Effect of α-difluoromethylornithine on polyamine and DNA synthesis in regenerating rat liver

Biochimica et Biophysica Acta, 696 (1982) 179-186

179

Elsevier Biomedical Press BBA 91021

EFFECT OF a-DIFLUOROMETHYLORNITHINE ON POLYAMINE AND DNA SYNTHESIS IN REGENERATING RAT LIVER REVERSAL OF INHIBITION OF DNA SYNTHESIS BY PUTRESCINE HANNU POSE) and ANTHONY E. PEGG

Department of Physiology, The Milton S. Hershey Medical Center, The Pennsylvania State Universi(v, Hershev, PA 17033 (U.S.A.) (Received April 13th, 1981)

Key words: Putrescine; Sperrnidine; Ornithine decarboxylase; DNA synthesis, Regeneration; (Rat liver)

The possibility that a-difluoromethylornithine, a specific, irreversible inhibitor of ornithine decarboxylase could be used to prevent the rise in hepatic putrescine and spermidine content following partial hepatectomy was tested. Administration of a-difluoromethylornithine at a dose of 400 mg/kg every 4 h reduced hepatic putrescine to < 2 nmol/g, but had only a small effect on the rise in spermidine seen at 28 h after partial hepatectomy. Such treatment also reduced the rise in DNA synthesis produced by partial hepatectomy by up to 70%. The inhibitory effect towards DNA synthesis could be reversed by administration of putrescine which increased the hepatic putrescine content to about 30-40% of that in the regenerating control livers. These results suggest that accumulation of putrescine rather than spermidine is needed for DNA synthesis after partial hepatectomy. They also suggest that part, but not all of the rise in putrescine normally seen in the liver after partial hepatectomy is needed for the enhanced DNA synthesis associated with liver regeneration. Experiments with lower doses of a-difluoromethylornithine showed that a substantial part of the rise in hepatic ornithine decarboxylase activity could be abolished without affecting either the rise in spermidine content or the increase in DNA synthesis after partial hepatectomy. Introduction It is well known that there is an increased accumulation of putrescine and spermidine in rat liver regenerating after partial hepatectomy [1,2]. Some evidence that these increases may be essential for the normal proliferation after this stimulus has been obtained by the use of inhibitors of ornithine decarboxylase to prevent putrescine synthesis [3-7]. These experiments used either 1,3diaminopropane or 1,3-diaminopropan-2-ol given as multiple injections [3,4,7], via continuous oral administration [5], or as a single dose combined with 1,1 '- ((methylethanediylidenedinitrilo)-bis(3aminoguanidine)), a potent inhibitor of diamine oxidase and S-adenosylmethionine decarboxylase [6]. Treatment with these inhibitors not only pre0167-4781/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

vented the accumulation of spermidine and putrescine, but also greatly reduced the stimulation of DNA synthesis invoked by partial hepatectomy. This finding suggested that increased putrescine or spermidine (or both) was needed for DNA synthesis and cell replication in this system, but some doubts remain. The amounts of these diamines needed to suppress ornithine decarboxylase activity are substantial. Toxic effects [8-11] related to exposure to the diamines themselves or to their oxidation products are difficult to rule out, particularly since it was not possible to attempt to reverse the inhibitory effect on DNA synthesis by administration of putrescine or spermidine. Recently, specific and potent irreversible inhibitors of mammalian ornithine decarboxylase have become available [12,13]. Unlike 1,3-diarnino-

