threc publications that complement each othcr. T h e publica~ionshave bcen sustained successfully a n d it is hopcd that thcy will be developed further. A quarrerly newsletter, produced by Dr. Christian Sccrri. serves to inform mcmbers of Chamber

activities. a n d thc monthly Malta Sciericc Rcports in "Thc Timcs", edited by Dr. Richard Mnscat. providcs space for thc mcmbers of the Charnbcr lo give visibility to their work in the e.ycs of the gencral public.

Togethcr the?; contribute substantially lo increasing science awarcncss and sciencc literacy especially anlong thc younger gcncrations. I t is known that thc Malta Science Reports arc read with considerablc intercst by prorninent members of thc reading public including high officcrs in the civil scnlice and diplomatic representatives.

T h c rcgular Business a n d Scientific fvlcctings havc bcen prominent activities successfully sustaincd for thc Iast two years. Many important a t ~ drclevant topics havc been covered ranging from basic or enabling sciences to science policy and industrial applications. O n e of t h e most riotablc events was the joint mecting that was held with the PUGWASH Conferencc on Sciencc and World affairs. This was the first public appcarrtnce of

I-Iis Esccllcncy Dr U :v;itsud Bon~iici,Prcsidcnt 01- lhc Kcpublic of Malta addressing the Chamber at its i ~ t a u g u n ~ i on o ~ i2R July.

1994, at the Universe of Malta. Sccn to his leH arc: f m f AE. Fclice. President of the Chamber, Rev I ' d 1.' Scrmcir~o111 Jon Rmlnr ofthc University. Mrs Mifsud Donnici and Prof V. Femto, Presidentelcct of the Chamber.

PUGWASH and of Nobcl Lmreate Joseph Rotblart after they had been awarded the Nobol Pcacc Prizc. Since thcn. the Malta Chalnbcr of Scientists has rcprcsentcd thc Pugwash Conference on Science and World affairs in Malta. Il is hoped that the role of the Chamber in this prestigious group will bc incrcascd. Equally important meclings on sciencc policy haw becn hcld in conjnnclion with thc Malta Developrncnt Corporat~on and other business leaders. Following such a meeting, an agrcemenl was rcached with Mcssrs. Sin~ondsFarsons Cisk L,td., for financial support for thc scientific nieclings. Hcnccforth. thcsc scientific meetings shalI be known as the Farsons Serics. The Charnbcr played a n imporlant part in the Malta Scicncc Week by providing public speakcrs and many members contributed in various capaciiics. I l is hopcd Lhat Ihe Chamber will participalc again ncst year.

Thc Chamber continues to grow and to establish itseIf on Ihc Maltese profcssional sccne. There are now 200 rncn~bcrsdrawn from all seclors of the national cconomy and f r o ~ ~ a11r scientific discipli~~es.

A p~~ofessional: rcgistcr of scicntists has been compiled for the first :in~e. This register will occupy an irnporlant position in Llzc ccrtiilcation of sciencc spccialisls in lhc contest of profcssional recognition within thc Europcan Union.

Furthcr g r o ~ l his possible by targeling new graduatcs for mcrnbcrship. Thcrc is scopc for undergraduates and possibly sixth form scier~ecstudents to be aFIiIiatd with the Chamber of Sclcnlists in a Junior Chnbcr. Also a Corporale Clwnkr desemcs i~rtcntionto su.st.aIn gronlh.

There haw been areas in which less success has been achieved, such as in promoting a m d t a t i o n and continuous professional development credits. This is important because shortly it will no longer be possible, in Europe, to use my professional .scientrfic designation without an appropriate national accrdtation proccss. Pcrllaps, m n t , sad wcnts in the industrial scclor serve to emphasisc the importance of this issue in a dramatic manner. The membership should be left in no doubt as to thc irnponancc of a properly conducted aarcditation system which will senle a!l members in enhancing their profmional standing in Miita and within the larger European context. The next council will undaubtedl~havc the onus of in~plernentinga professional accreditation syslcm lrnked to progranrtm for conlinuous professional development. The new council will also have to work lwdcr at the political level to ensure stable and sustairid funding for scientific rescarch and lo improvc the Iink between enabling sciences and social or cconornic developn~cn~, perhaps, through a National Formdatjon for ScienlxGc Resuch.

All thc abovc mentioned acriviks would not have boen pssible without thc efforl put in by tho.% members silting on various conuninees of the Chmntcr. Also nwiy compxuics have supported the Charnkr and spccial rnenlion should be oven lo thc Malh CounciI for Science and TecImology for a foundation grant of Lm 1,000 and Messrs.

Lowcnbrau Ltd. for cordially hosting council meetings. It is hoped that this slrpport together wilh addiCiona1 public and private SECIor financial support will be attracted in thc future.

Membership Full membership of the Malta Chamber of Scientists is open to all individuals possessing a first degree in a science related subject. Candidate membership is open to students reading For a science related degree. Membership fees are due on the I June of each year.

Yearly membership fees:

Em 10 for hll membership (Lm IS for a married couple both of whom are members).

Em5 For candidate members.

Benefits include:

1. Subscription to Xjenza 2. Right to attend all monthly scientific meetings 3. Quarterly newsletter'

Please n~ailor Fax your details to the: The Malta Chamber of Scientists, P.O. Box 45, Valletta B.P.O., Valletta.

TeI:/Fax: 343535

Xjenze 1996; 1:1 6 7

6

Communication Wind Energy in Malta Pius J. Darmanin and Edward A, Mallia Deportment of Phpics, University of Malta, Msidu Wind energq is an atkactivc dtcnlative sourcc of energy. It is becoming increasingly popular in northern munlries which llave a g o d wind resource. It is estjmatcd that Iherc are about 17.000 machines providing about 2.7 GW in munlries snch as D c t w k . Gcrrrwi~. S~vcdcn. China.

Tnda. Hawaii. and the U.S.A.etc.

Wind e n e r p is a renewable. frecly available. nonpolluting sourcc of cncrgy. Although the wind is notoriously variable, thc rncan encrgy available is generally reliable constant over long periods of time.

Thc wind cnergy resource of any counrry depends on two main factors; thc gencral rnctcorological conditions that cffect thc particular zonc. atid thc Land area available for farms.

7.31n/s and provides 235w/rn2. Both valucs are small when compared to the required input loading of 8 3 0 ~ / n lThc ~ . estimated annual wind encrgy at Luqa is 2944 k~-hr/m'.

Thc rangc of wind spccds obscncd. allow n typical WEC to providc clcctricity during only 62% of Ihe rime: it would be at a standsrill for an accumulated period equivalent to 4.6 rno~~ths. half of which would occur from July to September. Furthermore during the 7.4 months production pcriod. thc conversion elficiency varies from 8% to 46% as is &own in Tablc 1 :

%

Observations of wind speed and direction taken by thc McteoroIogical Oficc at Luqa dnring the period 19721991 have been analyzcd for a dcternlir~ationof wind cnergy in the Maltese Islands.

The Prevailing Wind Direction Thc prcvding nind direclion is Nodl Wcst (260"-340").

Wind in this sector blows for 4 1.4% of the time and conlributcs to 54% of the total energy contcnt. East ro South East wind (080"-160') blows for 18.8% of thc timc contributing 19% of the total encrgy. These two reciprocal scctors contain 73% of the totaI wind cncrgy.

Thc best locations for wind energy convertcrs (WECs) in Malta arc those areas exposed to the Nonh Westerlies.

Conversion efficiency

Time 26.1

0.2

]

1

%I

11.8 45.7 33.1 7.9

I

Remarks Wind sped low (4.5 mk - l l mb) Wind speed idd (ca. 1 1 rills) Wind s p e d high excess energy is spi llcd Wind speed too high

I I mk):

Wind speeds too high

'Tablc I . Kangc o l wind s p e d ubscrwd and coriwmon cRicicncics

Whcn the wind spccrl is higher than thc ratcd capacity of thc WEC, thc excess energy is purposely spillcd. keeping Ihc machinc producing at its maxinlu~nrated output. Onc typical WEC (irrespeclive of size) in thc Maltese Islands can achieve a meau annual ovcrall cficicncy of 3 1%. This means that onc 3MW WEC can producc 7 154 MWhr annually.

Wind Enera to Electrical E n e r ~ v Wind h~rbincscome in a range of sizes, the biggest, havc a rotor dia~ncterof loom, stand on a l00m high tower (Delimara P.S. stack I IOm) and can produce 3MW at a wind spced of I I rds. Sn~allerpraclicaI turbincs have a 42m rotor. stand on a 50m tower and can produce 0.5MW at the same wind speed. This rneans that cach largc machine provides as much cncrgy as 6 small ones.

These machines havc an clectrical loading ovcr 370w/lni which implies that they require an input wind c n e r p of about 830iV/m2. Only pan of Ihe wind kinetic energy. can be convcrtcd to practical use duc to aerodynamic and electrical constraints. It is necessary. therefore. to assume a @pica1 WEC to evaluale the amount of clcctricaI energy that can be produced.

In 1975. a German tcam who carried out a similar study, proposed a wind turbine farm of lOMW producing 22.000 MWhr annually. amounting to 7% of the encrgy generated in 1974/75. I t works out that the demand for clectricity then was 3 14 GWhr p a . Due to the increased demand, the same farm would loday producc only 1.4% of that supplied in 1994.

Wind Characteristics and Encrgv The mean wind speed, at lO0m abovc ground level, is 6 . 2 d s and provides i42w/m2. thc median wind speed is

It is intcrcsting to note that while thc lMallcse popu1arion has grown by 21% between 1975 and 1994, he elcclncity generated rose by 380% during the m n e period.

Electricit!: Demand During the period 1993-94 EncmaIta produced a total of 1506 GWhr of clectrical encrgy to meet dernand. Thus. a typical output from a 3MW WEC reprcscnts onIy 0.48% of the nationaI dcrnand for electrical encrm. i.c. 10 largc LVEC would supply ca. 5% of the dernand.

_-.-, -

The f&sibility of Using Wind Energy in Malta We suggest that the corrected Luqa observations proGide a sound basis for a =on on wind energy utrl~sar~ori In Malta Of course. the,obsenlations arc sitc specific. Thc search for the best s~rcsis still opcn. and r l can be tackled with more rcfincd methods of observation. It takes 12 large WECs to replacc the gas turbines in current usc, w h ~ l c81 would bc t~ceded lo replace cornptelely one of Dclimara's Steam turbines. Such a number of WECs w o t ~ : ~ a http&ct ~ ~ on +the eaesthetics of the %s asthd besf s k s are likely to be of high' S&MC.', 'fahd~c~poand ecological value.

AWfMtFve &es may be found in industrial zones '

-_..-

such as Hal Far which has a high exposure to the south sector. Such a site might take 5-10 large - ('3h;i'w) Wf@s %ui may still suffer from proximity to thc line of the main runyay at Luqa airport. For workcrs in the 'd tb&:WECs may produce an intolerable noise level. Thest considerations suggcsl of that wind encrgy should b$ given a lower espldfaiPdn. than rNore! Wrkd *&bar %f&hatives like domestic solar u'alcr heating and distributed and cenlralised power gerteration via phorc+vollacs.

Mormw, sdrious consideration should be given 10 WECs dedicated to 'hroduction of hyd&j$eri by electrolysis rather than to augmenting' pow& in the grid.

3 Your favourite Lager...The better Lager

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1

Communication

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A Case for Biological Zeros

Most ecological work on rocky shorcs cntails thc quantification of specics distributions and abundances relativc to some comenienr and accurale daruni point.

Thc most commo~~ly uscd arc tidc Icwls and chart data.

The problem arises when tides arc virtually absent. as in most of Ihc Mcditerrancan Sca or whcn accurate chart data arc not available.

In the Maltcse Islands therc is only onc dalunl point. locatcd in rhc Grand Harbour. Valletta (14" 30.64' E. 35' 53.60' N). A number of bcnch rnarks and ~rigonorr~etric stnrions of various ordcrs are fbund scattered all ovcr Ihc Islands. but thcse arc not alnrays located very closc to thc shorc so that lcvclling work from thcsc slations lo a particular sludg sitc may. become quite a laborious task uhich may exceed thc capabilitics of most ecologists.

Furthermore, some of these marks arc old and location data c for them are untraceable, while others have turned out to t inaccurate (William, E, personal communication. 1993). In fact. a project is currently underway by the Mapping Unit of the Planning Authority to recalibrate these stations and to establish ncw bench marks. A furthcr complicating factor is that difkrent charts make use of different datunl points. Thus, while Admiralty Charts use a zero point (Chart datum) which is the level of lowest aslronornical tide and the lcvel to which all bathyrnetric soundings are referred, all heights shown on tile official Government of Malra s u n q shcets use a datum point which is 0.5859ni abovc the Admiralty Chart datum and which is takcn to be mean sea level (MSL) for thc Maltcsc Islands. Additionally, there is also a Public Works Departmcnl (PWD) d a t m which is 0.4100m abovc the AdrniraIty Chart datum. It is not always clearly stated which datum points charts and maps are based on.

This state of affairs has resultcd in ficld workers having to resort to some othcr (possibIy less accuraltc) datuni point with which to relate all thcir data.

By far thc rnosc colnmonly used reference point is thc socallcd "biological zero". that is, the uppcr limit of phacophyceans of the genus Cystoseira in qumlity (cfr.

Boudouresque and Cinelli, 1976). Thcse shrubby brown algae form a widc, mainly infralirtora1. bclt on most Mediterranean shores and in most places stop forn~inga co~lsistentbclt at aboul rncan sea lcvcl. This has lcd

sornc workers lo utilisc specics of this gcnus as irtdicators of mean sca levcl. Different specics or combinations of species occur in differcnl parts of rhc Mcditerrancan. In thc Mallcse Islands the most commonly occurring specics at sca Icvcl arc ('. s/ric[n. ('. cottpre.ssn, '. holenrrcn and T . harhnin (Atrard and Giglio. 1990: Borg. 1992: Callcja. 199 1 ; Carniller~ and Flcri-Solcr.

I99 1 ; Vella. 1990).

Obi.iously. the position of this datum is so~uewhat subjective a i d cannot bc dekrmined with rhe smnc precinon as for suwcyed Icvels. which may bc accurate to the nearcst niilli~netrc.Nonctllclcss lherc SCCITLS to bc sornc rclationship betivecn this darun) and m a n sen Icvcl as any visit to the seashorc on a caln~day would suggcst.

I t should also be kepl in mind that thc zonation of thesc algac on thc shore is expected not to depend solely on physical faclors, but also on biotic ones. The fact that different species, with different tolerances. occur in different parts of rhe Mcditerrancan aud possibly also on differcut shores in the same geographical locality, also nlcans that "biological zero" may not be the same cvcqwhcre.

As part of a wider project aimed at studying the zonation patterns of rocky shorc cornolunities in the Maltesc Islands (Mallia. 1993). a11 attempt was made to quantib the relationship, if any, betwcen this "biological zero" and m a n sea Icvcl for the Maltesc Islands.

This was done by fixing a 4cm long brass stud in ;I pre-drilled hole by means of Araldik adhesivc at two stations. Qawra and Dahlet ix-Xmajjar. on the northeastern coast of Malta. Thc hc~ghtof tliis stud abovc biological zero was deterrnincd using a slightly modified version of the can and tube mcthod dcscribed by Eifion Jones (1980). Nest. thc hcighl of the studs abovc the MSL datum was determined using the lcvel and staff method and slarting from the nearest trigonometric station or bcnch mark. Thc difference betwcen thc two mcasurcn~cntswas thcn calculated (see Table I).

Thcse results show that although rhc biological zero docs not cni~icidcwith thc MSL datum, thc difference bctiveen lhc two is fairly constant. Tlic differcncc

'~resontAddress: Eriviromncntal Managcrncnt Unit.

Planning Authority, St Frarlcis' Ravelin, Floriana

A Case for BIo~wIc~IZeros

LOCATION

Qawa DaMet ix-Xmaijar 'fablc 1.

9

Height of stud above "biofogical zero" (bottle and tube) 0.87m 0.88m

Hcight of stud above MSL (level and staff )

Difference in heights

1.IOm 1.12111

0.23m 0.24m

Comparison of hcight data for thc two localitics.

bctween thc two localtics (0.01m) is very small and within the crror range of the can and tube method.

