Mutation Research, 232 (1990) 77-88

77

Elsevier MUT 02113

Fate of D N A lesions that elicit sister-chromatid exchanges P. M o r a l e s - R a m i r e z , R. R o d d g u e z - R e y e s a n d T. V a l l a r i n o - K e l l y Departamento de Radiobiologia, Instituto Nacional de lnvestigaciones Nucleares, Mexico D.F. (Mexico)

(Accepted25 February1990)

Keywords: Sister-chrornatidexchangeformation;Model; DNA lesions

Summary Using 3-way differential staining (TWD) of sister chromatids, the fate of DNA lesions involved in sister-chromatid exchange (SCE) formation was determined in murine bone marrow cells in vivo, after treatment with either mitomycin C (MMC) or cyclophosphamide (CP). Both MMC (2.6 mg/kg b.w.) and CP (7 mg/kg b.w.) induced an SCE frequency near the expected in the 2 subsequent cell divisions, but the frequency of SCE occurring at the same locus in successive cell divisions was substantially lower than expected. The results are compared with previous data obtained after exposure to v-rays. A model of SCE induction is proposed.

Although the biological significance and the mechanism(s) of formation are unclear, sisterchromatid exchange (SCE) occurs during DNA synthesis (Wolff et al., 1974; Kato, 1980a) and is produced either by DNA damage (MacRae et al., 1979; Schvartzman and Gutierrez, 1980; Nagasawa et al., 1982) or by inhibition of DNA synthesis (Ishii and Bender, 1980; Nishi et al., 1982). The precise nature of the DNA lesions involved in SCE induction is not known (Carrano et al., 1979; Cassel and Latt, 1980; Ishii, 1981; Kano and Fujiwara, 1982), but SCEs are produced by different kinds of lesions (Sahar et al., 1981). Moreover, not every DNA lesion is involved in or capable of inducing SCE (Reynolds et al., 1979; Sahar et al., 1981; Heflich et al., 1986), but there is evidence that SCE-inducing lesions persist for more than 1

cell division (reviewed in Tice and Schvartzman, 1982). Notwithstanding that 3,-rays are not considered good inducers of SCE, they are capable of causing an increase in the SCE frequency that persists several cell divisions after exposure (MoralesRamirez et al., 1984a). Furthermore, evidence was obtained that radio-induced DNA lesions result in SCE formation at the same locus in 2 successive post-exposure cell divisions in murine bone marrow cells in vivo (Morales-Ramirez et al., 1988). The aim of this work is to determine the fate of mitomycin C (MMC)- and cyclophosphamide (CP)-induced DNA lesions capable of eliciting SCE in the same in vivo system.

Materials and methods Animals

Correspondence: Dr. Pedro Morales-Ramirez, Departamento de Radiobiologla, Instituto National de Investigaciones Nucleares, Sierra Mojada 447, 2° piso, Colonia Lomas Barrilaco, CP 11010, MexicoD.F. (Mexico).

BALB/c male mice 2-3 months old produced in our colony and weighing 30 g were used in these experiments. Animals were housed in plastic cages with sawdust bedding, with controlled tern-

0027-5107/90/$03.50 © 1990 ElsevierSciencePublishersB.V.(BiomedicalDivision)

78

perature (20°C), and were fed with Laboratory Purina Chow for Small Rodents and water ad libitum.

was injected intraperitoneally twice; the first dose was 250 m g / k g b.w. at the beginning of the experiment and the second dose, 7 h later, was 2.0 g / k g b.w. The BrdU had previously been adsorbed to activated charcoal according to a described method (Morales-Ramirez, 1980; MoralesRamirez et al., 1984b). The mutagens in aqueous solution were injected intraperitoneally 30 min before the second BrdU dose. The MMC (Sigma) dose was 2.6 m g / k g b.w. and that of CP (Cyclofostan, Pharma Werk) was 7 m g / k g b.w. Bone marrow cells were arrested in metaphase by injecting intraperitoneally 15 m g / k g b.w. colchicine; mice were killed by cervical dislocation 2 h later.

Protocol The experiments were conducted according to an in vivo protocol to establish the fate of D N A lesions involved in SCE formation during 2 successive cell divisions (Morales-Ramirez et al., 1988). This protocol is based on the 3-way differential staining (TWD) of sister chromatids (Schvartzman and Goyanes, 1980). To obtain the 2 levels of bromodeoxyuridine (BrdU) incorporation into D N A necessary for TWD, BrdU (Sigma)

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Fig. 1. Rationale for the analysis of SCE induction during the first, second or both cell divisions after mutagen exposure, see text.