180

propane or 1,3-diaminopropan-2-ol, which act to reduce ornithine decarboxylase by an indirect mechanism involving either the induction of a protein inhibitor [ 14] or a repression of the synthesis of the enzyme [15], a-difluoromethylornithine is a direct, enzyme-activated, irreversible inhibitor [12]. In the present studies we have used this compound to interfere with putrescine production at early stages of liver regeneration and have examined D N A synthesis in rats treated with the inhibitor alone and the inhibitor plus putrescine. The results suggest that accumulation of putrescine rather than spermidine is needed for DNA synthesis at the early stages of liver regeneration, but that not all of the increase in putrescine content or ornithine decarboxylase activity is required. Materials and Methods Animals and treatments. Male Sprague-Dawley rats were maintained on Purina rat chow ad libiturn and kept on a 12 h light 12 h dark cycle. Rats weighing 170-250 g (see legends) were subjected to partial hepatectomy [16] under light ether anesthesia, a-Difluoromethylornithine was dissolved in 0.9% NaC1 and administered by intraperitoneal injection. Putrescine dihydrochloride was dissolved in 0.9% NaC1 and adjusted to pH 6.8 prior to injection. Animals not treated with a-difluoromethylornithine or putrescine received a similar volume of 0.9% NaC1 alone. In some experiments a solution of 2g a-difluoromethylornithine/100 ml water was substituted for drinking water. Chemicals. L-[ 1-14C]Ornithine (53 Ci/mol) was obtained from the Radiochemical Center, Amersham, Bucks, U.K. [6-3H]Thymidine (21.5 Ci/mol) and [methyl-aH]thymidine (43.6 Ci/mol) were purchased from New England Nuclear, Boston, MA, U.S.A. a-Difluoromethylornithine was a generous gift from Merrell Research Center, Cincinnati, OH, U.S.A. All other biochemical reagents were products of Sigma Chemical Company, St. Louis, MO, U.S.A. Analytical methods. Ornithine decarboxylase activity was determined by measuring the production of 14CO2 from L-[1-14C]ornithine as previously described [17]. Results were expressed as pmol

released/mg protein added in a 30 min incubation at 37°C. DNA synthesis was measured by the incorporation of labeled thymidine into DNA in a 30 min period [3]. Results were expressed as cpm incorporated/#g DNA. DNA was determined by reaction with diphenylamine [18]. Protein content was determined by the method of Bradford [19] with bovine serum albumin as standard. Polyamine content was determined by separation on an amino acid analyzer with fluorescence detection [20] and expressed as nmol (for putrescine) or /.tmol (for spermidine or spermine)/g wet wt. liver. The limit of sensitivity was about 2 nmol/g. Extracts for assay of ornithine decarboxylase, polyamine content and DNA synthesis were prepared as follows. Rats were killed by decapitation and the livers removed, weighed and homogenized in 2 vol. ice-cold 10 mM Tris-HC1, pH 7.5,/0.1 mM EDTA/2.5 mM dithiothreitol. A portion of the homogenate was removed, precipitated by addition of an equal volume of ice-cold 10% (w/v) trichloroacetic acid and used for determination of DNA synthesis or for polyamine content. The remainder was centrifuged at 4°C for 30 min at 105 000 × g and the supernatant used for determination of ornithine decarboxylase activity. Results

As shown in Fig. 1 (panel B), treatment with 200 m g / k g doses of a-difluoromethylornithine every 6 h was sufficient to substantially suppress the large rise in putrescine content in the liver after partial hepatectomy. However, such treatment did not prevent either the increase in DNA synthesis or spermidine content in the liver (panels A and D). Although these parameters were slightly lower in the drug-treated animals, only at 30 h was the difference marginally statistically significant. It is apparent from the marked rise in spermidine content occurring in the rats given a-difluoromethylornithine that significant ornithine decarboxylase activity must remain with this treatment schedule. Direct measurement of the enzyme activity confirmed this showing that 14-25% of the activity in control partially hepatectomized rats was present in extracts from those treated with a-difluoromethylornithine (data not shown). Therefore, higher doses of the drug were tested to

181 ¢

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Fig. 1. Effect of a-difluoromethylornithine on polyamine content and D N A synthesis in regenerating liver. Rats weighing 173-+15g were subjected to two-thirds partial hepatectomy and treated with a-difluoromethylornithine (11 II) by intraperitoneal injection of 200 m g / k g doses every 6 h commencing I h after operation. Control rats ([3 []) were partially hepatectomized and received intraperitoneal injections of saline. D N A synthesis was measured by the incorporation of radioactivity into D N A during a 30 min period after injection of 8~Ci [6)H]thymidine. Results are shown as mean -+S.D. for four to five animals for D N A synthesis (panel A), putrescine content (panel B), spermine content (panel C) and spermidine content (panel D). The differences between control drug treatment were significant (P<0.001) at all time points for putrescine content. The only other significant differences were at 30 h for DNA synthesis and spermidine content ( P < 0.05).