Thcrcfore. thesc resulls indicate that in the absencc of an accurate, lcvcited datum, the biological zero is a rehabIc substi1utc datum point for ecological work on rocky shores.

Rcccnt work in thc Maltese Islands has also shown that o~hcrorganisms might also be useful as indicators of sea level. For examplc. Azzopardi (1992) found that thc Incan shell apcnure of thc reef-forming, vermclid gastropod Derrdropoma petraeurn, which on Maltcse shores estcnds its range of distribution well into thc lou'cr mediolittorai zone, is lowest towards mean sea Icvel and increases on either side of it. Such organisms may be used as indicators on shorcs where a dcfinite C~ysto.wirabelt is missing.

References Attard L and Giglio MP (1990) Zonation patterns on a Maltese rocky shore: Qalet Marku. Unpublished B.Sc. disscnation, Faculty of Science. University of Malta; 167pp.

Azzopardi L (1992) Aspects of the ecology of verrrtetid gastropods on hlaltese r o c b shores. Unpublishcd B.Sc. dissena~ion.Faculty of Sciencc. University of Malta: vi + i63pp.

Borg R (1992) Zonation patterns mound the const of Qmrm peninstila. Malla. Unpublished B.Sc.

dissertation, Facully of Scicnce, University of Malta; ix + l64pp.

Boudouresquc CF and Cinclli F (1976) Le peuplenlent algal des biotopes sciaphiles supeficicls de modc battu cn Mdditcrrande occidentale. Puhhl. Slaz.

Zool. hrapoli, 40, 433-459.

Callcja M (199 1) A /gal zonation and epiphytes along a ~lloltese rocky coast.

Unpublished B.Sc. dissertation. Faculty of Science, Univcrsiry of Malta; vii + 136pp.

Camilleri MV and Flcri Soler L (1991) A corr~parafive sludv of the mncrophyfic nlgae of three sites siluated along the north-east coast of Malta.

Unpublished B.Sc. djssertation, Faculty of Science, University of Malta; iii + I26pp + appendices.

EGon Jories W (1980) Field leaching methods in shore ecology. In: The shore environment Vol I: hfethoch (Eds WF Farnharn. DEG Zninc and JH Pnce), pp. 1944. Academic P m , London.

MalIia A (1993) EDct of exposire on rocky shore zonation palterms in the Maltese lslands. Unpublished MSc. dissertation, F a d t y of Science, University of Malta vii + 242pp.

VcUa M (1990) A study of the variation between the alga! contmunities of an exposed and sheltered site along the Maltese coast. Unpublished B.Sc. dissertalion Faculty of Science. University of Malta; viii + 124pp Williams E (1993) Deparlment of Land Sunc-yhg, University of East London, Essex.

Review Article The Current Status of Predictive Genetic Testing for Cancer in Humans: Scientific, Clinical and Ethical Issues surrounding the p 5 3 gene.

Our cr~rrenl understar~di~lgof thc ruolecular basis of human cancers has raised a \,er_v critical issue which is ~harof predictive gcrretic testing for canccr in humans.

Chcr the past few years so much data has bccn forthcoming that it is limclg to revicu the situatiol~.

For many !;cars it has bccn h~pothesizcdthat cauccr must have a gcrictic componcnr. As long ago as 1914. Bovcri suggeslcd that an abcrralion in the genomc migh[ bc rcsponsiblc for malignant transfonnalion. Subsequent work has supponcd this theon: and we now define cancer 111 humans as bcing n gcncric disease at cellular. I c \ d Evidcncc suppo11ing the observatiori that cancer or (hc risk of canccr could be inherited comes from thc work of Mullvihil ct a1 (1997) arid Li ct a1 (1988) on familial cancers.

Alrr~osleven. fontn of canccr in hun~anshas bccn rcported to clusl.er in fandies. This can bc cxplained, either by thc inheriuncc of a ~nutalcdsusceptibility gcnc, or by chance i~ssociatiori artd shared exposures lo enwl-onmcr~tal carcinogens (Knudson. 1989).

Since the early 1'380s. extensive rcsearch world-wide has bccn undertaken in order to identifv the gcncs responsiblc for rualignanl transformation of morphologically rlormal cells through their subsequent mutations. Some of thesc genes have been idcnlified. Thcse include the hereditary rc~inoblastor~ia(Rb) gcnc, [he Wilms' tuniour (WT 1) gcnc. lhe ~tcurofibroma~osisIjpc 1 gene. the familial poIyposis APC gene, the Li-Fraumcni Syndrornc (TP53) gcnc, the malc breast canccr (AR) genc, the dclctcd colon canccr (DCC) gcne and thc recently discovered faruilial brcasUovarian cancer (BRCA-I) gene. and the familial brcast canccr (BRCA2) gene (Knndson. 1'389; Wooster er al. 1995; Miki, 1994: Stratton, 1995). Two recent srudies havc shown that mutations in the BRCA-I gene which predisposes worncn to brcast and ovarian cancer may also bc associated with :in increased risk of' prostate cancer in mcn (Hall ct al. 1990: ~Miki ct aI, 1994: Easton cl al.

1995). ~Vappingand identification of thcsc cancel- predisposing gcnes on diffcrcnt chrornoson~eshavc been facilitaled in rcccnt years by [he applicalion of new rnolccular biology tcclmiqucs. Thc polymerase chiiin rcaction has in f a c ~ revolutionixd the approach to gcnctic rcscarch ~hrough its ability to cyclically a~nplifvthe genomic regions of intcrcst. As a rcsull of thcse technical advanccs we have a

situation where marry gcncs h a w bccn idcnlificd and others mapped so rapidly. that our u~~dcrstanding of thcir biological significance has not kcpl pacc.

Tl~cre is cvideucc to irldicalc thal for many typcs of' canccr. including thc ruost common forms such as brcast. lung. colo~r and bladder. thcrc csisls not onI! an cnvironrncntal influcncc but also a hcrcditaq predisposition to thc dei~clop~iicnt of canccr. This can n o v bc clear'l>.illustratctl by thc classical csanlple of lung cancer as it is relalcd lo cigarctte smoking W h i l s ~ appreciating lhc evidencc that cigarcttc smoking is clcwlj Iinked to h e devclopnmr of lung canccr in nlosr instances. nor all snmkers ncccssarii): cle\.clop lung cancer. Also, thc mortality rate from lung canccr among non-smoking relatives of lung cancer patients (smokcl-s) has bcen shown to bc highcr than t l u r of non-smoking relatives of non-smokers uscd as controls (Garfinkc1 er al, 1985). Similar rcsulls wcre obtaincd whcn comparing rhc mortalily ratcs from lung caucer of s~nclungrelatives oP lung cancer patients to illat of srtmkers with no L';lrnil>. hislor). of lung canccr. So it appcars that gcrtctic irlfluc~~ccs Inus1 contrib~ltelo thc dcvelopnie~tlof ainccr. cven wtrcn thcre arc cleady dcfirrcd cn\:irontncnral factors. .4 genetic prcdisposition to brcast canccr has also bccr~ shown (Adami ct a1. 1980: Bain ct al, 1980). The exact role that hcrcdity plays i r ~thc prcdisposition to p;i~~icular brcasl callcers cannot be quanrificd. Lj.uch ( 197 J ) statcs llial approximately 10-15% of brcasl canccrs havc ;I hercdilav background. Clcarly. \voIncn who ham no fanlily history of brcast canccr may also dcveIop rhc discnsc. but thosc individuals from fm~ilicswith a hisrory of brcast cancer arc a1 somc increased risk. as st ion:^^ by Andersort (1972. 1971. 1977). In gcncral. rlw risk of thc same ncoplasm dcvcloping in closc rclativcs of a cancer paticnt is approximalelg lhrcc tirucs grealcr tha11 i r ~ control populations (Knodsen, 1989).

A frcqucnlly asked qucslion collccrning bolh Ihc canccr pat.icnrs and thcir rcIalivcs is:"M? n~othcr or my fdther. or possibly both. dicd of canccr. Docs this mean [hat I will certainly devclop canccr? Under thesc circurnstar~ces, what arc my pcrccntagc risks and what can be donc lo prcvcnt [he disease from dcvcloping'?" This brings us to the vc? essencc of Ihc subject of prcdictivc gcnctic tcsting for canccr i n humms.

p53 Gem

Gencs predisposing to cancer

At molecular Ievcl, it is thought that cancer is thc cnd result of an accumulation of gcnetic Iesions occuring in key rcgulatoq molecules. Tlus widely held conccpt is gaining in importance as more informalion on growth arresl and cell death in Ure regulation of ccll number bccomes uriderstood. The identification and fimctional role of these nlolecules are the subject of intense rcscarch.

Positional cloning technology has begun to accelerate identification of gencs that are responsible for familial cancers (Ruddon. 1994: Symonds et al, 1994). Over thcse last hvo years nlc have scen thc clolung of two ven; i~nponanl cancer prcdisposing gencs. BRCA-1 w k i , 1994) and BRCA-2 (Stralton, 1995: Tavtigian. 1995).

Gcncs that slow down cellular turnover, in other words growth inhibitors, arc termcd tumour suppressor genes (Knudson. 1989: Li. 1988: Wcinberg. 1991). Scventccn ycars haw passed since thc originaI discovery of a nuclear phospl~oprotein with a molecular mass of 53-kd that reactcd with antiserum from aninlals with tumours induccd by simian virus 40 (SV 40) (Litner ct al. 1979; Lane and Crawford. 1979).

Thc p53 gene is the most widcly altered gene in human canccr (Hollstcin et al. 1991; Dc Fromcntal and Sonssi.

1092). It is a tumour supprcssor gene which in its normal form codes for a 53-kd protein which binds to DNA and acrs as a transcription factor ro halt cells in the G1 to S transition of the cell cycIe (Clarke el al. 1993). Mutant forms lack this DNA binding activity and therefore allows for abnormal proteins formcd during malign an^ ccll transformalion to procccd in thc cell cycle. The p53 genc spans a moderately sized segment of DNA (20 Kilobases long). locatcd on the shon arm of hurlran chromoso~ne17. that is ultimately translalcd to a protein consisling of 393 amino acids contained in 11 csons, the first of which is uon-coding. Five elroIutionaty consened domains wirhin thc coding regions are regarded as essential to the funclional activity of p53 (Malkin et al. 1990).

In thc presence of DNA damage induced by gamma irradiarion or chcmotherapcutic drugs, intracellular Icvels of p53 risc and prompt rhc expression of a downstream gcne WAF/CIP I , whose protcin product p21 binds to qclin-dcpeudent kinascs and inhibits rhcir activity (Harpcr et al. 1993; El-Deiry el al. 1993). In this manner ccll cycle is arrested prior to DNA synthesis and thc celI is given thc opportunity to repair the darnaged DNA. If such rcpair docs not occur, thc presence of nornlal p53 induces the ccll through a pathway of apoptosis or progra~nmcdcell dcath (Harris and Holsteiu. 1993). The apoplotic pathway is still poorly understood (Yonish, 1992; Stcwart, 1994). The early chcmical cvenls that cause apoprosis have bcen so far hypothetical and lhcsc includc: increases in ionisable calcium in the cytoplasm, drops in pH, generation of frce radicals, and phosphorylation cascades. That p53 plays a cardinal role in thc early cvents of apoptosis has been shown by the work of Stewart, (1 994).

11

During the past seventeen years some 1300 nlutations have been reported in more than 55% of all sporadically occurring hlmoars (Nigro et al, 1989). In 1992. the p53 gene was given the honour of being the second most significant scientific trend of the year in the Time Magazine (Tirnc 1992). In 1993, p53 was nanlcd Molecule of thc Year in Scicnce (1993).

Understanding thc role of p53 as a cancer predisposing gene comes from the clinical and scientific work on thc Li-Fraume~uFandlial Cancer Syndrome, first describcd as a clirucal entity in 1969 by Li and Fraumeni, who notcd the associalion between young onsct sarcoma and other lurrlours in closc relatives (Li, 1988). Tlus syndrome is an autosomal dominant disorder thar predisposes individuals lo multiple forms of cancers occuting at a young age and in cIosc relatives. It consists of a sarcoma developing in a first degree relative before the age of 45 and a second first dcgrcc rclative who has developed any type of canccr under the age of 45 years or a sarcoma at ary agc Makin. (1993). Othcr characteristic falures of the yndromc includc thc occurancc of multiplc primary cancers in affmtcd individuals. the early age of the patient at onset of mast tumours and the autosomal pattern of inheritance of thc disordcr as determincd by classical segregation analysis Malkin.

(1992).

Conlponent tumours of the syndron~e include breast cancers, leukaemias. brain tumours and adrenocortical tunlours. A recent study by Kyritsis et al (1994) reporting on germline mulations in the p53 gene on a set of glioma paticnts is important in that it sustains the recent obsenlations that germline p53 rnutations may occur outside the classically defincd LFS famiIies (Frebourg el al. 1992; Malkin ct al, 1992; Toguchida er al. 1992). This obscnfation colnplicatcs the sccne of prcdiclivc genetic tcsting for the classical Li-Fraunleni Syndrome (Malkin cl al. 1993).

Other cancer predisposing genes whose hnctior~is that of gronlh inhibition include: the rctinoblaston~aRb gcne, WiInls Tumour Gene WTI, WT2, thc adeno polyposis coli gcne APC and BRCA-I/ BRCA-2 gcnes.

Studies on lhc rctinoblaslorna genc dcmo~lstrdte the correlation between hereditary nlutalion in llunour suppressor genes and genetic predrsposition lo dcveIop cancer. Patients wilh the hereditary form (about 40%) have a high risk of m u l t i f d retinoblasloma and othcr turnours in thcir family members and they wry pass the disease to their progeny as an autosomal Lrait. Thc same faturcs of hereditary c;tses of Rb can also occur in paticnts with no family history of the disease and are duc to new mutations in the g e m linc. The non-herdtary cam are unrfocal, m$.h XI oldcr average age of onsct (Vogel, 1979; Knudson et al, 1989). By analogy with retinoblastorna. some patients with sarconlas of bonc and soR tissues and no family history of cancer may be carriers of ncw germline mutations at the p53 locus (MaIkin et a1, 1990) .

Implications of Germline Mutations ObsenAonal studies in thc mutation spectrum of the p53 gcnc have been going on for quite some time (Ba~tcket al, 1992). Wc are now at a stage were the focus of currenr work is to correlate the significance of thesc murations with clinical outcome (Harris et al. 1993). The frequency of canccrs among carriers varies from 50% lo 90% up to the age of 60. Above the age of 60 years thc risk of developing canccr is said to be the same as that of the gcncral population of the same age who do not c a r r ~a mutation in the p53 gcne (Garbcr ct al. 1991; Srivastava et al, 1990: Hollstcin ct al, 1.99I ) .

This may be explained by considering tile f x t that lhc prcsence of a parlicular mutation might just be a rare poly~norphisrn and so thcre would be no biological significancc of this mutalion on cell growth. Therefore. any genetic tendency to develop cancer would ~nanifcst itself carly in lhose individuals whose mutations arc not just a rare variant of DNA. but would not do so in those individuak tvhosc mutations are of no significant functional activity (Lynch and Kursh. 1971; Ory.1993).

When discussing the relativc risk for devdopmcnt of carly onset breast cancer, the overall penetrance of gene carricrs in the Li-Fraumeni Syndrome is 90% by thc age of 50 years and thc nlmjority of cancers after cluldhood arc breast cancers. Outside thc Li-Fraumeni Syndrome families, germ-line p53 rnulations haw also bcen reportcd in patients who dcvclop muItiple primary cancers and in patients with a suong family history of cancer afkcting mdtiple tissucs (Frebourg and Fricnd. 1992; Malkin et al.

1992: Toguchida et al, 1992).

Biological and statistical issucs also surronnd survcys for gcrm-line p53 nuta at ions in population studies (Shapiro, 1089). The prcdictive powcr of a posilivc test for p53 is delcnnined by t h e e filctors: 1. the prevdence of p53 mutations in the study population.

2. the sensitivity (the probability of detecting a truc positive) of the test.

3. the specificity (probability of detecting a true negative) ofthc test.

