We evaluated the influence of near-ultraviolet light (UVA) on the cytotoxicity and genotoxicity of 7 polycyclic aromatic hydrocarbons (PAIl) in larvae of the amphibian Pleurodeles waltl. Benz[a]anthracene (BA), 7,12-benz[a]anthraquinone (BAQ) and anthracene (Ac) proved to be lethal at low doses (some ppb), and the following order of genotoxicity was observed: BA = BAQ > DMBA > DMA (9,10-dimeth- ylanthracene). Ac, AQ (9,10-anthraquinone) and DBA (dibenz[a,h]anthracene) were not found to be clastogenic.
In the larvae reared in normal conditions (subdued natural daylight/darkness alternation) or in continuous darkness, the BA derivatives were shown to be more genotoxic than BA itself: DMBA > BAQ > BA; BA (> 187.5 ppb) slightly increased the level of micronuclei in circulating erythrocytes, while DMBA was strongly clastogenic, in line with their reported carcinogenicity.
In other experiments, rearing media alone (i.e., water containing BA, BAQ or DMBA) were UVA-irradiated for 24 h, and then tested on larvae in the dark ( 'IR-UV/dark' conditions). Photodegra- dation of BA (50 and 100 ppb) gave rise to clastogenic products. By contrast, DMBA (12.5, 25 or 50 ppb) was destroyed by UVA, and we suggested that any potentially mutagenic photoproducts formed were not in sufficient amounts to yield a positive response in the newt micronucleus test.
Polycyclic aromatic hydrocarbons (PAH), pro- duced by the incomplete combustion of organic matter or synthesized by humans, are among the
Correspondence: Dr. M. Fernandez, Centre de Biologie du D6veloppement, UA-CNRS 675 affili6e ~t I'INSERM, Univer- sit6 Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France.
major environmental pollutants being found in the air, ground and water (Mix, 1984; Lioy and Greenberg, 1990). A large number of studies have been devoted to this class of compounds since the discovery in the 1930s of the carcinogenicity of some of their members (see Newman and Veera- raghavan, 1988).
The biological activities, and especially the mutagenic potential of many PAH, are thought to
32
be due to their transformation into entities which react with cellular macromolecules (proteins, RNA, DNA). As in other species, biotransforma- tion in the newt is carried out by a variety of enzyme systems (monooxygenases and hydrolases in particular) leading to the formation of metabo- lites that are generally electrophilic. The harmful action of these derivatives in the organism may be neutralized by enzymes such as glutathione-, glu- curonyl- or sulfo-transferases, forming water solu- ble conjugates which are excreted from the organ- ism (Marty et al., 1989).
PAH may also be transformed outside the organism without any intervention of enzymatic systems (Gibson et al., 1978). In the aquatic medium for example, transformations can take place in the presence of oxygen under the influ- ence of light (Katz et al., 1979; Wood et ai., 1979; Sigman et al., 1991). As it occurs in biotransfor- marion, phototransformation may either activate or destroy the initial compound (De Flora et al., 1989).
The aquatic larvae of the newt Pleurodeles waltl are particularly suited for in vivo study of the harmful effects of experimental (Fernandez et al., 1989) or natural aquatic pollution (Van der Gaag et al., 1990). Apart from toxicity, other noxious effects of waterborne pollutants, even the drinking water ones (Jaylet et al., 1987, 1990), can be evaluated using the newt micronucleus test. This cytogenetic test effectively quantifies ge- nomic damage consecutive to the action of clasto- gens or agents which modify the structure a n d / o r the function of the mitotic apparatus (for the mechanisms giving rise to micronuclei, see Hed- dle et al., 1991). Micronuclei (MN) are readily identified in newt larval erythrocytes and can be counted on blood smears. This method has re- cently been validated by the French Standard A F N O R (1991).
Otherwise, in earlier experiments, benzo[a]- pyrene (BaP) and pyrene were found to be ex- tremly toxic towards larvae (or embryos) exposed to sunlight. These two PAH are widely dis- tributed in the atmosphere and in the aquatic environment (although at markedly variable lev- els) (Osborne and Crosby, 1987; Lioy and Green- berg, 1990; Smith et al., 1991). This appears to be also the case for benz[a]anthracene, anthracene
and to a lesser extent, for dibenz[a,h]anthracene, as all these PAH have been found in drinking water (Jani et al., 1991).