determine whether a more significant effect on DNA synthesis could be seen. It is known that a-difluoromethylornithine is cleared rapidly from the blood of the rat [21]. Therefore, the drug was injected every 4 h starting 1 h after partial hepatectomy and its effects on ornithine decarboxylase, polyamine content and DNA synthesis were determined (Table I). It can be seen that such doses of 200 m g / k g substantially depressed the increased ornithine decarboxylase activity normally associated with liver regeneration, but the activity was still above that seen in sham-operated controls. Increasing the dose to 400 m g / k g did reduce ornithine decarboxylase activity at 28 h after partial hepatectomy to that seen in sham-operated controls, but even this dose did not reduce this basal activity (Table I). The results of Fig. 1 and Table I agree with those of Danzin et al. [22] who found that a-difluoromethylornithine was relatively ineffective in reducing liver polyamine levels compared to other tissues. As shown in Table I, there was a 13-fold increase in putrescine and a 55% increase in spermidine in regenerating liver. Treatment with 200 m g / k g a-difluoromethylornithine every 4 h had a substantial effect on the putrescine level which was increased at most 2-fold, but did not

TABLE I COMPARISON OF D I F F E R E N T DOSES OF a - D I F L U O R O M E T H Y L O R N I T H I N E ON ORNITHINE DECARBOXYLASE ACTIVITY, POLYAMINE CONTENT AND DNA SYNTHESIS IN R E G E N E R A T I N G RAT LIVER a-Difluoromethylornithine was given as an intraperitoneal injection of 200 or 400 m g / k g as indicated every 4 h commencing 1 h after partial hepatectomy. D N A synthesis was measured by the incorporation of radioactivity into DNA during a 30 min period after injection of 35 # Ci [methyl -3 H]thymidine. Rats weighing 225 -+ 25 g were used and were killed 28 h after partial hepatectomy or sham operation. This corresponded to the peak of DNA synthesis in these animals. Results are shown as mean ± S.D. for six animals. Treatment

Putrescine (nmol/g)

Spermidine (/~mol/g)

Spermine (~mol/g)

42-+ 1 I

6-+ 2

1.26--+0.03

0.62-+0.05

4-+ 2

Partial hepatectomy

346 -+ 91

77-+21

1.95-+0.14

0.59-+0.04

89-+ 18

Partial hepatectomy + a-difluoromethylornithine (200 m g / k g per 4 h)

107-+37 a

10-+ 5 a

1.89-+0.08

0.58-+0.03

75-+22

Partial hepatectomy + a-difluoromethylornithine (400 m g / k g per 4 h)

35-+ 14 a

1.77-+0.07 b

0.58-+0.02

2 5 ± 10 a

Sham operated

Ornithine decarboxylase (pmol/mg per 30 min)

a Significantly less than partial hepatectomy alone (P<0.001). b Significantly less than partial hepatectomy alone (P<0.05).

<2 a

DNA synthesis (cpm/tlg)

182

prevent the rise in spermidine or the increase in DNA synthesis. However, 400 mg/kg doses of a-difluoromethylornithine reduced DNA synthesis by 70% despite having very little effect on the accumulation of spermidine. These doses reduced the hepatic putrescine content to below the limit of detection (< 2 nmol/g). These results could indicate that production of putrescine is needed for the maximal response of DNA synthesis, but another possibility would be that there was an effect of the drug on this process not related to its inhibition of ornithine decarboxylase. In order to answer this question, putrescine was administered to test whether it could reverse the effects of a-difluoromethylornithine (Table II). Treatment with 300 #mol putrescine/kg every 4 h completely restored the rate of DNA synthesis in the drug-treated with putrescine abolished the small decrease in spermidine content produced by a-difluoromethylornithine and increased the putrescine content to 31% of that in the partially hepatectomized controls (Table II). A larger dose

of putrescine (500 #mol/kg) did restore putrescine to that seen in the regenerating liver controls, but this dose was not able to reverse the effects on DNA synthesis presumably due to toxic effects. Table II also indicates that neither dose of putrescine influenced the reduction in ornithine decarboxylase by a-difluoromethylornithine showing that the putrescine was not interfering with the uptake of the inhibitor. Several papers have demonstrated that adifluoromethylornithine is effective via oral administration in the drinking water and that this route of administration can be used to administer a large daily dose of the drug [23-27]. Therefore, the effect of this treatment was tested on the regenerating liver system by placing 2% adifluoromethylornithine in the drinking water 24 h prior to partial hepatectomy. This mode of administration was not by itself satisfactory for our experiments because as shown in Table III, the rats drank very little in the time period shortly after the operation which corresponds to that in