Even when sensitivity arld spccificily are vcry high (99%), the prcdictivc power of a positivc test is only 50% when the prcvalence of p53 mutations in the sunfc. populalion is 1%; i.c.. only one half of those with a positive p53 lcst actually arc cancer-prone individuals. Thc power of thc tcst is increased substanrially by studying populations with a high prevalence. preferably greater than 10%. In prcdictivc tcsting of s i b l i ~ ~ gand s offspring of cancer palicnts with a germ-line p53 mutation. lhc prcvalence of rnutarion is as high as 50%. Available data suggest that the prevalence of this gerrnlinc niutation might be 0.01% in Ihc general population, 0.1-I% among various canccr patients, and 5-10 ?A among young patients w,ith multiple primary canccrs (Li et aI, 199 1).

Mutation-screening Techniques The p53 protcin can bc detected immunohistochemically using monoclonal antibodies against t h s protein. During 13 years of work on this protcin. it has been demonstrated that its overexpression can be detected in a wide variety of human malignancies including cancer of the breast. colo11. lung, bladder. prostate and brain (Nigro, 1989).

The i~nrnunol~is~ochcmical techniquc has k n shown to fail to stain both prcneoplastic and neoplastic cclls carrying a mutation of the p53 gene. Conversely. it has been shown to stain cclls in a cancer family in which the p53 gene is normal (Barnes ct al. 1992; Eeles et al. 1993).

Normally. the p53 protein occurs at a very Ion concentration in cells because i t is rapidly dcgraded by cellular proteascs. Howcver. using monoclonal antibodics against the mutant p53 protein. canccr cells often dcmonstrale high levels of the abnornial protein which accumulates in ttic cells. In transilional ccll carcinoma of the urinay bladder. de~ection of pS3 p.rotcin accun~ulationlias been rcporlcd in up to 61% of invasive tunlours (SidransLy i l aI, 1991; 1992). The imrnunohistochcmicaI Icchniquc still remains useful for detecting p53 o~~ercspression.

It can bc perforrncd on biopsy ~natcrialas we11 as on exfoliated cclls such as in cervical smears. serous effusions and spulum.

Morphologmlly normal cells, overexpressing thc p53 protcin, are presumed to indicatc a prencoplastic stage of cellular dflerentiation.

Thcrc is no question that as far as Lcstmg for p53 gcnc nlutation is concerned. the prima? tools are thosc of molecular genctic techniqucs. Mutations within thc genc are widcly dispersed niainly between codons 130 and 290 and most of them involve the evoIutionary conservd domains. In particdar. at lcast three mutational hot-spots at codons 173. 248 and 273 have en~crged.Mutations at these hot-spots arc characteristically transitions at CpG dinucleotides. Canccrs originating from various specific tissue sites differ with respect to thc distribution and frequency of mutations at these hot-spots [Hollsteiri ct al.

1991: Caron de Frornental and Soussi, 1992). Prchclive testing for p53 genc nuta at ion involvcs testing of the whole genc. A numbcr of screening kchniques for thc detcction of point mutations are available and providc the approsimatc location of the mutation.

Current mctl~odologics use thc polyrnerase chain reaction (PCR) to amplib a particular scgInerkt of the gene being investigated. The most commonly uscd mutation screening techniques are: single strand conformational polymorphisn~ (SSCP) (Oritia et al.

1989), denaturing gel elcctrophorcsis (DGGE or a \,ariant CDGE) (Fisher ct al. 1983; Borrcsen et al, 199 1) and with chcrnical rnismatch cleavage (CMC/HOT) (Montandon ct al, 1989; Curie1 I . 1990).

The basic principles are as follows. I n SSCP, single strands of DNA havc a diffc~ent secondar?; conformation depending on their base composition. In DGGE, doublc stranded DNA denatures at dilrerent

p53 Gene

temperatures or concentra1ions of denaruranl. depcndent upon thc base pair compositiott. Thc CMCHOT tecliriiquc niiscs normal DNA with teH mutant and allows singlc strands from each sample to reanneaI. At lhe sitc of a basc mutation. a mismatch occurs arid can bc idcrilificd by a chctnical which binds to thc nlisrnatch and acts as a cleavage site for pipcridine.

Each tcchniquc has its advarilagcs and disadvanlagcs.

SSCP and CDGE are rapid, but each exon (or at the most. two esons together) of rhc p53 has to bc atlalyscd scparatcly. Both havc a sensitivity of ncarly 80%.

Howcvcr. it is unlikcly to bc a long lerrn solutio~ifor population screcriiog in sporadic canccr bccause it is not so uscfi~l for analysiiig largc PCR products where confor~iiational dirr'crcnccs bccorric insignificant.

CMC/HOT cari arialysc larger areas. but is laborious and uscs hazardous chen~icals.This Iattcr mcthod showed a h i g h scnsltivity ivhcn compared to thc othcr methods in a blind stud! of san~ples(Condie et al. 1993) but has bccn rcportcd lo miss G lo T nit~tations.

Ncivcr rncthods are noiv bcing sought, such as the analysis of Duplcs DNA by t,riplc hcIis forrnalion and is applicd lo the dctcction of p53 microdeletions to f'acilitatc DNA scrccning procedures (OIivas and Mahcr, 1993).

This n ~ ehod t csploits Ilie ability of ccrtain oligoiiuclcotidcs ro riionilcr DNA sequences iu the major groovc without requiring denafuration of the doubIe helical DNA targct a i d might bc dircctly applicd to gcncral scrccning of mutalions fleeting homopurine scr~llcllccs.

All lhc abovc scrccnirlg tcchriiqucs indicatc thc approxmalc srtc of a mutalion. The gold standard is dircct scqucncing arid this will probably be the mcthod of choicc if clicnts wish to havc a 100% rcassurarlcc that their p53 gcnc is normal.

II is rcconw~cndcdthat oncc a niutalion is found it should always bc scqucnccd and also corifinncd by at least one othcr rcchnique. such as rcslriclion c n q m e or allclespecific hybridization.

Once a mulation is identified. tests can show wirh 100% ccrl.aint: whclhcr a rclativc is a carricr of thc ~iiulatcd gene or not (Eefes et 4. 1993).

13

ncoplasia type 2A (MEN 2A) in thc rcl prolo+mcogenc on chromosone 10, @onis-Kellcr ct al. 1993). Multiple cndocrinc ncoplasia (MEN 2A) is an auloson~al dominantly inherited cancer syridrome comprising nieduIIary t.hyroid cancer (MTC): adrenal gland phaeochromocytomas and hyperparathyroidism. Almost all patients with MEN 2A develop MTC during childhood or early adolesence. Gcuetic tests have been applied in thc preclinical stare to scrccri for MEN 2A, permitling early lrcatrncnt (early curative thyroidectorny) in children predisposed to the disease (Celtncltcs ct al, 1992: Marsh ct al. 1994). This approach is now bcing perforrncd at sevcral c c n k s . including Washington University and Cambridge University.

A different sccnario is set up when discussing thc problem of individuals carrying pS3 mutations. There is lack of associalion between specific mutatior~sand tunlour histopathoiogy. A situation is created whereby duc to our currclil limitations in clinical diagnostic techniques, lumours cannot be effectively screened. In the casc of thc Li-Fraumeni Syndrome whcrc fanlilics inherit a spectrum of cancers namely sarcomas, brcmt, brain. acutc lcidcactnia. melanonia. germ-cell tnmours. bladdcr cancers and adreliocorticnl carcinonia, currclit screening mcasurcs have shown to bc ineffcc~ivc in predicring discasc in thc preclinicai state.

Proposed blood screening for leukacrnias and magnclic resonance imaging for brain lurnours have all proved to be unsnccessful for early dctection. Mammography screening for brcast canccr has been shown lo decreasc mortality in thc over 50 years age group (Shapiro et a1.1988: Shapiro. l989), but its cflicacy in wolncli under 50 is uilknown. With rhc reccril idenlification of lhc breast cancer genc BRCAl (Miki cr al, 1994), this approach to screening for predisposition tu develop brcast canccr cari change cornplelely.

Prcvcntiic rneasures such as chemoprevention rnay bc of' some valuc in certain cancers but, there is no evidence that il is of universal bcncfit. Chcrnoprevention studies should also include hormonal therapy such as the use of laiiioxifen in rhc tnnioxifen prevention trial for women at high risk of brcasl canccr (Cuzick and Baurum. 1985; NayficId, 199 I ) .

Earl! detection and prevention 'Thc goal of prcdiclivc gcnctic tcsting in hrrrnan cancer is lo be able to prcdict thc inheritance of a disease gcnc Ihal is going to lcad to a malignancy. and to initiate preventive measures before a person actually dcvclops canccr. Thc bcst cxarnple so far of early dctcction and prevcnlion is in the ficld of inhcri~ed cancer syndromes.

Thc inclusion of vitamins such as retinoids rnust also bc given due consideration in chcrnoprevcntion studies. In wornen who are carriers of a mutated p53 gene, the risk of developing brcast canccr bcforc the age o f 4 5 is 18 fold ovcr the gcncral populalion (Birch, 1992; Eastoti ct a], 1993; Sidransb et al, 1992: Eclcs el aI,1993).

Prophylactic subcutaneous mastectomy may not be an u~ueasonable preventive measure for brcast cancer in those paricnts carrying p53 mutations.

Since the localizalion of the multiplc endocrine neoplasia type 2A (Molc ct al.I993), it has bcen possible to dcvelop n gc~iclictest to screen for ~riutalionin rnultiplc endocrine

Thcrcforc, it may be coricIuded that so far. detecting individuals carqlng gemline p53 rnutations is not tcchrlically impossible. Howevcr. monitoring these

individuals. for early dctcction of the different turnours which ma? develop, is not yct possible (Garbcr ct al.

1991).

Gene Therapy

Genc therapy is the stable insenion of a furictional gcne into the genonic of a h o s ~celI lo altcr the finctioml capabilities of thc ccll or to corrccr a spccific genctic dcfcct. This technique givcs rcsearchers thc possibility to understand more about the regulation of gcne function and at the same tin~cfind its applicability as a therapcutic approach in thc trearment of cariccr (Foa and Guarini.

1993: Miller. 1992; Gottesman. 1995). Optitnimlion of both cficiency and safety of ttic ways in which a genc is transferred. is t l ~ ccrucial feature of all strategies seeking to csploit this technology.

coristn~ctiori of retroviruses that do not contain the repIicatior~gcncs. and in w.hich Ihc viral slructural gencs are rcplaccd by the ncw genes to be insencd illlo [he cclls.

A v e v plausablc biological model systcni is bcing currently pro~tiotcd. This includcs thc dirccr adniinislration of a rctroviral wild-type p53 cspression vector in orlhotopic hun~anlung cancer modcl in ndnu mice (Fu-jiwar.a ct al. 1994: Miller ct al. 1989) resulting i n growth inhibition of canccr cclls.

As is [rue of interesting studies, these data raise a scries of qucslions that shouId bc corisidcrcd in Suturc espcrimcnts. Although Ihc usc of a relroviral vcclor favours integration in rapidly dividing cells. can all thc growti supression bc attributed to trarisfccted cclls only'?

Sincc it is likcly tliat all canccr cells arc rranfcctcd, could the supression of g r o ~ also h be duc to a bystandcr clTecl" The p53 turnour suppressor gem is a primc cariditarc for (Frcc~nan el al. 1993). Does the growth supprcssion obscncd in transfccted ccIls result from the induction of gcne therapy (Takahashi et al. 1989: D'Amico ct al.

1992: Chiba ct al. 1990). Genetic lesions in ~ h p53 c gene apoptosis (programmed ccll death)? Do thc bystandcr arc the most cornnionly occuring char~gesfound in all cclls undcrgo the samc growth suppression drie to hwnan cancers (Vogelstein, 1990). SeveraI groups of indnction of apoptosis? Is therc a bystandcr cffcct in metastatic cclls? The bystander effcct is an obscn~atiorr scieniists wcre able to show that thc stablc transfcction (T,akahashi et al, 19921, or retroviral tra~~sduction whereby transduced cells have bccn shown lo inhibil ~ h c growh of nontransduccd neighbouring cells in cullurc (Fujiwara el al. 1993) of wild typc p53 gene into cancer (Cai ct al. 1993; Freeriian et a1. 1993). Thc molccular cells with a mutant p53 dramatically inhibits cell growth in cultured ccIls despite the possible prcsence of othcr basis of this bystander effcct is under imestigation. Docs generic lesions. Tlic signifkmce of this observation is this possible theraputic tool work in other sitcs of thc body for othcr p53 ruutations'! that any other getictic lesion necd not be corrected before an anti-turnour efffcct could be seen (Fujiwara et al, 1994).

It is apparent tliat thcre arc still sornc fimda~ricntal In-vitro gene therapy experiments starled in the late practicaI and clinical problcnis to bc addrcsscd. Lung h a t we arc secing the possible 1980s and il is only now L canccrs are rarely one ccll la?;cr thick and tlicy are rarcIy applicalion of in-vivo gene thcrapy using wild type p53 confined to a closed spacc. Fujiwara and co-workcrs gene (Carbone and Minna, 1994). New,s of thc first (1993) have shown that wild-type p53 is capabIe of' proposcd gene therapy for lung cancer to cntcr tiurnan rrullrilayer pcnctrarion inlo lhc rhrce-din~ensional trials has been reporled in the (Journal of the National structure of multicelhilar tumour spheroids. Of clinical Cancer Institute, Vol. 86 No5 March 2. 1994). This trial concern is h e size of Ihe tornour and the acccssibility lo is still pending approval by the U.S. Food and Drug rnetastatic sites. Thc potential toxic cffects aIso need Lo bc Administration on the safety of the relroviral veclors addressed. Retroviruses integrate stably into the gcnorne of replicating cells. Thereforc, it is irnporlarir to considcr bcing used (Anderson. 1992). In oncology, rescarch into the outcorne of genetically altered normal epilhelial cells. gene lherapy is mainly concen[rated on lung canccr.

Mutations of the pS3 Lurnour supprcssor gene are thc genetic ab~iorrnaIiliesmost frequently idenlified in nonTaking into considerarion a11 the available rcsults so far, small ccll lung cancer (Takahashi ct al. 1989). il is being proposcd that in ttic case of lung canccr. microscopically established tunlours in thc bro~ichial epitheliuni can bc eficiently infected with a retroviral Corivcntional methods of treatment have no1 resultcd in a significant decreasc in mortality from lung cancer and vcclor expressing wild-lypc p53 gerlc and that in-sihr relrovirus-lncdialed gene transfer may be a useful siralegy thcreforc this crcales a political and economic power to for nial~ipulatinggenelic abriorrnaIities of cancer cclls in assist researchers pursuing this novcl therapy. vivo (Rolh et al, 1994). Using currctit n~olecular Thc immediate problcrn wilh application of gene lransfer tcchniques it is possible to identify pre-neoplastic as wcll technology in patients is thc deIivey of the therapeutic as microscopical neoplastic cells before these cells display gene Lo sufficient numbers of tumour celIs to produce a the cytological and 1iistoIogica.l fcalures of i~wasive clinically obsen~ablc effcct. Therc is also the cancers (C'sson ct al.I99 1: Sozzi ct al. 1992; Kastan ct consideration of safety of vectors. When a retrovirus al, 1992). infecls a cell, its viral RNA is copied by the enzyne reverse transcriptase into DNA that enters the nucleus Much research still needs lo be done. At prescnt there arc and integrates randomly into the gcnorne of the host. ovcr 100 protocols accepted or u~idcrconsidera[ion by These natural events are exploited for gene transfer by various advisoty committccs for gene targclcd therapies

world wide. thc majority bcing in ll~cUnited Slalcs. Most are Tor carlcer in which the ethics are perhaps sirnplesl with the lonesl risk benefit ratio.

Ethics of Predictive Genetic Testing Issucs regarding ethics of prediclivc genetic testing are cmcrging as lhemcs of great conccrn. Over the past five years scientific publications have presented data ranging from scrccnirig for canccr prcdisposition (Markham et al, 1994) to screening for specific cancers (Kodish el al, 1994). genetic inrervcntion sludies moth. 1994) and rcccntlg prc-implantation diagnosis of inhcritcd predisposition to cancer (Kogan cr al. 1987; Handyside.

1993; Harper ct al, 1994).