79

Cell collection and processing After the animals were killed, the femurs were dissected, sectioned on both ends, and the bone marrow cells were obtained by injecting a phosphate-buffered saline solution (PBS solution A) into one end with a 26-gauge needle. The cells were centrifuged at 250 x g for 5 min, suspended in 0.075 M KC1, incubated at 3 7 ° C for 15 min, and fixed in 2 changes of ethanol-acetic acid (3 : 1) for 15 min each. The cells were then centrifuged and resuspended in 0.5 fixative. Finally the cell suspension was dropped onto clean chilled slides.

Differential staining of sister chromatids Slides containing the metaphase figures were dried for at least 24 h before staining with a slightly modified fluorescence plus Giemsa method (Perry and Wolff, 1974). Slides were mounted in 10 /~M Hoechst 33258 in a phosphate-citrate buffer (Goto et al., 1975) and placed beneath a 25-W black light lamp for 60-90 min. Then the slides were incubated in 2 x SSC at 60 ° C for 20 min, washed with distilled water and stained with 10% Giemsa for 5 min.

sions that did not induce SCEs in the first division did so in the second division after exposure to the mutagen (SCE-2). Finally, an increase in SCEs with the appearance of those occurring in the first division after the beginning of the experiment, that is, in the cell division previous to the mutagen treatment, is the consequence of lesions that elicit SCEs at the same locus in 2 successive cell divisions after the mutagen exposure (SCE-1,2). Results

Murine bone marrow metaphases from untreated (A) and mutagen-treated (B) animals exhibiting T W D are shown in Fig. 2. The cell from the mutagen-exposed animal displays more SCEs than the control cell.

A

Analysis and statistical methods The SCEs occurring in the first, second or both cell divisions after mutagen treatment were scored in 30 cells per animal, in at least 4 animals, following the protocol described in the Rationale (Fig. 1). The significance between data was determined by Student's t test or paired t test, using a Hewlett Packard 25 Microcomputer.

B

Rationale Considering that animals were exposed to the mutagen after the first cell division and before the second BrdU dose, the increase in SCE frequency at each of the 3 successive divisions, as analyzed in third-division cells, was interpreted as follows (Fig. 1). An increase in SCEs in the second division after the beginning of the experiment is a consequence of lesions that only induce SCEs during the first division after mutagen treatment (SCE-1). An increase in SCEs in the third division after the beginning of the experiment implies that le-

Ib

Fig. 2. Bone marrow metaphases from an untreated control (A) and a cyclophosphamide-treated(B) mouse, displaying 3-way differential staining and several SCEs.

80 TABLE 1 SCE FREQUENCIES O C C U R R I N G IN THE FIRST, SECOND OR BOTH DIVISIONS A F T E R THE T R E A T M E N T WITH EITHER M I T O M Y C I N C (MMC) OR C Y C L O P H O S P H A M I D E (CP) SCE/cell Division post-treatment 1

Control MMC CP

2

1,2

x + SD

DIF

x + SD

DIF

x ± SD

DIF

2.0±0.38 7.2±0.92" 10.9±2.6"

5.2 8.9

3.8±0.7 5.7±0.8* 8.3±1.0"

1.9 4.5

0.38±0.07 1.30±0.36" 0.84±0.24*

0.9 0.5

* p < 0.01, Student's t test. DIF, difference from control. 30 cells were analyzed per mouse, in at least 4 mice. Analysis according Fig. 1.

The frequencies of SCEs occurring in the first (SCE-1), second (SCE-2) or both divisions (SCE1,2) after the treatment with either MMC or CP

are shown in Table 1. These data indicate that the frequencies of all types of SCEs show a significant increase compared to basal values. The largest

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Fig. 3. Probability of SCE occurring in the first, second or both cell divisions after mutagen treatment, assuming that each lesion has a 0.5 probability of inducing an SCE, see text.