T A B L E II REVERSAL O F E F F E C T O F a - D I F L U O R O M E T H Y L O R N I T H I N E BY A D M I N I S T R A T I O N OF P U T R E S C I N E Rats were treated with a-difluoromethylornithine by intraperitoneal injection of 400 m g / k g every_ 4 h as in Table I. Putrescine (300 or 500 p, m o l / k g ) was given by intraperitoneal injection every 4 h at 15 rain after the drug. D N A synthesis was measured by the incorporation of radioactivity into D N A during a 30 min period after injection of 10 ,uCi [6 -3 H]thymidine. Rats weighing 230 ± 12 g were used and killed 28 h after partial hepatectomy (corresponding to the peak of D N A synthesis) or sham operation. Results are shown as mean ± S . D . for four to six animals. Treatment

Sham operated Partial hepatectomy

Ornithine decarboxylase (pmol/mg per 30 rain)

Putrescine (nmol/g)

Spermidine (#tool/g)

Spermine (p, m o l / g )

DNA synthesis (cpm/ttg)

5 4 ± 22

5+

I

1.04±0.13

0.81~0.01

2--5

305 + 107

77+33

1.87 ~'l).16

0.68±0.04

37 + 7

1.56±0.12 a

0.71±0.09

17+8 b

24 + 9 a

1.76±0.05

0.67±0.05

40±8

63 + 19

1.80±0.15

0.67+_0.02

25_+8 c

Partial hepatectomy + a-difluoromethylornithine

24±

8a

Partial hepatectomy + a-difluoromethylornithine + putrescine (300 # m o l / k g per 4 h)

23±

4

Partial hepatectomy + a-difluoromethylornithi ne + putrescine (500 # m o l / k g per 4 h)

25±

10 a

~a. Significantly less than partial hepatectomy alone (P
'<2 a

183 T A B L E III C O N S U M P T I O N O F D R I N K I N G W A T E R C O N T A I N I N G a - D I F L U O R O M E T H Y L O R N I T H I N E BY RATS A F T E R PARTIAL HEPATECTOMY Rats were given normal drinking water or water containing 2% a-difluoromethylornithine as indicated starting 24 h prior to hepatectomy. The operation was carried out at about 8 a.m. One group of rats was then treated with 300/t m o l / k g doses of putrescine as described in Table II. The average value for water consumption per rat is given for the time period shown. Treatment of rats

Drinking water consumed in period (ml/rat) 24 h prior to operation

12 h after operation

12-29 h after operation

29-41 h after operation

41-52 h after operation

Partial hepatectomy + normal drinking water

23

2

19

12

25

Partial h e p a t e c t o m y + 2% a-difluoromethylornithine in drinking water

22

2

13

10

20

Partial hepatectomy + 2% a-difluoromethylornithine in drinking water + putrescine injections (300 # m o l / k g per 4 h)

23

2

18

I1

24

which ornithine decarboxylase is greatly enhanced. Therefore, the oral treatment was supplemented by injections of 400 mg/kg per 4 h. The results are shown in Table IV and were basically similar to those found in Table II. Exposure to adifluoromethylornithine via injection and in the drinking water produced a slightly greater depression in ornithine decarboxylase activity than injection alone. Putrescine was reduced to very low levels, but there was only a very slight reduction in spermidine. DNA synthesis was reduced 65% and this effect could be substantially ameliorated by treatment with putrescine. Table IV also shows results obtained 52 h after partial hepatectomy. At this time, there was a 2-fold increase in spermidine and a 10-fold increase in putrescine when the regenerating livers were compared to unoperated controls. Treatment with a-difluoromethylornithine reduced the increase in spermidine to 55% and completely prevented the rise in putrescine. Such treatment with the inhibitor substantially inhibited the increase in DNA synthesis at 52 h after operation. Administration of putrescine restored the spermidine content completely, increased putrescine to 43% of that seen in the regenerating controls and significantly enhanced DNA synthesis (Table IV).