Tesring young cancer palicnts and their una&ected relalivcs for p53 gcnillirie mutations presents a number of difiicull clinical and clllical questions (Li et aI. 1991; Prosscr cl al. 1991). As far as clinical rnanagcmcnr is concerned. the first set of problen~sarises becausc of the unccnaint>, about the risks confcrrcd by germline p53 mulatior~s.Thc spcctnini of canccrs so far reponed in hrnilies wilh germlinc p53 murations have been discussed previously (Li ct al. 1988: Birch. 1992: Frebourg et al. 1992). Tllc range of canccrs include boric and SOH lissuc sarcomas. breast cancer. brain canccrs. acute lcukaernias, ~nelanoma,gernl-cell tunmurs, bladder cancer, adrcnoconical carcinoma and prostate canccrs. As more I'arnilics are s c r ~ n e dthis list of associated cancers will probably incrcasc evcn funher.

Therefore, considering llle limited potcrltial for scrce~ungand m l y delcclion of cancers in carriers of p53 genllline mu~ations, the quesliou of whether it is ethical to lesl asyrnptonlatic rncmbcrs of cancer f~miliesin whom such mutations lzavc k e n found, niust be addrcssod. Whal are thc social, ccouonuc and pqchological consequences resulting liom such teskng? What eff~livcrncasllres are available to carriers of the p53 n~utations?At present the grealesl knefit that can be derkod horn t&ilg is reassurance and relief from ansicty in thosc faniliy rnc~nbcrsfound riot to bc carriers of a mulatio~~.

Thcre are other benefits including abilily la plan oducalior~futurc carccrs and decisions on maniagc and child t w r ~ ~ i gtalurig , into account thc kno\\?edge of catmr predisposition.

Therc are concerns that p r d d v e tdng in tIus pinrlar scttirig \4111 incrcw: pcoplc's 'anxiety and nu! haw a negalivc cffocl on psychologicaI and economic issues. raising the question of sclalion of iudividuals for predictive lesling (Kash el al. 1991). The effecr on iife insurance prenuurr~is unclear. but individ~dswith a strong family hislory would have thcir prcmirlrns weighted and these would k reduced to a o n i d if a rclatiw of a person wilh a known mutation was shown not lo carry the genc (Ecla et al, 1993).

Privacy and confidentialityof test results in idemtied carriers of germline p53 mutations have both social and economic impact. Ernploycrs for csan~plc,m y be reluctant to appoint persons at high risk of developing cancer. The pqchoIogical

effecls of belongmg lo a family with high risk of canccr havc not k n studied and are no: understood (Murray, 1991: Sujansky et al, 1990; U.S. President's commission for the study of cthics. 1990). C)nc way to rcsolve this conslallation of issues is to gain more i.nsight inLo h e biological significance of these mutations in correlation with particldar patterns of cancer, LC., spcclfic nlutations causing specific cancers. At present, testing for p53 germline mutations is perfarmed only within the settmg of research protocols out it is expected Lhat prcdictive tcsling will become more widely available. Predictive testing shouId be preceded by thorough counselling nluch should includc psychological assessmerit and potential impad caused by a positivc rcsult. There should also k informed consent. A carefully planned long-term folIow up is also needed in order to &tan data on the life experiences of caniers of ~nutalions,as w J l as on cancer incidence.

Testing on children at risk is anothcr contentious issue and il should be reserved only for cases in which a distinct and immediate benefit can be obtained. Such testing is usually postponed until adolesence, when the iridividrnls can nrake their onn irifonncd docisions as to whether or not they wish to be lest&, (Edwards and M I , 1092; Wald and Law, 1992; Wald. 1993).

Advisory cornnillees involved in lhe design of p53 tesling programs, relate lheir recon~mendationsbased on lheir esperience wilh Huntington's Disease (Went, 1990: Ruggins ct aI, 1990: Lam ct al. 1988; Bloch and Hyaden, 1990; Ford et al. 1994; Tylcr ct al. 1992). Predictive testing for Huntington's Disease involves an area where prevention is riot possiblc and by using a set protoco1, individuals having prcdictive testing for rllis disease helps to minimise Ihe problcms cxpericriccd and allows the individuals to have tirnc to decide if Lhey rcally want rhe lest and for what reason. There may be many rcasons why individuals may wish to have a predictive test. In the case of Hunlington's Diseasc, SO% of individuals at risk said lhc): wa.nted rhc tcst for planning their fumre and thcir fanlily. and to rcIicvc anxiety.

This experience has a sigufiml rclevanm to the design of pj3 t d n g programs. We cannot assume lhat the impact of t d n g for gcnnlinc p53 mutations would have the same favourable outcome but there is dehitely growing support in favour of such tcst.ing (Snugel, 1993; Berg, 1991: Li et al, 1992).

Conclusion

Thc concencd effons from thc work of the Hurnar~ Geno~neProject wilt soon lead to thc identification of many more genes rcsponsiblc for hereditary diseases. including cancer (Watson, 1990). The gcnctic data thal is presently available and that will bc rnadc available. in the near future, on populations and individuals is the subjecr of debate by all involved in this line of research. Issucs such as confidentiality, the right to know or not to know, non-discrimination anlorig carriers, insurance. disease specificity, availability of treatrnenl, inl~critariccpaltcrn

and genc penetrancc and cspressivitj,. fenrure prominantly in the ethics of any genetic scrccning program (Huggins ct al. 1990).

The aim of this rcview was to update the present sccnc of canccr genetics and to promotc thc intercst sccn over the past few ywrs on the concept of canccr genetics clinics.

At prcserlt genc rnarkcrs for the most contmonly occuri~rg canccrs have been idcnrilicd. i.c. bo\vel. breast and ovarian carlcers (Fishcl ct aI. 1993: Brouncr ct a1. 1994: Miki. 1994: Woostcr. 1995). This has gcrrcratcd a parricular inkrcst both from the general public and from clinicians dcaling with cancer patients, Morc information is bcirig dcmanded on thc availability of gct~clictcsts. cancer risks. screening nlcasurcs. prcvcntion and treatment.

Sincc 1989 the concept of canccr genetics clinics was alrcady being proposed (Hoskins. 1989). To datc therc arc more than 20 cancer genctics clirlics sel up in Europe. In thc United Kingdom aIonc rhcrc arc abou~6 ccntres. One such centrc is at the Royal Marsdcr~Hospital wherc a ger~cticcancer clinic is hcld which also offers a cancer gerlctics screcning senrice (personal comrnunicalion.

Eelcs RA). Clients attending these clinics havc acccss to inforrnation on their own gcrietic risk for canccr as we11 as to highly spccialised screc~~ing tcsts avaiIablc lo datc.

Ths clinid siguficana: of being cilher a BRCAI genc carrier or a BRCA2 genc canicr is at presenl st111 poorly undcrstd. Large epidemiological studies have to be carried out to rcally assess thc contribution of thesc gcncs lo caIuccr dwelopmcnt in farnitid and sporadic cases (Claus cl d.

1994).At prescnt, 10% of breast canccrs hat a slrorig family history and it is only possible to i d e n i i a definitc do~ninanl pattern of inherilana: in approximately 1% of individuals.

However. it is also possible that thesc genes codd account for as littlc as 1-IS'% of breast cancers. Since br~astcancer is the commonest turnour occurring in women world wide. one can appra-iate the potenlid burdcn rhat indwiduals can place 011 cancer genctics clinics, givcn thc current slate of inforrnation.

There are as many questions as answers in thc area of p53 cancer gene predisposition. in parriculnr, the at risk groups need to bc bettcr dcfincd and foIIow-up of known cxriers is nccded. Thc p53 gcnc is offcring thc niosl pronlising operings in the field of corrcclive genc thcrapy bur it is unrealistic to think that their cffcctivencss would be i~mnediatc.

The advanccs in science and niedicinc llavc brought about yet another clhical issuc ro thc sccnc. This is the erhucs of preirnpiatitalion diagnosis of inherited predisposition to cancer. it is very Likely dut couples wil1 seck prenalal diagnosis to prcvcnt passing a mutant genc on to their children. Is the idea of termbaling a pregnancy after a diagnosis by convenlional mans (chorion vilIus sampling) acceplable? Mcthods for diagnosing genetic dcfects in early embnos beforc in~planlalionare being developed and heavily supported by hvcslmcnts et A, 1994).

The challcngc which we now k c is how to rclatc all the inforn-tation we have so far on cancer predisposing gencs into elrccrivc scrccning and intcncntional programmes (Hawdrd ct al. 199 1). Effcctke studies also havc to bc 1.1ndcrrake11 to dcfir~cthe cost effeclivencss of' population screer~irigand thc health gain to bc anticipated l'rom cancer predisposilior~screening.

Acknowledgenrents: Sincere thanks go to Prof A Cuschieri. Prof A E Fclicc. Dr C A Sccrri. and Dr F Portclli for giving tnc Lheir acadctnic and practical support ro hc nblc lo initia~cthis line of Cancer Rcsearch a l Thc Unrvcrsity of Malta.

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19

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Commentary Graphs and their Spectra

From the contributions to confercnccs and journals, it is cvident that most of the rcsearch in rnathcmatics is directed towards the development of Lhe theoretical aspect of its various arcas. In this sense rnathetnatics is an art fathomed and pondcred on for thc sake of its own bcauty. Ir has dctcrmined the direction of most of rhe significant research in other fields. Being an i~rconiparablc human achievement. niathematical creativity has fathered our understanding about the Jiaturc of man and his universe. Invariably, apparerrtly fulile results find inlniediatc applications in various ficlds.

Graph thcory starlcd as a~nusirigbrain teascrs from rhe days of EuIcr, who showed, in the eighteenth century, that the sewn Kiinigsberg bridgcs cannot be crossed just once in a closed routc, and of Guthrie and De Morgan who. in the nineteenth ccntuq. qnestioncd the number of colours rcquircd to colour a rnap. Since thcn it has been taught under various guises including operational rcscarch in Inanagement studies, network theory in cr~ginccringand algoritlinls in co~nputerscience. It has bccorne a valuablc forecasting tool in several applications in indust5 and cornmcrce.

Since 1939. chemisls invcstigaring molccuIar orbitals noliccd a relationship bctn'cen thc cnergy levels and the stability of a molecule. Tlle Hiickel molecular orbital theory gives an approximation of thc x-n~olecular orbilals of a rnoleculc by expressing them as a linear co~nbinatiouof thc atomic prc-orbitals (Cotton. 1963; Coulson and Rushbrook. 1940; Zivkovic. 1972). Whcn Schrodinger's cquation, that detcrnlines the ~uolccular orbital cncrgies. is simplified. the resulting cqualion is l11e characteristic cquarion Der(hl--A) = 0. where A is the adjacenq, matrix of the graph whose structure is the urnc as [hat of (he nloleculc bcirig co~isidered(when hydrogcnsuppressed. as in the case of hydrocarbons. which is thc caw most corn~nonIyquoted in chcrilistry litcmturc) (Spinltcr. 1964). Thc speclrurn of a graph is thc solution of the characteristic equation of its adjaccncy rnatrix and the gen values in the spectn~~n dcscribe the energy of thc orbimls of'thc nloleciile whosc structure is tlic same as that of the graph..

The invesligation of Graph Spectra is one of the forcrnosl problcnls of current rescarch in thc thconf of graphs. Scvcral i~nportantresearchers havc addressed [be problem and thc thcory is sufiicicntly advanced so that statc-ofqhe-llrt books synthesizing rcsults havc

already appeared. In spite of this. there is amplc scopc for furlher research. Onc of the best books that offers a survey of the work done in this area up to a fcw years ago. is Spectra of Graphs by Cvetkovic and co-workers.

1980. When Cvctkovic was about to prcsent his Ph.D. thcsis on the spectra of graphs, an established physical chemist, I. Gutman showed a keen interest in his rcsults.

Hc realised that for a class of molecules the cigcnvalucs in thc specmm of a graph wcre precisely the energy of tlic orbitals of the rnolec~.ilcwhose structure is the same as that of the graph.

The .zero of the energy scaIc is that for no interaction bctwccn the separate aton~icorbitals with~nlhc molecuIe (Karplus and Porler, 1970). Thus the zero eigenvalues, h=O. deterlnine the non-bonding molecular orbitals (NEtMO) responsible for the instability of a molecule.

The NBMO are described by the corrcsponding linearly independent kernel cigenvectors of A (Zivkovic, 1972).

The solutions also shed lighr on the clectron density distribution in a ~nolccule(Cotton, 1963). So the results of graph spectra are of great interest in chemistry.

The mathematical analysis of an cxlsting molecule in tcrms of its spectrum may be ulvestigated if the bonds beiween t! aloms arc known. Thc data is represented as an adjacency lisl expresing h e neighbouring atoms of each atoni in the nloleculc or as a (0-1) adjacency matrix A in which an r - j cntp is 1 if thc corrcsponding atoms arc bonded within thc ~nolocule and Acre otherwise. The vanishing of the dcterniinanr of ,l is an indication of instabil~ty.Even the characteristic oqualion itself, Det(U-A)+, where A is the adlaccnq matnx of the graph whose structuuc is the same as that or tlic ~noleculebeing considered gives a number of rcsults abor~tLLlc structure of the molccde.

A graph whose ad-jacency matrix has the number zcro in its spectrum is said lo be singular. Aithough some rmlls regardug bipartite graphs havc already appeared in the litcrature, identification especially for ,arbitrary graphs, is complcx as with increasing order the number of basic structures responsible for thc singularities increases cnorn~ously.

As highly reaclivc tnoIecules are dficult lo prepare and lo isolate. and vcry mvstablc. it would be useful to predict their possiblc stnlcture.The charactcrisation of singular graphs is d I I an open qucsljon. 111 this rcspect the author has had sotnc succcss in characterising minimal configurations (Sciriha. 199%). A systematic search for the minimal

22

configurations in singular graphs that give rise to a singularity was carried out on the grounds that a linearly dependent set o f t row vectors of A. the adjacency matrix of the graph, is present. The linear combination between the t row vectors is termed a kernel relation and is denoted It. It is noted that the coefficients of the kernel relation are the ordered entries of the corresponding kernel eigenvector in the nullspace of A . An algorithm was set up to identlfy the non-isomorphic singular graphs for successive values of t. This theory becomes more and more cumbersome when applied to large systems. One can practically not avoid the use of an appropriate software package, such as MATHEMATICA, used in programming mode, for values o f t larger than or equal to 5 The first stage was to determine the core, x,, which is the subgraphs induced by the vertices corresponding to these t row vectors of A. From the core x,, a minimal configuration ( r , ~ , , t t ) is "grown" by adding a periphery. a set of vertices adjacent to those of the core, until the number of zero eigenvalues in the spectrum of the resulting graph G, called the nullity of the graph G, is reduced to one. In the case when the core has one zero eigenvalue in its spectrum then the minimal configuration. r', is the core itself. Such a graph has been called a nut graph as the periphery is empty (Gutman and Sciriha, 1995). The core X, and kernel relation R, of the minimal configurations T, thus obtained will be unique. There may be several peripheries for the same core and kernel relation so that there is a many-to-one correspondence between the structures of minimal configurations 1 and the pair (xtR). Such minimal configurations are the subgraphs found in all singular graphs. The 61 minimal configurations for t less or equal to 5 have been characterized. Several properties for larger t have also been established.

The core may shed light on the charge density distribution in a molecule. The vertices of the core have been shown to correspond to atoms that are charge rich in some ions and molecules. These are the atoms that are llkely to be involved in chemical reactions.

As diEerent minimal configurations may be "grown" from the same core, a maximum configuration may be set up by including all the peripheral vertices used to build up the minimal configurations from a core X, satisfying the relation R,. In this way the largest graph with the number zero in its spectrum satisfying a particular kernel relation R, with all the vertices of the periphery joined to those of the core and themselves inducing a null graph, is obtained. This configuration may have more than one zero eigenvalue.

Corresponding to each zero eigenvalue is a core and a particular kernel relation (Sciriha, 1995a).

The graph may be enlarged further by including edges between the vertices in the periphery and also by joining

Graphs and their Smxtra

arbitrary graphs to the vertices in the periphery. In this way the order of the graph may be increased indefinitely without upsetting the core X, or the relation It. So will the rank of the graph which is equal to the order less the nullity.