Thus, it is important to evaluate on aquatic organisms the consequences which might result from the light-mediated transformation of such PAH, inasmuch as some of them are known to be carcinogenic in the newt (Arffmann, 1964).
In this study, we investigated the influence of UVA on the toxic and genotoxic effects of 7 PAH in vivo, and we attempted to discern relationships between certain chemical modifications of the basic structure and biological effects. We showed that the biological activities of benz[a]anthracene and derivatives may be modified by lighting con- ditions.
Material and methods
Chemicals (degree of purity and abbreviation) Samples of anthracene (98+%) (Ac), 9,10-
Biological assays For each tested substance, several groups of 10
or 15 animals were formed from the same hatch- ing. Larvae at stage 53 (rough of the fifth toe on the hind feet) were reared at 20°C_+ 0.5°C in synthetic water containing different concentra- tions of the substance under test (or DMSO alone to a maximum of 0.5 ml/1) as well as food (live Chironomus larvae). The synthetic water used corresponds to the medium recommended by the French Standard for acute toxicity assays in the trout Salmo gairdneri (AFNOR, 1985).
The rearing medium (water + chemical) and food were renewed daily for 6 days in the case of a toxicity assay, and for 8 days (or 12 days) in the case of a genotoxicity test. At the end of the 8-day period of exposure, blood smears were taken from each of the treated and the control larvae by intracardiac puncture and the level of
MN was determined after counting 1000 erythro- cytes per smear.
Animals treated with BAQ and their controls were punctured at 2 different times: some ani- mals underwent the puncture after 8 days of treatment, and others after 12 days.
Lighting conditions Four experimental conditions were used. (1) 'UVA' conditions: the larvae were UVA-
irradiated throughout the duration of the experi- ment. They evolved in plastic boxes (10.5 × 31.5 × 17 cm) without cover in a water column of 4.5 cm thickness. The UVA light was provided by two 15-W tubes (TL 15 BLB; Vilber-Lourmat) that had a spectral output ranging from 320 to 400 nm (maximum output at 365 nm). The lamps were linked to the cover of the thermostated enclosure and the larvae were exposed at 45 cm away from the light source. Irradiance, measured with a radiometer (UVR-365 nm, Cofralab) was 250 / z W /c m 2.
(2) Normal conditions: the larvae were alter- nately subjected to subdued natural daylight and to darkness (day/night rhythm). Illuminance, measured with a photometer (Polycontr61e 94, Chauvin et Arnoux), never exceeded 175 lux and radiometric measures confirmed the absence of UV radiation.
(3) Darkness: animals were reared in darkness throughout the duration of the experiment.
(4) Irradiation of the rearing medium: water (1.5 1; 4.5 cm thickness) containing the chemical was previously exposed to UVA for 24 h in the experimental boxes (bottom 45 cm away from the lamps). The larvae and food were then added to the irradiated medium and the experiment was carried out in darkness. This procedure was re- peated daily over a period of 8 days. These exper- imental conditions are abbreviated as ' I R - U V / dark' in the text.
Expression of results The levels (%0) of micronucleated erythrocytes
from n animals in a group were ranked in ascend- ing order. Median, lower and upper quartiles were determined. The F-pseudosigma (a resistant analogue of the standard deviation) was calcu- lated according to Hoaglin et al. (1983). Data
33
from experimental and control groups were com- pared using three statistical tests: Student's t-test (two-tailed), the Wilcoxon test (two-tailed) and the McGill et al. (1978) method. When Student's test was not appropriate, a situation that hap- pened in 20 cases out of 51, interpretations relied on the two other tests. However, when no firm conclusion could be drawn (on account of a dis- crepancy between the statistical tests) a question mark has been plotted in the column 'Results' (Tables 1 and 3).
Moreover, empirically a result is considered as weakly positive if the median of the treated group is below twice that of the control group, when the difference between the two medians is significant.
Results
In previous toxicity assays, larvae were exposed for 6 days to increasing concentrations of each PAIl and to UVA (total dose: 130 J/cm2). The following order of toxicity was established: BA > BAQ > Ac > DMBA > DBA > DMA > AQ (re- suits not shown).
The results obtained in the genotoxicity tests following exposure of larvae to both PAH + UVA (total dose: 173 J / c m 2) are shown in Table 1.