81

increase was observed in SCE-1 after treatment with MMC or CP. The frequency decreased to one third or one half in SCE-2 and SCE-1,2 was 2 and 10 times lower for MMC and CP, respectively, than SCE-2. Fig. 3 shows different chromosomal appearances depending on the cell division in which the SCEs occur, as well as the expected probabilities of occurrence. The probabilities are estimated assuming that each lesion has a 50% probability of manifesting itself as an SCE, and that there is neither repair nor transformation of the lesions. The relative frequencies of SCEs in the different post-treatment cell divisions are estimated from Fig. 3 as follows. Probability of first division after treatment SCEs = p F + p G = 0.375. Probability of second division after treatment S C E s = p C = 0.125. Probability of first-second division after treatment SCEs = p H = 0.125. This means that the expected relative frequencies are 3 : 1 : 1 for SCEs occurring in the first, second or both divisions after mutagen treatment. It is pertinent to comment that, due to the characteristics of the protocol, each SCE occurring in the 3 cell divisions of the experiment remains registered in the third-division cells, although the lesions that induce the SCE had been diluted by cell division. The relative SCE frequencies obtained for each cell division after treatment with either MMC or CP, and the same parameters estimated for 7-ray exposure from previously reported data (MoralesRamirez et al., 1988), are compared with the expected relative frequencies in Table 2. These values were estimated with respect to the frequency of SCE-1, from the average of relative frequencies of each animal. The proportion between the frequencies of SCE-1 and SCE-2 after treatment with MMC is very close to the expected value (3:1), and with CP it is slightly different (3:1.6). This difference may be caused by some CP metabolites which remain in the blood for long periods producing SCE in the second division post treatment (Domeyer and Sladek, 1978). These data suggest that the probability of MMC- and CP-induced lesions to be expressed as SCEs is approximately 0.5. The response to "t-rays was completely different: the SCE-2 frequency after irradiation did not increase compared to the non-irradiated control,

TABLE 2 R E L A T I V E SCE F R E Q U E N C I E S O C C U R R I N G IN T H E FIRST, S E C O N D O R BOTH DIVISIONS A F T E R T H E T R E A T M E N T W I T H E I T H E R M I T O M Y C I N C (MMC) O R C Y C L O P H O S P H A M I D E (CP)

Expected a MMC CP "t-Rays b

1.7 Gy 2.9 Gy

SCE-1

SCE-2

SCE-1,2

3 3 3 1 1

1 1.1 1.6 0 0

1 0.53 0.15 1.10 1.05

a According to Fig. 2. b From previously published data (Morales-Ramirez et al., 1988).

and, as shown in Table 2, there was a directly proportional increase in SCE-1 compared to SCE1,2 after mutagen treatment (Morales-Ramlrez et al., 1988). This suggests that -/-ray-induced lesions that elicit SCE are quite persistent and very tenacious (i.e., lesions always are involved in SCE formation) in inducing SCE. The relative frequencies of SCE-1 and SCE-1,2 are hypothetically 3:1. However, the obtained values were 3 : 0.53 and 3 : 0.16 for treatment with MMC and CP respectively. A possible explanation is that some SCE-inducing lesions could have been repaired during the interphase previous to the second post-treatment division. But the lesions that induce SCE-2 would have had the same opportunity of being repaired, consequently, the proportion of SCE-2 and SCE-1,2 should be near 1 : 1. However, results were 1.1 : 0.53 and 1.6 : 0.16 for MMC and CP exposure respectively. Considering that the fate of the lesions that induce SCE-2 differs from that of lesions which induce SCE-1,2, in that the latter were involved in an event of SCE before the second division, it is likely that lesions involved in SCE are prone to be repaired either during or as a consequence of the SCE. The propositions previously mentioned, and implicit in Fig. 3, are valid although differences in sensitivity to SCE induction by mutagens may exist between the BrdU-substituted and the unsubstituted D N A strands. The present protocol permits an evaluation of the relevance of BrdU incorporation to SCE induction by mutagens, by

82 TABLE 3 SCE F R E Q U E N C I E S IN G R A Y - W H I T E (GW) A N D B L A C K - W H I T E (BW) C H R O M O S O M E S IN T H E S E C O N D DIVISION POST TREATMENT WITH EITHER M I T O M Y C I N C (MMC) O R C Y C L O P H O S P H A M I D E (CP) SCE/cell (x ± S)