In 1993 M. Ellingham published a paper showing that he has addressed the problem of singular graphs from a different point of view (Ellingham, 1993). He also built a singular graph G from "basic subgraphs". These he defined as non-singular graphs of order r where r is the rank of the adjacency matrix of G. It has also been interesting to investigate graphs of small rank. As the order of a graph is increased the rank is kept fixed or under control at small values by adding vertices which increase the nullity, that is the number of zero eigenvalues in the spectrum. Thus as new vertices are included more minimal configurations are discovered as subgraphs. For very small rank only complete k-partite graphs are admissible. allowing only vertices of the same type, that is vertices having the same set of neighbouring vertices. For rank greater than or equal to 4 more minimal configurations contribute to the nullity.

As the set of minimal configurationscharacterizing singular graphs a being collected and their properties studied. certain common properties are already apparent. In fact several conjectures have been made. the valid@ of wluch will be investigated. One such conjecture is on the common value L of the coefficient of 1 of the characteristic polynomial Det(W-A) where A is the adjacency matrix of the graph, for minimal configurations grown from the same core for the same kernel relation. It is conjectured that L may be expressed in terms of the properties of the vertexdeleted . subgraphs of the corresponding minimal configuration (Sciriha, 1995b).

J.J. Seidel who is one of the pioneers of the development of Graph Spectra stressed his belief that the eigensolution holds the secrets to the understanding of graphs and the systems they model. The aim of this work is to unravel some solutions of significant importance.

References Cotton FA (1963) Chemical Applications of Group Theory. InterScience, 117-182.

Coulson CA and Rushbrooke GS (1940) Note on the method of Molecular Orbitals. Proceedings Cambridge Phil. Soc, 36, 193-200.

Cvetkovic D, Doob M and Sachs (1980) Spectra of Graphs. Academic Press (London).

Ellingham MN (1993) Basic subgraphs and graph spectra. Australasian Journal of Combinatorics. 8.

247-265.

Gutman I and Sciriha I (1995) Graphs with Maximum Singularity. Graph Theory Notes of New York.

Sciriha I (1995a) On the Construction of Graphs of Nullity One. Proceedings of the 15th British Combinatorial Conference, International

Conference held ar Stirling University.

Sciriha 1 (1995b) On the Cocficient of h in thc Characteristic Polynomial of Singular Graphs.

SpiaIrer L, (1964) The atom conuccuvity I T U L ~ ~ and X ils

J. I,Yietn. Doc., 4,26 1-274. ~ h ; ~ c t e r i s lploynornial. ic Zivkovic T (1972) Calculations of the non-banding triolccular orbitals in the Hiickcl Thcoq . finafico C'herrricn Acta. 44, 35 1-364.

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Research Article Preliminary Data On The Occurrence And Distribution Of Shallow Water Marine Sponges (Porifera) Around Maltese Coasts

Summary. Data on the ecology of the MaItesc Porifeni is lacking altogether. Evcu docunlentcd basic information 011 the occurrence of coniniercial sponge spccies i n Maltesc ~ d s t a waters l is nna~ailablc.This sludy prcscnls the rcsults of a four year diving survey airned at studsing rhe occurmice and distribution of st~allowwaler spongcs sround lhc Maltese Islands. In all. 33 spccies of Porifera have been identified. most of which are new rccords for the Malresc lsla~ids.Information on the barhyrnctric distribution and abundancc of thcse species is given. Although an exlensivc area has bcen covcrcd in our survcy. wc havc not recordcd any comn~crcialsponges. It is therefore likclv that thcsc species do not occur IocaIIy. at lcast in shallow inshore nfatcrs.

Kqwords: Porifcra. sponges. Mallcsc Islands, spongc disease. species lists, chcck-lists, sponge fishcrics.

Sponges are a ubiquitous coniponcnt of thc marinc benthos. Some species havc considerable cornrnercial irnportancc as their fibrous skcleton is the familiar bathroonl sponge and t h q have been exploited by n u n since antiquity. Until recently thcre has been a thriking fishery for spongcs in the Meditcrraneari centered mi~inly on Greece. Tunisia m d Turkey (FAO. 1994). Despite both their con~ri~crcialimportance and illtercsting biology, therc is a lack of cven the mosr basic biological and ecological inforniatiori on Ihe Maltesc spongc fauna.

In July 1990. a F A 0 rcgional workshop was held in Malta lo discuss thc situation conccrnirig lhe occurrence of a discase which had afnictcd both commercial and non-cornrncrcial species of sponges since 1986 (FAO. 1994). Ttus disease had a large cconornic impact on thc Mcditcrrariean cornrnercial spongc fisheries, having pracdcally eliminated this iridnst~y in sonlc countrics (VaccIct, 1991). Studics made during the past four ycars have suggestcd that the disease is duc to a bacterium which normally plays a part in Ihc digestion of the spongin skelctori of dead sponges by secreting a collagenase enzyme. In coriditions stressfid to sponges, ttus bacterium becomcs virulent and also atlacks thc skelenl tissue of live sponges (Vacclet et ui., 1994). At the time of the Malta F A 0 workshop, the authors wcrc asked to supply dala on the local occurrence of conl~nercialsponges arid on the incidence of the spongc discasc. The only information avaiIable conccrning spouge fishcrics in Malta were thc rccollectio~lsof sorile local fisher~ncn (see below). A lilerdhm search for inlormation on thc local conunercial and noncorntiercia1 sponge fauna rcvcaled only a singlc publication, which recorded ninc non-commercial spwies of Porifcra (Micallcf and Evans. 1968). These identifications arc suspect. howcver. as thc identlilcarion source uscd by thc authors is a scnli-popular guide to Mcditerranean marine lrfc which oriIy fearures a handfill

of'the spccies occurrilig in the Mcditerranean. and in ariy case, idcnlificalion of spongcs is dillicult withoul detailed histological examination. The lack of local infornzatio~i on co~nmercial sponges arid of a local sponge f i s h ? contrasts with rhc important sponge fishcries in scvcral nearby Mcditerranean countrics such as Italy. Tunisia arid Greece. Howcvcr. local fishcrrnen from W i d iz-Zurriq co~lIirmed that forcign lishing boats had in thc past collccted comnlercial sponges off Filfla Island. From the fishermen's descriplions of the di\,ing gear uscd by the sponge fishcrmcn at thal tirnc. such an activity nwst have been carried out scrvcral decadcs ago. There is also a documented record in local newspapers of thc 1890s co~rccning comercia1 sponge fishrig activities in Malla (J. Inguanm. personal conlri~~uca~ion.

1991).

The lack of basic data on h e local sponge h u m , such as an accuratc vies lid. pronlptcd thc autllors to initialc a s w e y having Lluce primary aims:

1. To compile a checklist of the shallow water Porifcra of h e Maltese Idands and to provide basic c c o l o g d infornia~on on thcir bathymcuic dislribution and abundaicc.

2. To cslablish whether any commercial spongcs csist localIy. 3. T o nlonitor thc local occurrence of tile sponge discasc.

As a preliminary to this snrvcy, J. Vacelet (Station Marine d'Endoume, MarseiIIc) together with onc of us (JAB), carried out five dives at different sites around mainland Malta to assess the irlcidcnce of the spongc disease amongst local sponge populations, lo record thc niost cornrnonly occurring non-comlnercial spccics, to gain expcriencc jn their ficld identification arid to search for cornnicrcial specics. Data on the iricidencc of thc

ShaIIow Water M a h e Sponges

sponge disease amongst non-commercial sponges obtained during this prelilninary survey have been published in Vacelel et a!. (1994) and FA0 (1994).

Materials and methods A total of 3 1 SCUBA dives wcre carricd out at depths ranging from zero to 45m in 17 diffcrent localities around thc Maltese Islands (Figure 1). In vicw of the indications of a past spongc fishery off Filfla, nine out of the 3 1 dives were madc off this island. During most dives, divers working in pairs moved undenvatcr along 6-metre wide belt cransccts at predetermined bearings.

Thc length of thesc transects varied depending on the depth of the water at the site concerned. All sponges cncountcrcd in the transects werc idcntificd and recorded irr silu where possiblc. but specimens were also dlccted for later identification in the laborato~.An estirnate was made of tlic abundance and a particular look-out was kcpt for dimscd individuals and conlrriercial species. A Tcw spcciniens which arc iriclrrded in our spccies list werc obtaincd from sanipks coIlectcd by traw3ing at 401Wn1 off maidand Malta (Tables I and 11). whilst others were callccted during other studics. Most of the idenlifications to species level haw bccn chcckcd by J. Vacelel of the Station Marinc dlEndourne, Marseille.

Francc. The collection has bccn deposited at thc museum

25

of the Departrklent of BioIogy, University of Malta.

Results In all. 33 species of sponges have becri idcntifid, most of which are new Iocal rccords. Table I gives a classified list of the species recorded while Table I1 provides data on the localities surveyed.

The most abundant species in shallow (1-15m), e,vposed waters appeared to be Sarcoirng~sspino.nrla and Ircinin vnriabilis. The latter also occurred, although less abmidantly, at depths of 20-25111. In rnorc sheltered shallow waters (24m). cspccially along the rocky headlands of several idcts, Chondrilla n u d a appcarcd to have the highest abunchice. In sciaptulic environments throughout tl~e 15-35111 dcpth range. Crawbe cramhe and Agelos omides had Lhe highest abur~dancc.Ihrrdrusia ren~forrnis and Pekosia jkiJorntis w r c conunon in the 5-25rn depth rangc in some of Lhe sites suneyed. At FiMq (~acospongia scn1rri.r was the most abultdant s-pccies in the 20-35m depth.

No commercial sponge spccics were encou~ireredduring this survey. Only single individuals of non-co~nri~crcial spougcs apparently afflicted by the spongc disease were encountered during dives carricd out in 1993 and 1994.

MEDITERRANEAN SEA

Fipurc 1: Map of thc Maltcsu Archipelago showing the localitics suncycd for sponpcs.

Berg J.A. and Sdrembri P.J

26

Species

Site

Clathrina clathrus (SCHMIDT)

M6

Leuconia sp.

Petrobiona nmssiliana (VACELET) ,Sycon elegrrns (BOWERRAVK) Ute glabra (SCHX~IDT)

Xghaj ra MI3 M12 Xghajra

dcanrhellrr scum (SCHMIDT) Agelas oroides ( s c ~ h r l a r )

M6 MI. MS. M9. M10.

M12. Ml3. C l , F1 MI. CI M2. M4 CI MI. M I 0 M2, M3. M4. M6. F1 M6. MI3 M2. M3. M1, M6. M7. M10.

MlO. MI 1, G2 MI. M2.M4. MG. M7. M8. M9. M10.

M11, M13. C l . C2 M 1, M2. M4, MS. M6.

M9. MIO. M11. M12.

M13. CI. GI. G2. F1 M3 MI MI MI Off Ras il-Wahs CI M I , M8. M9. M13. C1 M 1, M2. M1, M5.

M6. M8, M9. M10, M12, M13. Cl. GI M9. M10. F1 M6 MI, M5, M6. M8, Off Qa~nrnieh MI. M2, M3. M4, M5. M6, M7. M8, M9, MIO. M I 1.

C1, GI. G2, F1 Off Qammieh Off Qarn~nich MI, M9. MI0,MI I M12. GI

Class CALCAREA Subclass CALCINEA I Subclass CALCARONEA

Class DEMOSPONGIAE

Anchinoe sp.

Anchinoe pa1ipertn.r (ROW E R B ~ K ) Aplysrnn rrerophoba (SCHMIDT) .4xlnef/nverrucosa (ESPBR) Batzella rnops (TOPSEKT) Cacospongia scdaris (SCHMIDT) Cacospongia rnollior (SCHMIDT) Chondrilla mmila (SCHMIDT)

Chondrosia renijorntis ( N M D O )

f>icryone//aincrsa (SCHMIDT) Dysidea cf. fragilis (XIONTAGU) Dysirlea sp. fisciospongia sp.

Haliclonn sp.

Ircinra dendroides ( s c r ~ w n ' r ) Ircinio oros (SCHXIIDT) Ircinia variabilis ( s c ~ x r m ~ )

Oscarella lohularis (SCHMIDT) Petrosia/iciformis ( POIRET) Raspnciona aculenta (JOHNSTON) S'arcolragus spinosula (SCHMIDT)

Scopalina lophyropoda ( s c ~ h m - r ) Siphonochalina sp. spirntrelh cunctnlrix (SCHM~DT) Tefhyoaurantium (PALLAS) --

-

-

-

-

-

Tablc I . Classified list ofspccics rccordcd. l'hc sitcs whcrc thc spccies wcre recorded are indicatcd by a code corresponding lo lhar in Fig. 1 Sites indicated by thcir actual name wcrc not surucyd by SCUBA diving but thc spccimcns wcrc obtained from olhcr workcrs.

ShaIlow Water Ma*

Code

Name of site

MI

Ahrax Point

M2

M3 M4 M5 M6

M7 MR M') M I0 MI 1 MI2 MI3 C1 Gl G2

F1

I

1 /

27

Smn.m!s

Date of divels

May '90 Aug '90 Mellieha Bay Aug '9 1 Sept '9 1 Oct '9 1 St Paul's Bay Oct '94 Qawra Point May '90 Qawra reef Sept '9 1 Scpt '9 l Sliema May '90 Zonqor Point May '92 Muusar Jau '93 Dcl~tnara Aug '92 W i d iz-Zurricq Aug '9 1 Aug '9 1 Ghar Lapsi Mag '90 Anchor Bay Oct '90 Cirkcwwa May '90 Aug '9 I Irqicqa Point Oct '9 I Imgarr ix-Xini Oct '91 Hondoq ir-Rumrnien Aug '93 Filfla Aug '92 Aug '92 Aug '92 Aug '92 Aug '92 Sept '92 Sept' 92 Scpt '94 Sept '94 OfT Qanrniieh Aug '92 Off Ras il-Waku Mar '93 Xg haira AUE'90

Max. depth

Bottom type

25m l51n 15m 20m 15m 15m 351n 27m 27m 30m 15x11 35m 30m 30m 35111 15, 15tn 30m 2.5111 30n1 25m

BedrocklBonIde~lPosiiionioocennica nleadows BedrocklBouIdersll"osii1onia uceonicn meadows BedrocklBouldcrslPosidoniaoceanica meadows BedrocklBouldersPosicloniooceauica meadows BedrocklBoulderslPos~doniaoceanica mcadows BedrocWPosidonia oceanica meadows BedrocWPosidonia oceanica meadows BedrocWPosiilorria oceanica rneadows BedrocWPosidonia uceanica rneadows BedrocWPosidonia oceanica meadows BcdrocWPosidonia oceanica meadow Bedrock/Boutders/Pusii1oniaoceanica rneadows BcdrocWPosidonia oceanica meadows BedrocWosidonia oceanica meadows BcdrocWPosidonia oceanico rneadows BedrocWPosidonia oceanica meadows BedrocklBouldcrslPosidonia oceanica meadows BedrockiPosiilonio oceanico tncadows BcdrocklPosiilonia occanica meadows BedrocWBoulderslPo.sidoniooccanica nleadows BedrocWosidonin oceanica rneadows

IGm 301n 30111 25111 25m 25m 3Om 2Sru 30m 25m 4Otn 1501n 0-0.5111

BcdrocWBoulderslPosiclonio ocennica meadows BouldersISatid BouIdcrs/Sand BoafdersISand Bcdrock/Bouldcrs/Sarid BedrocklBoulders/Sand Boulders/Sand Bedrock/Boulders/Sand BcdrocklBoulders/Sand BouldersISand BcdrocWSand Sandmud Mediolittoralillvver infralittoral Bedrock

'Tablc 11. Details olthc sitcs suwcycd lor spongcs.

Concluaior~v As cxpccted, the majority of sponges recordcd during this study belong to the class Dernospongiae. Due to llle search and sanlpling tnelhods employed in this study. snlall sizcd epiberithic and epiphytic specics such as [hose found in meadows of the scagrass l'osidor~in oceauica tnay h a w been ovcrlooked. As a rcsull. our spccics list is cotisidcrably shortcr than for other parts of thc Mediterranean (see for example Pansirii arid Pronzato. 1985: Carballo and GarciaGomez, 1994) and many rnorc spccies no doubt occur: there are currcntly about 554 known spccies of triaritic Porifera in thc Mediterranean (Pansini, 1990) Ne~c~Ihclcss, on the basis of our rcsulls we arc able to slatc that: 1. The shallow watcr sponge fauna of the Maltcsc lslands appcars to be similar to that of olhcr parts of the Mediterrancan.