Larvae coming from the spawns of two females were treated with BA (experiments 1 and 2). At the maximum tolerated concentration (3.125 ppb), animals from the two hatchings reacted in a simi- lar way and the relative increases in micronucle- ated red blood cells (RBCs) were equivalent: the response was only weakly positive. On the other hand, experiment 1 carried out with DMBA clearly demonstrated that the clastogenicity of this carcinogen increased almost linearly with concentration, despite the fact that half the lar- vae died at 50 ppb. Moreover, the positive result found in the first experiment with the lower dose (12.5 ppb) was confirmed by the second: once more, larvae from the two females showed similar reactions.
The offspring of a single couple was used to test BAQ: concomitant exposure for 8 days to UVA (173 J / c m 2) and 12.5 ppb BAQ led to the death of larvae, whereas after treatment with half this dose (6.25 ppb), BAQ was found to signifi- cantly raise the MN level in circulating erythro-
34
TABLE 1
F R E Q U E N C I E S OF M I C R O N U C L E A T E D E R Y T H R O C Y T E S (PER 1000 CELLS) FOLLOWING E X P O S U R E OF LARVAE TO BOTH PAH A N D U V A (173 J / c m 2) ( ' UVA' CONDITIONS)
Compound Exp. a Concentrat ion Number (Abbreviation) ppb ~ M of larvae CAS No. Mol. weight
* F-pseudosigma (see Hoaglin et al., 1983, p. 40). a Experiment number. b Toxic dose (corresponding to LCs0 , 8 days). • Larvae treated for 12 days with BAQ + U V A (total dose: 259 J /cm2) . - , negative result; + , positive result; (+ ) , weak positive result; ?, questionable result; ? c, negative with the McGill method (the median confidence intervals of the control and treated group overlap), but positive with Student 's t-test (the means of the two samples are significantly different: p < 0.05) and the Wilcoxon test (the null hypothesis, H0, is rejected: p < 0.05); ? o, positive with the McGill test (the median confidence intervals of the two samples do not overlap) and Student 's t-test, but negative with the Wilcoxon test (H 0 is accepted: p > 0.05),
35
TABLE 2
F R E Q U E N C I E S OF M I C R O N U C L E A T E D E R Y T H R O C Y T E S (PER 1000 CELLS) F O L L O W I N G T R E A T M E N T OF LAR- VAE IN N O R M A L CONDITIONS
Compound Exp. Concentration Number Micronucleus frequency (%0) Result
F R E Q U E N C I E S OF M I C R O N U C L E A T E D E R Y T H R O C Y T E S (PER 1000 CELLS) F O L L O W I N G T R E A T M E N T OF LAR- VAE IN DARKNESS
Compound Exp. Concentration Number Micronucleus frequency (%c) Result
See footnotes to Table 1. ?, questionable result (positive with Student 's t-test, negative with the McGill and Wilcoxon tests).
36
cytes. Over a longer p e r i o d of t r e a t m e n t (12 days) the c las togenic po tency of the chemica l fell (sign of sl ight toxicity).
Final ly , among the last four a roma t i c hydro- ca rbons assayed in the p re sence of U V A (Ac, D M A , A Q and DBA) , D M A induced a signifi- cant inc rease of M N at a non- toxic concen t ra t ion , while the t h ree o the r s d id not.
Thus, f rom the overa l l da t a ( U V A condi t ions) i the genotoxic po tency of P A H was shown to dec rea se as follows: BA-- - B A Q > D M B A > D M A .
In o t h e r exper iments , la rvae were exposed to B A (or to a der ivat ive) in n o r m a l cond i t ions or in darkness . So, a f te r 6 days of t r e a tmen t , B A at 1250 or 2500 ppb led to a loss of weight of the animals , w h e r e a s at a 200-400- fo ld lower concen- t r a t ion (6.25 ppb) , B A led to the d e a t h o f U V A - i r r a d i a t e d larvae. The toxici ty of the two B A der ivat ives was also m a r k e d l y r e d u c e d ( > 20-fold) when they ac ted in the absence of U V A (resul ts not shown).
The resul ts of the mic ronuc leus tests achieved u n d e r no rma l condi t ions ( subdued na tu ra l day- l i gh t / da rknes s a l t e rna t ion ) and those ca r r i ed out in da rkness a re l is ted in Tab les 2 and 3 respec- tively.