Control MMC CP

GW

BW

1.7±0.45 1.4±0.65 a 2.3±0.89 a

2.2±0.39 0.5±0.17 a 2.1±0.57 a

Average of individual SCE frequencies less basal SCE frequency. MMC: p < 0.02; CP: NS, Student's paired t test. a

comparing the frequencies of SCEs occurring in the second division post treatment in the chromosomes descending from the unsubstituted strand (black-white; A in Fig. 3) and from the BrdU-substituted strand (gray-white; B in Fig. 3). As shown in Table 3 the SCE frequencies of these types of chromosomes differ significantly in the cells treated with MMC, indicating that the incorporation of BrdU into DNA predisposes to the induction of SCE by MMC. The cells treated with CP did not show a difference in sensitivity. Discussion

Although it is well established that the DNA lesions involved in SCE production can persist from one generation to the next, no clear knowledge about the ultimate fate of these lesions or the biological consequences of lesion persistence is available. However, lesion persistence has been related to neoplastic transformation (Marginson and Kleihues, 1975). Because SCE occurs during or immediately after DNA synthesis (Wolff et al., 1974; Kato, 1980a), the study of persistence a n d / o r repairability of lesions involved in SCE induction has been focused on the capacity of lesions to persist during either interphase or DNA synthesis and cell division. There is evidence that UV-induced lesions eliciting SCE can be repaired during the G a phase of the cell cycle (Nagasawa et al., 1982; MacRae et al., 1979). Furthermore, the total or partial persistence through G 1 of lesions induced by different agents has been demonstrated in human

lymphocytes (Lambert et al., 1983, 1984; Hedner et al., 1984; He and Lambert, 1985). In murine salivary gland cells in vivo, evidence has recently been obtained that y-ray-induced SCE in early G 1 is half of that induced in late G~ (Morales-Ramirez et al., submitted for publication). Using different protocols, an increased frequency of SCE occurring long after in vivo treatment with mutagens has been reported, in both non-proliferative (Stetka et al., 1978; Raposa, 1978; Huff et al., 1982) and proliferative cells (Latt and Loveday, 1978; Morales-Rarnirez et al., 1984a; He and Lambert, 1985). Particularly, CP and MMC are capable of inducing SCE in human lymphocytes (Raposa, 1978; Littlefield et al., 1981) as well as in rabbit lymphocytes (Stetka et al., 1978; Huff et al., 1982) long after the in vivo treatment. In Syrian hamster fetal cells in vitro, there is evidence that the increase in SCE induced by MMC and other chemicals persists after several divisions or is partially reduced. In this system the UV-induced SCEs were completely reduced after a few cell divisions (Popescu et al., 1985), suggesting that beside the reduction caused by successive cell divisions the lesions are actually being repaired. Additional evidence of persistence was obtained by combining different protocols of in vivo treatment with mutagens either before or after BrdU incorporation (Conner and Cheng, 1983; Conner et al., 1983; Takeshita and Conner, 1985). Based on this system, a model of SCE quantification was proposed (Conner et al., 1984). However, this method requires the determination of SCE frequency after and before BrdU incorporation which in turn could affect the response to SCE induction by mutagens (Allen et al., 1978; Popescu et al., 1980; Ockey, 1981; Natarajan et al., 1983; Morales-Ramirez et al., 1984a; Morgan and Wolff, 1984). More convenient methods of persistence determination are those in which analysis of SCE occurring during successive cell divisions is accomplished in the same cell, i.e., frequencies of single and twin SCEs in tetraploid cells or symmetric and asymmetric SCEs in third-division cells. However, those methods have some disadvantages already discussed (reviewed by Tice and Schvartz-