2. There do no1 appcar to be any largc commercial sponge beds in Maltese shallow coastal waters thar could bc ecoriornicallv exploited. and indeed. no commercial sponge specics were recorded.

We suggest that filfure work on the locaI marine sponge fauna should aim at quantif)ing species abundance, corirpiling a more conip1e;c species list and at i d a r w i n g he more important species itivolved in intcrspecific relationships with other marine plants arid animals.

Acknowledgements This work has beell partly finamed by the Marine Resources Network of the MaIta Council for Scicnce and Technology WCST), the Department of Agriculture arid Fishcries of the Goverriment of Malta. FA0 programme T C P M / 8 8 5 3 (C), and by research grants from the thiiversity of Malta. We are indcbted to Proi J Vaalet of

28

[he Station Marinc d'Endoun1c. ~Marseille. France for idenrihing most of thc specks and for supporlir~g us with his specialist knowledge. We are gratcful to Mr Constmtinc Mifsud and Mr Adrian Mailia for donat.ing several sponge spccirnens which they collected in thc course of their work. We would also like to thank Mr Aldo Drago and Mr Anton MicalIcf for logistic assisiancc. a ~ dMr Konrad Pirotta and all thc other divers. especially members of (he CaIypso and ATLAM sub-aqua clubs. who havc participated in Ihc survcvs.

References F A 0 [Food and Agriculture Organisalion of the United Nations]. (1994) The smugglc against rhc cpidenuc which is decimating Meditcrraaean FA 0 'lechtiica/ Reporl sponges.

F1/7TP/H/IB/88S.I:Rornc. FAO: v + 39pp.

MicalIef H and Evans F (1968) The rnarine .fauna of ddcll~n.Msida: Malta U t i i v e r s i ~Press: vi + 26pp.

&ty J.A. and Sahembri P.J

Pansini M (1990) Biogeographic and s$rnatic notcs for a n up-dating of the Mediterrancan spongc fauna.

In: D Bellan (cd.).Sprcialion and hiageup-aphr; in the Mediterranean Sea, France: Perpignan.

Pansini M and Pronzato R (1985) Distribution and ecology of epiphytic Porifcra in two J'o.xidoriia Delile meadows of Ihc Ligurian and ocear~ica6.)

Tyrrhcnian Sea. P.S.Z.N.1.IWn-incr k'colo~v.G(1): 1-11, Vacelet J (1991) Slatut des epongcs co~nmcrcialcsen Mlditcrrancc. In: C F Boudor~rcsqueel a!. (cds.).

Les especes marine.r a proleger en Adkdil ~ r r a ~ ? k e : pp. 75-42; Francc: G.I.S. Posidonic.

Vacelcr J. Vacclct E. Gaino E and Gallissian M F (1994) Baclerial attack of spongin skclck~n during the 1986- 1990 Medifcrranean spongc disease. In: R W IM Van Soest el aJ. (eds.)

Sponges in finrc and space, pp. 3 5 5 - 3 6 2 .

Rollcrdam: A A Balkcma.

Research Article

-

The Cytotoxic Activity of Cucurbitacin E and Busulphan on Ovarian and Stomach Cancer Cells In Vitro: A Comparative Study

Keywords: I:'cballircnr ela1e1-rrrt~; I'ucur-bifaceae: cucurbitacin E: busulphan: ovarian canccr ccl1 line: s t o m c h canccr

Cucurbitacin E and orher cucurbitacins arc highly oqgcnated lrilerpencs which are found soIeIy in plants groupcd undcr the C'ucwbi~nccoe family. including fihnlliu~n clnlc~rirrrr~I,. (thc squirting cucumber).

E~~hcrltr 1,111 c h r t ~ ~ L?, lis a local medicinal pla~ttwhich has bcen used rn folk mcdicinc as a caihartic (Cini, 1991) and as an cnietic (Lanfranco. 1975). It has also bccn used in dropsy (Pcnza. 1969) and in rhc rrcatment ofjaundicc (Cini. 199 1).

Espxi~nenls;on ~ h juice c of dtc plant haw s h o n ~(hat ~ it is cCl;cctivc in the ~rearrncn~ of constipation, oodc~na,sinusitis, and thc p~~cvcnlion of livcr disease (Yesilada el a/,. 1988).

Ho~cver. ir Im k n found that the juicc has a low l h c r a ~ u l i cindcs ( F a m v o r t k 1992), but thiit ir conlains cocrhit~cim.includrng cuarhitacins B and E. which have :u~lilun~our aclivity. Dcspitc this. when individual cucuhiracins i w c tested on various norma1 cclls. the cell viability w s not alfectcd (Gallily el crl.. 1962).

Cytoloxicih (Gitler el a / .. 196 1) and rnetnbolic studics (Shohat P I 01.. 1962) were performed on Sarcoma 180, Lcllrc Erhlicti ascitcs carcinoma and Sarcoma Black usin:: cucurbitacins D. E arid I in m i x . Thcrc was a highcr c>totosic cliect shown by thesc compounds on Sarcoma l8fl than on Ihc olhcr two cell lincs. Metabolic srudies showcd that in Ehrlich ascites carcinoma cells. rhc o q g c n uptakc of cells was morc sensitive to rhc aclion of cucurbitaci~lsthan thc anaerobic glycolysis. I1 was obscrved that the inhibirion of ~ h coxidative metabolism of the canccr cclls by the cucurbilacins was related lo that obscncd by hydrocortisone. 'This may bc duc to the fact that ttic cucurbitacins havc a steroid-like struclure which may influence thc permeability of the

membranes of the cells and nlilochondria. Cornbinalion therapy with cucurbilacins and X-rays on transplanled tunlours in mice (Shohat el 01.. 1965) was less effective on Ehrlich turnour than Sarcoma BIack.

Cucurbitacins B and E showed an effect on c u h r c d human nasopharj~igeal carcinoma. and Sarcoma 37 implanted intramuscularly into right hind legs of CAF, mice nlicn Ihese compounds were injccled inrraper~toncally.

Cucurbitacin E (Figurc 1) can exert its cytotosic cffml cither 011 Ihc cell metnbrane (GalIiIy el al., 1962) or on the DNA in thc nuclcus of the caucer cclls (Kupctlan el 01.. 1973j. 'Thc cucurbilacin side chain is important for thc obscwcd c! totosic aclivity (Kupc11m el a/.. 1970).

0

Busulphan IdcavingGroup

1-1 0

0 I

Lcaving Group

Cucurbitacin E

Figure I : Structul-cs of Busulphat~and Cucurbitacin I 7

30

Busulphan (Figurc I) is an alkylating agcnt. which cscrls its action by joining two guaninc residucs on two strands of thc DNA. lcading to cross-Ii~lking.This. in urn. prevents the uncoiling and replication or the DNA ~noleculc.thus halting the niul~iplicatioriof rhc lumour cells (Rogers et al.. 1976).

Scveral studies have been carried out on the ccU lines alrcad!, mentioned using different cucurbitacins. but thc effcct of thcsc compounds on ovarian and stomach carlcer ccll lines has not been studied. The prescnt study was thcreforc undertaken to cxarnine any possible effects of cucurbitacin E on ovarian and sromach cancer cell lines and also to compare thc cffect of cucurbitacin E with that of a widely used c!totoxic agent. busulphan.

Esperimcnlal Procedures Cucurbitacin E was prcparcd by solvcnt estraction of the fruit of Ecballitcnr eiatrritrnr (Lavic er 01.. 1958). collected from Marsascala (Malta). 52%) ( n . 1 ~ )of the pure compound was oblained. Its pnrih was co~firmed using five analytical methods: UV arid IR spectrophoromct~.MelLing Point Dclerminalion. TL.C and HPLC against a knokvn standard. A spccinlcn is dcpositcd at the Institute of Agriculture, Univcrsih of Malta. From a stock solution of 1 . 8 s l 0 - " ~ .I in 10 dilutions were prepared.

Busulphan (hfy1cran6 Wcllcome. West Susses, U.K.) 500rng tablcts wcre ground in a mortar and then dissolved in RPMI 1640 medium to make a final stock solution of 4x10'".

1 in 10 dilutions were rheri prcparcd.

Single stomach (SNU-1) arid ovarian (OVCAR-3) cell lines wcre obtaincd from the Department of Anatomy University of Malta. These cell lines were culrurcd and subcultured to propagatc the ccll lines. The cclls were grown in RPMI 1640 lrlediurn in sterile Nunclon@> culturc flasks and iricubatcd at 37째C iu 6% C02.

Subculturing was performed evcry seven days (Frcshncy, 1988).

A cell suspension was obtained by dclachirig :he nionolayer from the flask using trypsin and resuspcnding the cells in RPMI mcdium. Complete ccll detachment was visualized under a s l O O rnagrlificalion nlicroscope (Diavert Leitz-Wetzlar). 20ml of RPMI nlcdiunl was added lo cach flask (x2) and the celI suspension was nixed. A ma11 samplc was withdrawn and cells werc counted in an Improved Ncubauer h a e ~ ~ i o ~ ~ o n using lcter the method described in the Sigrna Ccll Culture Catalogue ( 1994).

The drugs wcre added on day 0. Twenty-eight tubes ivere used in all. Irnl of ovarian cell suspension was added to each of fourteeu tubes while lrril of stomach ccll suspension was added to anothcr fourteen tubes. Thc coriccntrations of cucurbitacin E used were: 1.8sl0'~hrf, 1.8~10-~M and 1 . 8 ~ 1 0 ' ~The ~ . concentrations of

me Cytotoxk Activify of CucurbitacinE and BuruIphan busulphan used werc: ~ X ~ O - ~4x10-% M, and 4 x 1 0 ~ ' ~ ' ~ .

2nd of [he tlircc solutions with differcrit concctrlratioru or cucurbitacin E wcre addcd to sis tubcs conraining ovarian cariccr cclls, and to sis lubes conhining stomach canccr cells. Thc samc procedurc was repeated for busulphan. The rest (LC. four tubes) actcd as thc control tubes in which 2 rnl of RPMl rnedinnl ucrc itddcd to the cancer cell suspcnsion.

From days 7 to 11, the number of viablc arid non-viablc cclls was countcd using a haernocy~ornerer and 0.4% trypan bIuc for [he sraining of non-viablc cells (Frcshncy.

1988). Tllc expcri~ncntwas followd from day 7 10 1 I. as it was obsencd in our laboraton that therc was no significant lethal effcct on the cancer cells froni day O to day 7. The fivc-day period. day 7 to 11. was sufficient lo provide i ~ ~ o r m a t i o on t i lhc cylotosic actiiity of both cucurbitacin E and busulphan on thc rwo canccr ccll lincs. A p r c l i n l i n a ~study had shown that rhc decrcasc in percentage ccIl viability was not significant aAcr day I I. Thc percentage cell viability was calculated using ihc number of viablc and non-viablc cells ob~aiiicd.Thc four results wcrc used to obtai~ian averagc perccntagc cell viability. Thc counting of non-viabilc cclis was necessary to dctcrmine thc LCro. which is the concentration of cytotosic compound rcquircd to kill 50% of thc cells in suspension. Thc dccreasc in cell viability should depend on the cylotosic activity of the compounds and not on thc limi~edenvironmcnlal. factors. which include nutricnt availabiliy in thc 111cdiurn and the conditio~isinsidc Ihc ~ncubator. The cell counts for thc tubes treatcd with thc cytotosic conipounds wcre adjusled by taking Ihc average ccll count in thc controi tubes to be 100%.

Rcsults Tumour Cell Growth Inhibition. Figurcs 2 to 5 show the percentage log cell viability against number of' days. for ovarian and stomach cancer cells. both trcated with cucurbitacin E and busulphan.

As can be observed from Figure 2. at 1.8x10'%. cucurbilacin E showed a lower ternii~lalpercentage log cell viability than at 1 . 8 I~O ' ~ M alrhough . ceH viability, for the latter, was markedly reduced, Figure 3 shows that the idibition of tumour growth is higher with increasing busulphan concentration. At the two lower concentratiotls (4x IO-'M and 4 x I O - ' ~ M )of busulphan used. a rapid fa11 in cell viability was cvident after day , decrease was observed 10 while at ~ x ~ o - ~a Mrapid after day 9. Figure 4 shows that the effect ot' cucurbitacin E on storiuch cancer cells varied with the three different concentrations used. At I . 8x 1 0 ' ' ~ cucurbitacin E, there was a rapid decrease in cell viability betweeu day 8 and 9 but a slow steady fall thereafter. At 1.8xl0-% cucurbitaciri E, there was a linear decrease in cell viability. At 1 . 8 ~ 1 0 " ~ cucurbitacin E. there was a slow decreases in cell viability after day 10. Stomach cancer cells treated with

The four different celldrug combinations wcrc compared using the Probit analysis. The d8ercnccs in the trends for these combinations were found to be statistically significant (Pc0.05, T-4).

4 x 1 0 " ~ busulphan (Figure 5) showed a stepwise decrcase in viability with time.A considerable decrease in cclI viability after day 8 was observed at lhe three concentrations of busulphan used, but no furthcr effect at 4xl0'% and 4s l0%l was obsen~edafter day 10. At 4 ~ 1 0 " busulphan ~ a higher cytotoxic eff'cct was obscncd than at the lower concentration of 4x10-''~.

Ciicurbiracin E showed a higher cytotoxic effcct on the ovarian cancer celis than busulphan. It is worlh noting

100

O/o Log Cell Viability

\

I I Concentrations o f Cucurbitacin E : L

10

0

I

I

I

I

1

2

3

4

I

I

6 Time (Days) 5

\

I

I

I

1

7

8

9

10

II

Figure 2: 'l'hc Perccntage Log of rhc Adjusrcd Ccll V~ahilivagainst Tirnc for ovarian cancer colls cxposed to lhrcc conccntrntions of Cucurhitacu~E (Concentrations: I I 8x10".

2 1 8 x 1 0 ~ ' ~.1. = 1 8x1 O'"M).

% Log

Cell Viability

-

Concentrations o f BusuIphan: --I +2 +3

5

6

Time (Days) Figure 3. Thc Pcrccntage Log of the Adjustcd Ccll Viability against Tirnc for ovarian cmcer cells csposcd Rusdphan (Concclr~rations:1 - - 4 x 104M.2 4 s 10" M, 3 = 4 x 1 0 . " ~ ) .

.--

to

thrcc conwntmtions uf

7?te Cutaroxic ActEviv o f Cr~cr~ditacin E and Busrrl~han

32

% Log Cell Viability

C o n c e n t r a t i o n s of C u c u r b i t a c i n E :

-+1-

10

0

-t2

jr-3

I

I

I

I

I

2

3

4

I

1

5 6 Time (Days)

I

I

I

I

7

8

9

10

I I

1:igurc 4: 'Thc I'crccntagc Log ol' 111cAdjusted Ccll Viabil$ against Time for stomach cancer cclls exposed to thrcc conccrr~rationsot' Cucurhibcin ?I (Chtlcentmt~on.s: 1 1.8 s I O ~ M2. . 1.8 s 10-'M3 1.8 x 10%l).

-

100

% Log Cell Viability

-

10

1 0

Concentrations of Busulphan: --I + 2 +3

I

I

1

I

I

I

1

I

I

I

1

1

2

3

4

5

6

7

8

9

10

11

Time ( D a y s ) Figurc 5: Graph of thc Perccntagu Lee of thc Adjustcd Cell Viability againsl l'irnc [or stomach cancur cclls cxposcd to thrcc cor~ccntrat~ons of tlasulphnn (Conccnrralions: I - 4 X ~ O - M , 2 4 s 10-%. 3 = 4 x 10"%)

that at the highest conccntralion of both q-totosic cornpunds used (1.8~10% cucurbitacin E and 4 ~ 1 0 %

bnsulphan), a similar pattern was obsenled: clraractcrized by no fi~rthercytotoxicity at day 1 I . In thc stomach cancer cells, a grealcr percentage ccII d a r h was obtained with busnlphan than with crlcrlrbitacin E.

for the ovarian cancer cclls at day 10, and a minimum LC5(,of 0.5269M for the stomach cancer ceIIs at day 7.

For busulphan, the minirnum LCW ior ovarian car~cer cells was 9 . 1 4 ~ 1 0at- day ~ ~ 9. while that fur the stomach cancer cclls was 2 . 1 4 s l 0 - on ~ ~day 10.