B A sl ightly inc reased the level of mic ronuc le - a t ed RBCs at concen t r a t ions ranging from 187.5 to 750 ppb (exp. 1, Tab le 2). Since B A at 93.75 ppb (exp. 2, Tab le 2) or 100 ppb (exp. 3, Table 3) y i e lded negat ive results , the lowest eff icient con- cen t r a t ion (LEC) in bo th these expe r imen ta l con- d i t ions (i.e., in the absence of U V A ) was rela- tively high, and in any case far above (60 t imes) the L E C in U V A condi t ions .
Wi th r e f e rence to D M B A , here again a d o s e - r e sponse re la t ionsh ip was es tabl i shed . The re to , the c las togenic i ty of D M B A (12.5 ppb) in the da rk was s ignif icant ly h igher than tha t r e c o r d e d for the same concen t r a t i on in U V A condi t ions ( c o m p a r e exp. 2 in Tab le 1 and exp. 2 in Tab le 3: these expe r imen t s were accompl i shed s imul tane- ously f rom a single spawn).
TABLE 4
INFLUENCE OF PAH PREEXPOSED TO UVA (22 J/cm 2) ON LEVEL OF MICRONUCLEI ('IR-UV/DARK' CONDI- TIONS)
Compound Exp. Concentration Number Micronucleus frequency (%0)
In darkness conditions, BAQ at 50 ppb gave a positive response (Table 3) while at half this dose (25 ppb), BAQ led to a questionable result. In fact, a similar conclusion could be drawn from the McGill and the Wilcoxon tests, opposite to the Student test ones, from which the means of the control and the treated groups were found to be significantly different.
In another set of experiments ( IR-UV/dark conditions), PAH solutions were preexposed to UVA for 24 h (22 J/cm2). When solutions con- taining 50 or 100 ppb of BA were UVA-irradiat- ed (experiments 2 and 3, Table 4), such media proved to be genotoxic towards larvae reared in the dark. As a dose-dependent elevation in mi- cronucleated RBCs was recorded, this situation was at variance with previous observations (Ta- bles 2 and 3). On the other hand, the clastogenic- ity of DMBA was abolished at the concentrations tested (12.5, 25 and 50 ppb): once more, the results were found to contrast with those previ- ously obtained in normal or in darkness condi- tions. Finally, only the larvae exposed to BAQ in the actual conditions reacted approximately like those treated in the dark: the noxious effects were obvious at 50 ppb and their magnitude was little affected by prior irradiation of BAQ.
Therefore, the assessment drawn from experi- ments related to BA and its two derivatives may be summarized as follows: in UVA conditions, the genotoxicity of chemicals was found to be BA = BAQ > DMBA. In the absence of UVA (i.e., in normal or in darkness conditions), this order was reversed: DMBA > BAQ > BA. Lastly, in IR -UV/da r k conditions, the clastogenicity of BA was enhanced, that of BAQ was nearly unaf- fected and that of DMBA was abolished at the concentrations tested.
Discussion
Whatever the lighting conditions may be, the effects we observed result from a complex set of processes taking place inside the organism along with interactions between larvae and the sur- rounding environment.
In spite of their low aqueous solubility, ranging from 10 -4 to 10 -9 M (Sigman et al., 1991), and their hydrophobic character, the PAH present in
the rearing water (partially dissolved in trtie solu- tion, solubilized as micelles and adsorbed in or- ganic suspended particles) are markedly concen- trated (by both direct and indirect routes) in the tissues of the larvae within a few hours (Grinfeld et al., 1986). So, active biological concentrations of the pollutant(s) can be reached in our experi- mental conditions.
Investigations related to the in vivo and in vitro metabolism of PAH have been carried out in the newt with the well-known promutagen and carcinogen BaP as a reference chemical (Marty et al., 1989; Marty et al., in preparation): identified BaP derivatives have been shown to be similar to those found in rodents. Analogically, it is conceiv- able that in the newt larvae a n d / o r in the rearing medium, some derivatives of the PAH studied here may be identical to those generated in ro- dents by metabolic processes a n d / o r those aris- ing from their chemical oxidation (Boyland and Sims, 1964, 1965; Boyland et al., 1964; Sims, 1964).