83 man, 1982). Besides, by scoring symmetric and asymmetric SCEs, the persistence of lesions induced by MMC (Ishii and Bender, 1978) and methoxypsoralen plus UVA (Latt and Loveday, 1978) has been reported, but the opposite was found by scoring singles and twins (Linnainma and Wolff, 1982). All the previous protocols are based on the analysis of SCEs by means of 2-tone differential staining. However, SCE could be underestimated if it happened in the same locus, as has been proposed (Stetka, 1979). The protocol used in the present study permits an evaluation of the persistence of lesions eliciting SCE, and of how often SCE occurs in the same locus. Also, with this protocol, each cell records the SCE events occurring in 3 call divisions without the dilution of events caused by cell division. Another advantage is that it allows the determination of SCE induction in vivo from very low basal values (Morales-Ramirez et al., 1987) and without the uncertainty of the effect that culture conditions and substances could have on SCE induction (Kato and Sandberg, 1977; Morgan and Crossen, 1981). The most important disadvantage of the present protocol is that the mutagen must be administered after the incorporation of BrdU for 1 cell division, and although the amount of BrdU incorporated during that cell division is lower than that required in the common protocol, the presence of BrdU could affect the results. However, the interaction between incorporated BrdU and the induction of SCE by mutagens can be evaluated by comparing SCE frequencies in the chromosomes of third-division cells descending from the BrdU-substituted (gray-white) and unsubstituted (black-white) DNA strands present at the moment of mutagen exposure. The present results indicate a sensitization of the BrdU-substituted strand to SCE induction by MMC. No effect was observed after CP exposure (Table 3). As shown in Table 2, the induction of SCE by -/-rays, although low, is provoked by a very persistent lesion which is very tenacious (i.e., it always is involved in SCE induction) in inducing SCE in successive divisions (Morales-Ramirez et al., 1988). Schvartzman et al. (1985), using a similar experimental design in human lymphocytes in vitro, did not find any response after y-radiation

exposure; they observed, however, that both UV and MMC cause lesions which are persistent but not tenacious in inducing SCE. They did not observe lesions capable of inducing SCE at the same locus in successive divisions after mutagen exposure. Although their data differ from those in the present study, they coincide in that the occurrence of SCE at the same locus is much lower than the expected value (Table 2). This fact, plus the observation in the present experiment that SCE-2 frequency is similar to the expected, suggests that the lack of tenacity of lesions in inducing SCE in compatible with the possibility of their repair, either during or as a consequence of the SCE event, because the original lesions might be modified during SCE formation and then repaired. An interesting point inferred from the extreme persistence and tenacity of ,/-ray-induced lesions and supported by the present study is that SCE occurs at or near the site of the lesion. This contradicts the models which proposed SCE as the result of multiple lesions. Multiple lesions only open the possibility but do not determine the actual occurrence of SCE so that the tenacity (capacity of a lesion to induce SCE always) mentioned above does not fit into this alternative. Besides, a multiple lesion alternative implies that the number of SCE depends on the number of lesions necessary to cause enough delay (Painter, 1980) or conformational stress (DuFrain, 1981) during DNA synthesis, but the dilution of damage caused by cell division would not permit a complete tenacity even if all lesions persisted. Estimations of SCE per lesion are from 1 : 200 (Sahar et al., 1981) to 1:20000 (Reynolds et al., 1979; Heflich et al., 1986). However, it is important to consider that the cell sample in which SCEs were estimated may not be equivalent to that in which the lesions were estimated. The cells in which the SCEs were scored could be those receiving less damage, those which repaired damage efficiently or both, and were therefore able to survive and divide. Other possible explanations for the difference in the proportion of SCEs to lesions are that SCEs are induced at specific sites of the genome (Latt, 1974; Carrano and Wolff, 1975; Bostock and Christie, 1976; Hsu and Pathak, 1976; Carrano and Johnston, 1977; Crossen, 1983), or that they

84 are produced by a rare type of lesion. Division delay has been proposed to be responsible for SCE induction (Shiraishi and Sandberg, 1980) and has been compared with the situation in Bloom's syndrome, in which a very high frequency of SCE (Chaganti et al., 1974) is associated with a longer duration of cell division and particularly with a delay in D N A duplication (Hand and German, 1975). In addition, the induction of SCE by suboptimal growth temperature was related to division delay (Kato, 1980b). These observations support the multiple-lesion model proposed by Painter (1980). However, these data do not clarify whether division delay induces SCE or SCE production causes delay, or whether their relationship is only circumstantial. The fact that in the present study, the duration of cell division in both the mutagen-treated and the untreated cells was the same but the SCE frequency was not, suggests that the duration of cell division is not related to SCE induction. Considering our results, that each lesion seems to have a 50% chance of inducing or not inducing an SCE, it is possible that SCEs resulted from a process similar to that proposed by Holliday to explain D N A recombination (Dressier and Potter, 1982). In this model, 2 alternatives are possible, one of which is a double-strand exchange. Although this model was focused on D N A recombination, it has been proposed that a similar process could permit the synthesis of damaged D N A (Lavin, 1978). The evidence obtained in the present study and in a previous report (Morales-Ramirez et al., 1988) suggests that (a) MMC- and CP-induced lesions involved in SCE have at least a 50% probability of inducing SCE; (b) SCE is related to a process which permits the cell to synthesize D N A in the presence of lesions; (c) lesions involved in SCE are prone to be removed either during or as a consequence of SCE; (d) SCEs occur at or near the site of the lesion. These observations allow the proposal of a model for SCE induction and for its biological significance; this model is based on Holliday's recombination model (Dressier and Potter, 1982) and on the postreplication repair model (Lavin, 1978) and has some points in common with propositions previously reported (Comings, 1975; Kato, 1977; Shafer, 1977; Ishii and