From thc vaIues onc may notc that cucurbitacin E has a greatcr aclivity on ovarian cancer cclls than busulphan the rninirnum median lcthal corlcerltration for thc two (niLCso E < tnLCSo B). On thc other hand. different cell lirics 1reale.d with the two qotoxic drugs. busulpha~ishoweda grcatcr effect on the stomach carlccr Cucurbitacin E showed a minimum LC50 of 2 . 7 2 ~ 1 0 - ' ~ cells although this occmcd on day 10 as opposcd lo the

Minimum Median Ldhul Concentrution. Table i shows

Ovarian Cancer Cells

I It value*

Stomach Cancer Cells

Cucurbitacin E

0.7575

0.8795

Busulphan 1nI.C50

9 . 1 4 3 ~ 1 0M -~

2 . 1 4 x 1 0 - ~M

R value*

0.9006

0.9540

'I'ablc I . 'Table showing the m l C 9 for thc two different compounds and ccll lincs. ' N l R valucs arc takcn at p0,05,rr = 4.

rrlLCFrlof cucurbitaci~lE which was found on day 7. 'The LC5(,for cucurbitacin E-treated stomach cancer cells is too high (0.5269M) to be considered as an effective compound.

Discussion Since cucurbitacin E has been shown lo have an effect on DNA by alkylation (Kupchan cr (11.. I973), and on the ccll membrane by [he proccss of pinocytosis (Gallily ef 19621, it was of interest in this study to compare ils cffecls on calicer cells with thosc of busulphan and to draw sornc conclusions from the results obtai~ied.

(I/.?

T ~ m o u Cell t Go~uIhInhibition. The results show that thc in vilro cflotoxic effect of cucurbitacin E was best observed on ovarian cancer ceIl Lines wlulc busulphan sho~vcda greater effcct on stomach carlccr cells.

If one considers that cucurbitacin E is taken up by the tunlour ccll by a ratc limiting process, there might be sulficicnl uptake a1 low concenlrations to have an aIkylating effcct on the DNA. This might explain the greater cylotoxic effect observed for cucurbitacin E on ovarian caricer cells a1 thc lowest concentration ( 1 . 8 ~ 1 0 - % ) uscd. Whcther [his effect is due to the process of pinocylosis is still to be detcrrnined.

Howcvcr, this process is grcarly influenced by high cucurbilacin E concentrations, whcrc an increase in the uptakc of fluid inside the celI leads to cell blistering and eventually cell dcatli. This was observed by Gallily and co-workers (1962) on four cell lines, using elatericin A and 0. At high col~centralions. the effect on thc ccll rncnlbrarie is marc pronounced.

not appear to affect the plnocytic acrivity of thc turnour cells since cell morphological changes were not observed. Thc high ratc of ce11 death observcd for thc high concentration might be duc to lhc effects on the DNA by alkylation.

At the lowest concentration ( 1 . 8 ~ 1 0 of ~ ~cucurbilacin ) E

used,a small effcct on the stomach cancer cell line was observed, probably due to the limited amount of drug in solution. However, at the highest concentration (1.8x10%Q a tnarked effect on cell viability was observed. It can be concluded that stomach cancer cells showed marked resistance towards cucurbitacin E, as was shown by the ovarian cancer cclls toward busulphan. On the other hand, busulphari showcd cffcctive cy~otosicily in the stomach canccr cclls. Although there was a great reduction in the viable count at Lhe highest concenlratioti ( ~ x ~ o - ~used.

M ) the 4 x 1 0 " ~ concentration showed a lower end-point. However, at these hvo conc~ritrations, after day 10. no firrthcr significant inhibition was obsenfed.

It wouId appear that the activity of tlic cytotoxic compounds on stotnach cancer cclls does not depend on thc pinocy?ic activity but on the alkylating cffcct on the DNA since busulphan had a greater activity lhan cucurbitacin E on thesc cancer cells.

It can be concluded from these results that cucurbitacin E lacks thc pronounced alkylating effcct of busulphan but the latter lacks the pinocytic activity of cucurbitaciri E. It might also be postulated that cucurbitacin E iricrcascs the uptake of busr~lphan(and other alhylating agcnts) while the latter exerts its effccts insidc the cell. The effect of cucurbitacin E on the permeability of wII tnenlbrancs (Shohat er a/., 1962) could be due 10 its steroid-likc structurc which is sirniliar to that of Ilie cell rncrnbrane. It should aIso bc strcsscd that cucurbitacin E has an effcct on both cell lines. although a tuini~nalone on the stomach canccr cclls.

Minimum Mediun Lethal Concentraizbn. The mcdian lcthal coucentrations (LC50) for cucurbitacin E on the ovarian and ston~achcancer cells show that for tlic ovarian cclls the LCso was quitc satisfactory and hence nierits fi~rthcrattention while for stomach canccr cells the high LCso indicates a lack of sensitivity of rhese cells for the cytotosic con~pound.

For bnsulplia~ithe LCSo, for the ovarian canccr cc1k is ) ~ 4s10'%, BusuIphan, at a conccntratiori of 4 s 1 0 - ' ~and qnitc high and so it can be regardcd as iricffective for the did riot havc an cffect on tlic ovarian cancer cells. lreatrnent of ovarian cancer. In fact the mLCro for This is known as the tumerostalic effcct. At these two busdplian is 3361 times greatcr than that for concentrations. insignificant cytotoxicily nmas cucurbitacin E in thcsc cclls. However, the low LCso for obsenled until day 10 aftcr which a decrease in cell stomach caricer ceIIs suggests lhat it can bc used. On the viability was obscrvcd. At ttic highest concentration C busulphari ~ ~ is much smaller than contrary, tbc I ~ L for ( 4 x 1 0 . ' ~ ) uscd. thc same cffcct was observed until that for cucurbitacin E, i-e. the mLCr) of the Iattcr, being day 9 after which there was a better response.This about 2 . 4 6 ~ 1 0 times ~ grcatcr Ihan the rnLC5,, of ~nigtit be explained by the fact that busulphari did busulphan.

34

To substantiate the above finding, further iuvestigations would have to be carried out to determine the cstcnt of thc activity of the two cytotosic agents in vivo, fo deterrnirlc morphologicaI changes and to detect any DNA aberrations.

Acknowledgments Thc authors are indebted to Prof D Lavic. Head of Department of Organic Chemistry in the Weizmann lnstitutc of Science. Israel for thc authentic sample of cucurbiracin E, and lo Dr G Pcplow of the Department of Chemistry, University of Malta. for thc consultation in the use of analytical methods for thc determination of thc prcpared cucurbiracin E.

References Cini G (1991) Faqqus il-Hmir li faqqas it-Trnintas IrbighaL L-Eghjun. Lehen il-Movimenl Civiku Xoghra fiICV?. 9.4.

Farnsworth M (1992) Napralert (SM) Issue 2: Departmen1 of Medicinal Chemistry arid Phannacognosy. College of Pharmacy, University of Illinois. Chicago, U.S.A. Freshney RI (1988) Culture of Animal Cclls: A Manual. of Basic Technique. Alan R Liss, Inc., New York.

132-134.

Gallily R. Shohat B. Kalish J. Gitter S and Lavie D (1962) Further Studies on the Antiturnor Effect of Cucurbitacins. Cancer Resenrch. 22, 1038.

Gitter S, Gallily R. Shohat B and Lavie D (1961) Studics on the antiturnour effect of Cucurbitacins. Cancer Research, 21, 516.

me Cvtotoxic Adivifv of Cuarbltacin E end Busufphan Kupchan SM and Tsou G (1973) Thc Structure and Partial Synthcsis of Fabacein. Journal of Organic Chemistry, 38, 1455 - 6.

Kupchan SM. Smith RM. Aynehchi Y and Maruyama M (1970) Tumour Inhibitors. LVI. Cncurbitacins 0. P and Q. the Cytotoxic Principles of Brandgea higelo vii. Journal of Organic Chemistry. 35, 289 1 .

Laniia~lwG (1975) Duwa o Semm il-Hxeiiex Maltin. In: Edi-izjoni Klabb Kolba Mallin ValIctta. Malta. 33- 40.

Lavie D. and Szinai S (1958) The constitucrils of Ecbnlli~inreluteririm L. 11, aElatcnn. Jortrnal of' the American Chernicnl Socic(y, 80, 707.

Penza C (1969) Flora Maltija Medicinali. Progress Press, Malta, 29.

Roger H and Spcctro R (1976) An Inlroduc~ion to ~\dechnrrisrns in Pharrrracology and Therapeutics.

Firs/ Edtion. William Heimmann Medical Books Ltd., Great Britain. 383-385 Shohat B. Gitter S and Lavie D (1962) Antitumour Activity of Cucurbitacins: Mcrabolic Aspects.

Cancer Chenrotherapv Reports, 23. 19.

Shohat B. Gilter S, Lwy B and Lavic D (1965) Thc cornbiried cffect of cucurbilacins and X-ray trcatmcnt of transplanted tumours in inicc. Cancer Research. 25, 1828.

Sigma Cell Culture Catalogue (1994) Sigma Chen~ical Company. 205-206.

Ycsilada E. Tariaka S. Sezik E and Tabata M (1988) Isolation of an anti-irfflanunatory principle from the juice of Ecballiurn clatetium. Journal oJ tVn,ural Products. 51, 504.

Research Article Motor Vehicle Accidents: Analysis of Casualty Department Data, St. Luke's Hospital, Malta.

Summary: Mdor vehicle accidents WAS)referrcd to St. Luke's Hcspital CasualQ D q m t n ~ ~were k t mdyml. There were 616 MVAs ref& to hospitd during the year an incidence of 170 per 11)[),000 of the popkition. There were 3 accident rnte peaks.

1l;llncly.oiie wly 111the morning a second around nud-day, and a Lhird m u n d 6 pm. The majority (55%) involved young pcrsoris under the age of30 ycats. The risk of hospiralisation was highest for nlalcs in the 20-29 age group (4801100.000). Nmrly one third r q u i r d Impitahsttion fbr m r c scrim injurics.Thisanalysis crnptkuiscs the need for urgent rnt.lasures to bc taken to rwfuce thc rntc of MVAs nnd axcialed ri~o.clrbidity.

.

Keywords: lriotor vc1iiclc accideiits !Vdtz St Lukc's HospiraL ernergenq?adnussions

Tllc numbcr of motor vehiclc accidents is increasing at an alarming rate. This has resulted from a nuruber of factors. In Malta. there is currently one car for cwry IWO residents (scc Brockdorff 1995).

Morcovcr. Mallcsc d r i w r s havc bcconlc rnorc ~nobilc.using their car illorc frcqumlly, and younger i l l agc as a result of thc increasing purchasing power of lhc individual.

Sludics rclating to thc incideacc and pancrn of car accidcnts in Malla arc riot rcadily available (see Canlillcri 1969). 111 h i s sl11d1,. an analysis of MVA casuallies admilled to the C:asoaIly Dcpartrne~llor St. L.ukc's Hospiral ovcr a pcriod or onc year (1994) was cnrricd out with a ~ ~ i c itov dctcrnlinc pallcrns which wcre signiiicantly rclarcd to increased traflic accidenls.

Jan Fcb Mar AP~ May Jun Jul Aug S ~ P Ocl Nov Dec

Total 37 45 38 34 21

70 97

13 44 34

Male 26 29 22 24 I6 48 62 26 26 20

Female 11 16 16

10 5 21 32 15

42

28

16 13 14

61

41

19

I'ablc I : Motor vchiclc i n j u y adrnittancc ro SILH. !)I sex 1994.

Ratio M/F 2.36 1.8 1 1.37 2.10 3.20 2.28

1.94 1.65 1.69 1.70

2.00 2.16 rnonth and

Materials and Methods Thc registers of the Casualty Dcpartmcnl. St. Luke's Hospital. werc examined Tor tllc vwr 1994. TIic was obtalncd. follon.lng ~r~formatiou Agc. scs, placc of rcsidcncc, datc and r i m of accidcnt. and outconic (whether discharged or lra~lsfcrredto a hospital ward). Dala was stored on a data-base and analysed nsir~g n stalis(icnl analysis packagc (Mnrulgislics Statgraphics Plus). Risk of adnlissior~to SLH for injury for MVAs was defined as: ,Swnhcr ofpersoru nd~nrrieddrmng a dejined t m e ,f'rrnrhcrofpersons rn thar oge,:wr bracket rn the Malrese poprrlatron

Results

Thc number of ~njuricsresuliing from motor vehicIc accidcnts adniittcd to Sl. Lukc's Hosp~taI(MVAs) pcr wonth vaned from a low of 21 in May lo a high of 97 in July (TabIc 1, Figurc 1). As cxpcclcd. Ihcrc is a cons~dcrablc rlialc preponderance. wilh malc.~en~alc ratios ranging froni 1.4 to 3 . 2 . Thcrc docs riot seem to bc a sasonal distriburiori in this ratio.

Jan Feb MarAprMayJun Jul Aug Sep OctNovDec

Female

Male

Months

1:igure 1: Monthly variation in the number nf admissions to SLJI casualty dcpanmcrrt.

Therc was a preponderance of accidents in the early hours or the moniing. In ruaIes. the pcriod from midnight till 4.00 an accounted for 28.5% of all accidcnls. Therc was a second pcak during latc

36

Motor V e b k k Accldentr

morning and another around 6 pm. In fcmalcs. thc periods bctween 4 and 6 pnl accounted for thc highest relative proponion of accidents (Figirc 2. Tablc 2).

-c-Male

+Frniale

Therc wcrc significant variations in the incidence of hospitalimtion ratcs belwcen thc days of the wcck, with Sundays showing thc highest incidcncc (21%) (Table 4)

Time of day

'IY Y IS\ L Figure 2. Variation in (hc ~lumbcrof admissions to SL.H by timc of day Nolc thc prnmincnt a r l y niorning peak affecting mainly male individuals. Olhcr pcaks occur a1 around mid-day and around 6

I

1751178560

383

I82709

1214

5%

'I'ablc 3: MVAs: risk of Hospi~lisalion.by agc and scs.

I

Males [Females 1 Tohl No. % % ]NO. %

Injuries Number

NO.

I

Per cent

Monday

Tuesday Wednesday Thursday Table 2: Number of pcrsorls Iiospitaliscd following MVAs. by lirnc of occurrcnce.

The majority of accidents involved young persons undcr thc agc of 30 - in fact, 64% of injuries invoIvcd persons in this age group. with a sharp drop after the age of 30 years (Figure 3, Table 3).

Friday Saturday Sunday Tablc 4: Varialion in nunrhcr ol'injurios. by day of wcck

A measure of thc degrec of sevcrih of thc MVA may be obtained from an analysis of thc discharge rates after attending the Casualty Department: obviously. those transfcrred lo one of the hospital wards would accorinl for the more severe cases. Table 5 shows that nearly 29% of all MVAs were referred. Thc proportion of males was slightly higher than that for fcmales. but this was not slatistically significant.

1 Males I

-c-XMex

+ Fr.nrdlr3

,%Re

6 n)

Discharged Referred Total % Referral

25 1 104 355 29.3

Females

147 49 196 25.0

1 Total 404 153 55 1 27.8

Figurc 3. varialion in admission ro Slh by agc and scx. Notc thc sharp pcak for nialcs agcd 18-20 ycars.

Tablc 5

There were 185 MVAs involving persons undcr thc agc of 20 years (33%), and 171 bctwe.cn the age of 20 and 30 years (23.4%). For thc 20-29 age group, the risk of hospitalisation was 4XO/lOO,OOO in males and 1991100,000 in fcmales and was only slightly less for Ihe 10-19 age group (373 and 181 respectively).

Table 6 gives an anaIysis by area of residence, for localities where Ihc total nii~nberof MVAs was 10 or morc. Whcn expressed as a proportion of the total

Proportion of MVA pailcnts rcfcrrcd I;or Purtlicr inveslignt~onsand/or lrcatrncnt (Notc: Kclativc risk - 1.24. SE(KK! 1.22. 95% confjdcncc intcrval for corrcctcd Kr 0.38 ,- 1.84. cA for cqual risk 0.96. N.S.).