In this work we showed that in the presence of UVA, the toxicity of BA, BAQ and DMBA was higher than that of the corresponding tricyclic compounds (BA > Ac; BAQ >> AQ; DMBA > DMA). Furthermore, the addition of a benzene ring at the site of the C1-C 2 double bond of Ac, AQ or DMA gave to the tetracyclic hydrocarbons (BA; BAQ) a genotoxic activity lacking in their parent compounds (Ac; AQ), or enhanced it (at equimolar concentrations, DMBA was much more clastogenic than DMA). On the other hand, the pentacyclic hydrocarbon (DBA) was much less toxic than BA; moreover, it did not induce mi- cronuclei in the experimental conditions under which it was tested.
One may assume as Greenstock and Wiebe (1978) did, that in the rearing water, the hydro- carbon can give rise to singlet oxygen (10 2) by type II mechanisms (energy transfer from a pho- toactivated molecule of PAH to a molecule of oxygen). This latter could react rapidly with sensi- tive regions of the PAH (such as the mesocarbons of Ac, BA or DMBA); oxidized PAH might then undergo further transformations. However, type I mechanisms (involving O 2- a n d / o r PAH "+ formation) cannot be ruled out (Smith, 1989; Sigman et al., 1991).
38
So, under UVA conditions, the biological ef- fects observed in the newt might be due to vari- ous reactive species. It is known, for example, that radical species a n d / o r the reactive forms of oxygen play an important role in cytotoxicity and mutagenicity processes (Chesis et al., 1984; Wei et al., 1989) as well as in PAH-DNA adduct formation (Bryla and Weyand, 1991). The mecha- nisms by which some chemicals and radiations cause carcinogenesis are also believed to be me- diated by free radicals. Moreover, experimental data so far available provide substantial argu- ments in favor of an involvement of radical species in both the initiation and promotion stages of carcinogenesis (see the review of Sun, 1990).
At any rate, the results obtained in the newt micronucleus test are in general accordance with those of carcinogenicity tests in newts (Arffmann, 1964) or even in rodents (Soderman, 1982; Kier et al., 1986; Tierney, 1988). Nonetheless, it can be pointed out that no firm conclusion has been drawn with respect to anthracene in rodent tests, while this chemical has been shown to be non- carcinogenic in the newt. On the other hand, even though DBA is known to initiate tumors in mice (DiGiovanni et al., 1983) and in newts (Arff- mann, 1964), this PAH has not been clastogenic on larvae, as mentioned above. A better under- standing of this unexpected result may be pro- vided by our continuing investigations.
In normal lighting conditions, we showed that BA had a weak genotoxic action with no clear-cut dose-response, while DMBA was strongly clasto- genic. These results are in general accordance with those obtained in vivo in the mouse, after analysis of metaphase plates: BA (as well as the 3,4-diol of BA, precursor of the diol epoxide of the 'bay region') was not found to induce signifi- cant chromosome aberrations in bone marrow cells, whereas DMBA (or the 3,4-dioi of DMBA) induced numerous abnormalities (Ito et al., 1988). However, these authors showed that BA was much more mutagenic than DMBA in S. ty- phirnuriurn (TA 100) in the presence of PCB-S9. It is conceivable that in vivo, as reported by Tierney (1988), certain BA metabolites inhibit the cytochrome P-450 dependent monooxygenase sys- tem, or that certain enzymes such as cytosolic NADP-dihydrodiol dehydrogenase reduce the
mutagenicity of the diol epoxide(s). Furthermore, even though BA appears not to have been tested in the rodent bone-marrow micronucleus assay, DMBA yielded positive responses (Mavournin et al., 1990) in this in vivo test and a dose-effect relationship was demonstrated by Tinwell et al. (1990). A similar correlation was also found in the newt micronucleus test.
In darkness, we found DMBA and BAO to be more clastogenic than BA itself, indicating that substitution at the 7 and 12 positions of BA of two hydrogen atoms by methyl groups or oxygen atoms leads to a marked enhancement in geno- toxicity compared to that of the parent com- pound.