Bender, 1980) and criticized (Stetka, 1979; DuFrain, 1981). The process described in Fig. 4 begins immediately after D N A duplication (Kato, 1980a) as a consequence of a lesion which does not permit the synthesis of the daughter strand (B), but induces the recombination of the strand opposite to the damage with the strand of the same polarity (C, D). The single-strand exchange crosses the zone where the lesion is located (E), and opens the possibility that the gap in front of the lesion could be filled based on the complementary strand recently synthesized (F). This complex would be solved according to Holliday's recombination model (G) to produce the intermediate (H), which has been made evident by electron microscopy (Potter and Dressier, 1976; Thompson et al., 1976). Finally, depending on the axis of cleavage of the intermediate, it could give rise to a single-strand exchange (I) or a double-strand exchange (J). Evidence of the exchange was obtained by sedimentation of unifilarly substituted D N A (Rommelaere and Miller-Faures, 1975; Moore and Holliday, 1976; Resnick and Moore, 1979). The cleavage of the Holliday intermediate could affect a segment of the strands involved, and in the case of the damage strand, the lesion could be removed. This event is associated with a double-strand exchange (SCE) and could explain how the lesion could be removed when an SCE occurs (J). Although the model is described for damage involving 1 D N A strand, it is applicable to interstrand crosslinkings, if the lesion is previously cleaved during D N A synthesis (Shafer, 1977). The behavior of y-ray-induced lesions which are involved in SCE production does not fit completely with this model because these lesions are apparently non-repairable and very tenacious in inducing SCE. This implies that 7-ray-induced lesions are very homogeneous but quite different from those induced by the chemical agents. Maybe the difference is related to the size of the lesion, i.e., a crosslink DNA-protein, as was previously proposed (Ishii, 1981), because a bulky adduct could always be involved in SCE formation but not easily removed. It is possible that the chemical agents induce different types of lesions and some of them are more easily removed during or as the result of

85

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SCE formation. This could explain the higher efficiency of removal of the CP-induced lesions involved in SCE production compared to the MMC-induced lesions as is inferred from the difference in SCE frequency induced by these mutagens in both divisions after treatment (Table 2). From this point of view, the difference in persistence of the lesions can be explained on the

basis of their proneness to be removed during cleavage of the Holliday intermediate. The model proposed here could explain the indirect induction of SCE by the previously suggested process, that is, by affecting enzymes involved in DNA synthesis (Ishii and Bender, 1980), in DNA recombination (Deaven et al., 1978; Marshall et al., 1983) or in postreplication repair

86 (Nishi et al., 1982), b ec a u s e a c c o r d i n g to the model, the 3 p h e n o m e n a co u l d be i n v o l v e d in the S C E event. Also, the p r o p o s e d relationship of S C E with mutation, previously analyzed (Carrano and T h o m p s o n , 1982), could be e x p l a i n e d if the cleavage of the H o l l i d a y i n t e r m e d i a t e or m o r e specifically the r e m o v a l of the d a m a g e is error-prone. T h e study of the response p r o d u c e d by m u t a gens that i n d u c e different kinds of lesions in this e x p e r i m e n t a l system will p e r m i t v a l i d a t i o n of the pr e se nt co n cl u s i o n s and the model, c o n s i d e r e d as a w or kin g hypothesis.

Acknowledgements W e wish to t h an k J o r g e M e r c a d e r M a r t in e z , A n g e l Reyes Pozos, Perfecto A g u i l a r Vargas, F e l i p e Beltrhn Bibiana, a n d E n r i q u e F e r n h n d e z Villavicencio for their excellent technical assistance; Dr. D a v i d Alcfintara a n d Dr. M a t i l d e Bre~aa for their c o m m e n t s a b o u t the m o d e l ; Miss Isabel P r r e z M. for English c o r r e c t i o n a n d E f r a l n L a g u n a s for illustrations.

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