I

1

XIA1,ES FEMALES TOTAL % EXPECTPOPn

Possible factors accounling Tor this unnecessary loss of lifc incIudc the following: a , the marked tendcncy for Maltese youths to indulge in late night entcrtairunent. particularly on a wcekend, and, in thc summer months. throughout ~ h week. c b, thc increased tendency to drink, which is now being offered in bulk (Disco clients are being offered thc option of paying a once only fcc of tmIO.OII and drink alI they can). c. increased availability of molor cars for younger age group drivers. Iticreased afflucncc has been a rrigior factor in this phenomenon. in fact, one of thc most obvious findings in this study in con~parisonwith the study carricd our by Carnilleri rclating to traEk accidents in 1967 is thc Dresence of h e early morning pcak of accidents which was nonexistent at that timc.

I'ablc 6: llospilalisation following MVAs: by plact: ofrcsidence

population residing in the particular locality. i t is seen Ihar certain areas of residence are associated with a grater than expccled incidence of MVAs. For instance, Floriana, Marsascala. St. Juliaos. Santa Lucia, Msida and St. Pauls Bay (Bugibba) were associated with a MVA rate of more than 3 per 1000 population. whcrcas at the other end of the scale, Paola. Rabat, Qormi, Mosta. Zejtun and Hamrun had less than 1.5 per 1000 (X2 = 64.5; P < ,001).

Discussion This study represents a a analysis of motor vehicle accidents prcsented at SLH during a period of one year.

There were 616 MVAs treatd during this year, representing 170 per 100,000 populalion. It is important to bear in nlind that this represents only a proportion of MVAs and does not include the considerable number of minor accidents that do not rcquire hospitalisation.

It is of interest to note that whiIc Junc and July saw peak MVA rates, August was a relatively quiet nlonth , . . .- ..-.. . .. . . (wit11 45 M V A Inclcients. less man tne numtxr In February). Thus the dislil~ction between the "busy" summcr months and the rest of thc year is not so clcarcut. Thc rcasons for this low incidcnce in this relatively busy month are not clear. and could be due to random variation from year to ycar. Analysis over a numbcr of ycars would bc rcquired bcforc an analysablc pattern can cmcrge.

Thc timc distribution of MVAs calls for comment.

There were three distinct peaks seen in the rate of accidents, namely (a) early hours of [he morning, (b) around mid-day, and from 4-7 pm. WhiIe the daytitne accidents might be correlated with the increase in motor trafic on the roads, the early morning peak is more likcly to be associaled with a youngcr age group.

The day-to-day variation was significant, wirh lowest incidence during Monday-Wednesday and ir~creasing for the resl of [he wcck with a peak on Sunday (which inchdcs thc Saturday night / Sunday morning MVAs).

Whether thcrc csists an accident-prone personality is > . * . . .. . * -.- > IronI n:-,--> rrruana (nmtuvi amicun to prove. A rcccnr. stuay et al, 1989) has shown that you.ng male persons involvcd in iraflic accidents are rnc,re likely to show impassivity, ad~~enturousness, n: iivete, excessive trustfulness, and dcpressio~i - faclors relating to the control of emotions.

C

",!.-,KL:

It is to be noted that whcrever statistics over a long period of time have been analysed, the conclusion is confirmed that there is a tendency for accident death rates to increase over all ages, but the most strilung increase is likely to affect youths aged 15-24 years of age (Millar & Last, 1988). These findings havc bcen confirtncd in Malta in a recent study (Gala, 1992).

Any preventative aspects must take this into consideration. and special effort should bc made to investigate thc factors that could possibly be responsible for thc escalation of this specific type of MVA. 'I he value 01 weanng seat belts In preventmg senous

injury has been ernphasised again in a number ofstudies.

For instance, Orsay et al (1988) conclude that "safety belt wearers had a 60.1% reduction in severity of injury, a 64.6% decrease in hospital admissions, and a 66.3% decline in hospital charges", etnphasising thc increasc cost of u~edicalcare required for non seat-belt wearers.

The higher than expected incidence of MVAs associated with certain localities is of interesl.

Residents at Floriana, M'Scala St. Julians. Santa Lucia.

Msida and St. Pad's Bay (Bugibba) arc 2-3 timcs more likcly to bc involved in MVAs than rcsidcnts of Paola, Zcjt~rn,Rabat, Qormi, Zurrieq or Hanlnrn (Table 6). It is to bc emphasised Lhal these are residential addresses and not accident localities, and therefore do not

'

-$ ; - :.:

-

..

necessarily relate to local road or tr&c condifions. although it is reasonable to argue t h l residents of high density W c areas are more likely to be involved in car accidents than tbose residing in m r e rural areas.

More study would be required to confirm these findings #mi to ferret out those factors rrssociated witb this &crea& accident rate. t

While tbh study is reslicted lo only ode year's W A S . the pattern of accidents found is likely to be repeated.

Efforts to rcduce tbe number of W A S must stad with an analysis of the factors that lead to thcse accidents.

While it was not the aim of this study to tease out these factors. serious efforts must be made by the relevant authorities to ensure that the multiple factors involved in dar accidents as highlighted in the daily prss (see cg. Cauchi M.N.1995) are analys&dand dealt with.

References

Brockdorff PV (1995) Facts. Figures and Myths on

Malta. The Sunday Times, February 12.6.

Cauchi MN (1995) Road Canrage: Need for Action 17te Sunday Times, March 5.39. illeri W (1969) T d c Lnjuries in Malta some considerations and s u g g d o Hospital Gazette. iv, 19-27.

G (1992) A Young man's Premature Mortality in Ma1 4 I, Veilahti J.Asplund P. Siniwo J, Laitinen L. Kosken~duoK (1989) A sixteen factor t g for predicting automobile driving accidents of young driversAccid Anal Prev. 21.4 13-4 18. r WJ and Last JAd (1988) Motor vehicle ugffic accident mortality in Canada. 1921-1984. Am, J. Prev. Med, 4(4) 220-230.

Orsay EM, Turnbull TL, Dunne M, Barrett JA, Langenberg P and Orsay CP (1988) Prospective study of the'effect of safety belts on morbidity and health care msts in motorvehicle accidents.

J.A.M.A.. 260,3598-3603.

-

d

*,

I

I

Current Research Profile Dr. Robert Martin Borg Ph.D. (Dalhousie) Deparlttrent of Clhernishy, University of,Llallu, A~fsida,Xfcdta.

Roben M. Borg was born in Slierna in 1956. He attcnded secondary school at St. Aloysius College, B'Kara, followcd bv h ~ oyears at the (old) Junior College, Valletta. In 1974. he joi~icdthc B.Sc. course at thc University of Malta, and subsequently read for a Masters degrcc under the guidance of Professor Victor Ferrilo. The rille of his M.Sc, dissertation was "The Ano!ysrs of the Oxuzolone Derivutives oj'dmino Acids h.v tim C:i~ron1atograpt7y-1tfn~vs Spectroscopy", This work also led ro his firs1 academic paper.

His sclec~ion for a Canadiar~ Cornrnonwcalth Scholarship in 1979 cnabled him to read for a P11.D. in chcrn~stv a[ Ddhousic University. WalXas, Nova Scolia. Thc ncsl four years, undcr the rnentorship of Prof'cssor Donald R. Arnold. determined the direclion his acadc~niccarcer would takc, as a passion for organic photochemisrry was kindled. Indeed. to this day, the titIc of u s Ph.D. thesis: "Rndicnl lons in Phorochetnrstty" best describes his current rcscarch intcrcsts. These involve thc sludy of thc light-induced generation of chargcd organic molecules, and rheir subsequent reactions.

Having graduatcd from Dalhousie in 1983, he spcnt the ncst five ycars as a post-doctoral fcllow, the first two at Ihc University of M q l a n d a1 College Park followed by threc years at thc Univcrsi~yof Toronto both posts held with prornincnt photochernists. His research effons in this pcriod cul~ninatcd in sevcral publications, including the award of a U.S. Patcnt, which describes a novcl proccss by n'hch water-soluble polymers used cstcnsively in thc oil indusrr?, can be ~nadcfluorcscent, a feature that rrlakes thcir quantitative use easier. and thus might prevcnt pollulion by avoiding waste.

in a British journal within six months of its acquisition.

Since then, another paper has been publishcd. with one more ~nanuscript on the way. These publications (described beIow) are also thanks to the hard work of the cxcellent graduate studcnts currently reading for a Masters dcgret: under Dr Borg's supervision.

Current Research Interests a) The photochemical reaction between cyan* aromatics and alkenes in acetone solution.

It has been s h o w prcviously by Arnold and co-workcrs (1984) that the irradiation of the dicyanobe~ucncsin the presence of 2,3-dimethyl-2-butene (telramethylethene, TME) in polar acetonitrile solution yields substilution products as a result of photoinduced electron transfer, whilst in lion-polar benzene solution, no reaction is observcd to occur (Scheme 1).

Scheme 1 A new mode of pholorcactivity of thc dicyanobenzencs with TME, when acetone is uscd as a solvent, has recently been repond (Borg, 1994). Irradiation of 1.3or 1.4-dicyanobenzene in the presence of TME, in acetone solution. affords qano-substituted 2,3-dihydro2,2.3.3-tetramcLhyl- Ikl-indcn-l-ones in good yields.

Thc orfho isomer of dicyanobenzene provcd relatively

In 1988 Dr Borg returned from Nonh America and joined thc Depan~nentof Chernisty at thc University of lMa1l.a as hll-tilnc lecturer. where hc still is today (now as senior Icclurcr). Hc tcachcs organic chemistry, nuclear magnetic rcsonance (NMR) and mass spectroscopy. and photochemislry on thc 8.Sc. and M.Sc. courscs.

I n 1994. thc Depanment of Chcn~istywas granted snff~cient funds to purchasc a 25(liMHz NMR spcctrornctcr. This csscntial instrument gencratcs some controversy duc to the high running costs of its snperconducting magnet (thc only onc in Malta), which requires cryogens (liquid heliurtl and liquid nitrogen).

Howevcr, by having access to this instr~mlenl(which is i n use by rcscarch sludents practically 365 days a gear), Dr ~ o r was g able to publish the results of his research

5-CN 6346 6CN 6896 7-04 trace

Evidence for rhe irnines being intermcdiatcs, as wcll as

for thc involvement of a charge-transfcr interaction in the reaction n ~ ~ h m i was s n ~also p r m n t d .

These ncw pholor~ctio~lr arc significant. sincc they may provide a general synthetic n~cthodoIop lo indenonc derivatives wluch are known to possess biological activity. or could scnc as qnthetic prccmors to natural products such as steroids (Galatsis. 1994). Ongoing studies in the laboratory of Dr. Borg are thercfore exploring the full scope of this reactton. so as to deterniinc its synthetic utility. This work is curren~lyk i n g ~ i e out d by an M.Sc. student. Ms Mariclla Berry, whose first research efforts at tllc undergraduate lcvcl w x c responsible for thc inuplemcntalion of this pro-jccl.

Refer~ncw Borg RM. Bcn-y M. and Mangion D (19911 Thc photochc~iucalreaction bctwccn dicyanobewsnes and 2.3dimeUlvl-2-birtene in acetone solution: A potential route to subsw~tod2.3-dihydro-IH-indenI ~ n c s7elrcrhedr.on ixlters. 35(45), 3485.

Bolg RM. Amold DR arid Cameron TS (1984) Ra&cal ions in photochemisp. 15. The photosubs2ltution rcactiori behmn dicyanobenr.ncs and alkyl olefins C,'nrr. J. C h t ~ i . 62, .

I785-1802 Galatsis P, Manwell JJ. a i d Blachcll .IM (1094) hdenonc synthesis. Iniprovcd synthetic proloco1 and cffcct of substilulion on thc intrmolccular Friedel-Crafts aqlalion Cat!.J. C'lretn..72. 1656-1659.

-

h) The photochemistry (electron transfer) of l,l,op tetraphcngl-1 ,(el>clienes The discovcy by Neunteufel and AnloId in 1973, t h ~ UIC l irradiation of an acetonitrile solution of I. I-chptle~qMhene and elmron Vamfer photosensitizers such as 1.4-

diqanobenzene yields thc tc~&dromphdlalene ring systcm via a f o r n d [4*2e] ~ycloadditior~reaction (Schcme 3) led to a pleLhora of activity in this arm.

f o n ~ ~ i oof'n dic expected 14et2el cycloadducr in Iugh chemical yield and stercosclectivity (Schcrr~c4).

I

J

Schernc 4 In Dr. Borg's laborator?, studics have been cdcnded to othcr teuaphenylatcd dienes of v q i n g m&ylcne cllain length, with v e 9 irncrmng results. These will be presented at the American Chenical Sock& National mcctitig in Orlando, Florida this summer, with Ihc full paper due to bc subnlitkd for publication shnnIy t h c r d c r .

(Thcsc: resulls are n i a i ~ i rhc l rcsult of thc Iurd nark of :in M.Sc. student Dino Mangon. who is currcntlv in the process of writing his Qssertntion. Dino will shortJy bc leaving for Canada. where he has been awarded a gradi~atc fellowship to read !or hs Ph.D. - also at Dalhousic University under the pidance of Don Arnold!l.

References Arnold D R and Neurlteufel RA ( 1973) Radical ions i r ~ pholochcmistry. 1. Thc I , 1 -diphcn~lcthylcnc cation rad~cal.I. .4n. C ' k ~ i .SOL:..05. 4080.

Anlnld

DR Borg RM. and Albini A (1% 1) Photosensitized (electron trans fer) [2c+4c] dimcrimtion and cross-cycloaddilion of phcnylated olefins: trapping the itltermediarc. .I. (.'hem. Soc., Chetn. C'otrrmun.. 1.38.

Pvlangion D, Borg RIM. and Errington W (1905) Thc photosensitized (electron rransfcr) (4e+2cl I , 1,K.Xintramolccu[ar cycloadditiorr of tclraphcny1-1.7-octadieric; X-ray crystal stmcturc cl f 4a.93-trans-4a. 10-trans1.2.3.4.4a.9.9a. lO-octahydro-9.Y.10triphe~ylanthraccnc ./. Chettr. Sot., ( X m .

C'otnttrrrn..1963. c) Chemical Actinometry

Scheme 3 Despitc Llic cxlensive 1uture of Ulc abwc studics. the [4e+2c] qlcloadditiorl reaction described abovc has nwcr b a n applied lo an inlramoloclllar system and it was dccided to investigare these r e a ~ ~ i o nfurther. s Indeed. in a preliminary report (Mmgiolt 1995) it has recenlly k e n demoriwated that the 1.4-ch~yanobc1v.cnc-sensllij.4 irracliation of 1,l.S.S-telmphcnyl- 1.7+cta&ene leads to thc

Although this is not a research arca (as such, this article w,ould not bc con~plctcwithout mentioning anothcr M.Sc. pro-ject nearing completion. This involvcs lhc constniction and subsequent use of an electronic ac~inometer.This is an irtdispcrwiblc instrument in pholochc~nistry.and is uscd to quantify photochernicaI Irarisfnrnuations (such as those dcscribcd abovc) 10 thc amounl of light input into Lhc systcm i.c. n ni~~surernentof the qrmnhtt; eflciency of thc photoreacrion. This project is in ~ h ablc c Ilands of Nick?

Gingell, who has almosl cornplelcd thc construction stage, in a highly professional rnanncr. Hc will soon bc cnibarking on sortic important measurenlents on thc pliocochernical sysrems dcscribcd in A) and B) . above.

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Semnd,lheutdityofthestudywiIlbe~in tams of the balance betwen t k di.itms udlided and the LkeWood of beneZn Third. it mua be that the objediws of tke c q m k n f could nol have bem achieved by h e use of less stresdirl proocdures; h e fad that a prbcular procedure lm been used in prcwollsly publistled work will not in itself suffice.The cditols ln'licve h a t scienti* apply skid M criteria to heir wnk, and wiU have no diRin~hyin meding thcrc

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Felice AE (1992) Molearl:~ Epidaniolgy of Haemoglobin, and the Molecular biology of N d and Abnormal Glohin gene e r p z s i o n In: Collected Papers 0;'A R Ellul-Micalld and S Aorini). pp. 357-39 1. Ilniversrty Press. Malta David Rawn J (1989) Biochernis~ty Neil Pattemon Publishen, North Carolina bfuscat 11 Papp ?r.I and Willncr P (1992a) Antidepressant-like cffecu of dopamine agonists in an animal model of dqraqion.

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