However, as it was also proven in this work, in I R - U V / d a r k conditions the deleterious effects of BA or DMBA are modified when they have been previously irradiated. It is known that photooxi- dation of BA and DMBA generates 7,12-epidi- oxy-BA and 7,12-epidioxy-DMBA respectively (Cook and Martin, 1940; Wood et al., 1979; New- man and Veeraraghavan, 1988). In aqueous solu- tion, photolysis of these endoperoxides gives rise to a variety of compounds including 7,12-BAQ (Wood et al., 1979). The ascorbic acid-Fe 2÷- oxygen system also leads to the formation of this quinone (Boyland and Sims, 1964; Boyland et al., 1964). It should be pointed out that benzo[a]- pyrene, photooxidation of which does not give rise to an endoperoxide (but to the 6-oxo-BaP radical; Osborne and Crosby, 1987), also leads to the formation of BaP quinones in aqueous medium (Katz et al., 1979). In this respect, as demonstrated by Zahn et al. (1982) in the sponge Tethya lyncurium, light-activated BaP binds to macromolecules (proteins, RNA and DNA) in the dark. The BaP binding ratio to DNA proves to be strongly correlated to the concentration of this PAH. On the other hand, with non-il- luminated BaP, only a very low association (if any) is observed in darkness.
In the newt, the clastogenic effects observed with BAQ at 50 ppb (irradiated or not) in the dark support the idea that if any photoconversion of BAQ takes place, it occurs at a very slow rate, or that the photoproducts are themselves clasto- genic. Moreover, it may be assumed that some of the genotoxicity of irradiated BA could be due to
39
BAQ. O u r resul ts also suggest tha t the B A derivat ives arising f rom metabo l i c processes and those ob t a ined by pho to t r ans fo rma t ion are quan- t i tat ively a n d / o r qual i ta t ively different : in fact, be low 187.5 ppb, non - i r r ad i a t ed B A was devoid of c las togenic effects in darkness , whereas i r radi- a ted B A gave a m a r k e d posi t ive response at lower
doses. Wi th respec t to D M B A , our resul ts indica te
that , for the most par t , the P A H has unde rgone p h o t o t r a n s f o r m a t i o n in the rea r ing m e d i u m fol- lowing the 24-h pe r iod of i r radia t ion . A m o n g the pho top roduc t s , 7 ,12-ep id ioxy-DMBA, ident i f ied in aqueous solut ions ( W o o d et al., 1979), has been pos tu l a t ed to be devoid of mutagenic i ty on Salmonella typhimurium (McCoy et al., 1979). On the o the r hand, it i nduced morpho log ica l and biological a l t e ra t ions in chick cu l tu red cells (Warshawsky et al., 1977). However , inject ion of the e n d o p e r o x i d e into mice was not found to induce tumors (Cook and Mar t in , 1940). It is thus l ikely that f rom init ial concen t ra t ions of D M B A _< 50 ppb, po ten t ia l ly c las togenic pho top roduc t s ( B A Q a n d / o r o t h e r der ivat ives) (Wood et al., 1979) a re not p r o d u c e d in sufficient amounts to give a posi t ive response in the newt micronucleus test.
Expe r imen t s with h igher concen t ra t ions of D M B A , U V - i r r a d i a t e d and then tes ted in the dark, a long with o the r exper imen t s in which scav- engers of active oxygen species are a d d e d to the rea r ing med ium, ~re in progress . In any case, qual i ta t ive and quant i ta t ive analysis of photo- p roduc t s would have to be p e r f o r m e d in o r d e r to verify our hypotheses .
W h a t e v e r the processes involved in toxicity a n d / o r genotoxic i ty may be, the resul ts ob ta ined to da te on the biological activit ies of P A H in Pleurodeles waltl prove that some hydrocarbons p re sen t in na tu ra l waters can become highly nox- ious (at concen t ra t ions as low as a few ppb) especia l ly when they act in the p resence of UVA. In addi t ion , var ious P A H in the p resence of light (UVA, sunl ight or visible ar t i f icial l ight) are ter- a togenic a n d / o r kill the newt embryos (Fe rnan - dez and L ' H a r i d o n , in preparat ion ' ) . So, the pho- todynamic convers ion of such pol lu tan ts deserves cons ide ra t ion and eva lua t ion as, for example , this may dis turb aquat ic ecosystems.
Acknowledgement
W e thank Dr. J. Van Poucke (Labora to i r e de ModUles et Logiciels en Ana lyse des DonnEes, Univers i t6 Paul Sabat ie r , Toulouse , F rance ) for his va luable advice on stat is t ical analysis.
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