Discussion

The present work confirms previous indications [22,23,28] that a-difluoromethylornithine, although a potent inhibitor of ornithine decarboxylase activity and polyamine accumulation in other cells and tissues [22-31] is relatively ineffective in inhibition of liver ornithine decarboxylase in vivo. Since the compound is an excellent inactivator of liver ornithine decarboxylase in vitro [12,32,33], this lack of inhibition in vivo may relate to inefficient uptake of the inhibitor, a high intracellular concentration of substances such as L-ornithine or putrescine which interfere with inactivation [12,33] or the rapid synthesis of new enzyme [1,2]. The rapid clearance of a-difluoromethylornithine [21] and the insensitivity of hepatic ornithine decarboxylase made necessary the administration of very large doses of the inhibitor in order to reduce hepatic polyamine accumulation. These doses amounted up to 4 g/kg per day in the rats receiving the drug both in the drinking water and via intraperitoneal injection. Such doses are not unreasonable for the type of biochemical investigations carried out in the present study since adifluoromethylornithine is remarkably non-toxic [23] having an acute LDs0 in rodents of more than 5g/kg [21]. However, doses of 2 g / k g per day are known to reduce the gain in body weight in grow-

184 T A B L E IV E F F E C T O F O R A L A D M I N I S T R A T I O N OF a - D I F L U O R O M E T H Y L O R N I T H I N E ON P O L Y A M I N E A C C U M U L A T I O N A N D D N A SYNTHESIS IN R E G E N E R A T I N G R A T LIVER Rats were exposed orally to the drug by substitution of 2% a-difluoromethylornithine for drinking water commencing 24 h prior to partial hepatectomy and continuing until death. The drug was also given by injection of 400 m g / k g doses every 4 h as in Table II as noted in the Table. Other details of putrescine treatment and measurement of D N A synthesis were as given in Table II. Results are given for 28 and 52 h after partial hepatectomy as indicated and are shown as mean ± S . D for five to six animals. Treatment

Exposure to a-difluoromethylornithine

Unoperated control

--

Partial hepatectomy, 28h

--

Partial hepatectomy, 28 h

400 m g / k g per 4 h injections only

Partial hepatectomy, 28 h

Oral exposure 400 m g / k g 4 h injection

Partial hepatectomy, 28 h + putrescine ( 3 0 0 / ~ m o l / k g per 4 h)

Oral exposure 400 m g / k g per 4 h injection

Partial hepatectomy, 52h Partial hepatectomy, 52 h Partial hepatectomy, 52 hr, + putrescine (300 # m o l / k g per 4 h)

m

Ornithine decarboxylase (pmol/mg per 30 min) 26 -+

Putrescine (nmol/g)

Spermidine (/lmol/g)

Spermine (/~mol/g)

1.09± 15

0.74±0.10

3± 2

1.95-+0.20

0.75±0.12

50-+13

2-- I a

1.87-+0.14

0.82±0.06

25-+13 a

2+

Ia

1.78±0.09

0.84±0.05

18± 6 a

33± 9 b

1.97+0.15

0.80±0.08

42 + 10

53±23

2.23±0.24

0.72±0.07

41± 8

1.69-+0.12 ~

0.78---0.03

19-+ 4 ~

2.47-+0.12

0.72±0.04

30-+ 5

5

6-+ 2

4 5 1 ± 54

115±51

27±

14 a

16 -+

9a

16±

3a

2 5 3 ± 121

Oral exposure + 400 m g / k g per 4 h injection

31 ±

4c

2-+1 c

Oral e x p o s u r e + 400 m g / k g per 4 h injection

29 ±

9c

2 3 +- 9

DNA synthesis (cpm//~g)

Significantly less than partial hepatectomy, 28 h (P<0.001). b Significantly less than partial hepatectomy, 28 h ( P < 0 . 0 1 ) . Significantly less than partial hepatectomy, 52 h ( P < 0.001 ). a

ing rats [22,23] rendering such treatment unsatisfactory for following the role of polyamines in liver regeneration measured over a long period by the gain in liver mass. Many studies have now confirmed the reports of Mamont et al. [29,34] that growth of mammalian cells in culture is stopped by a-difluoromethylornithine once endogenous spermidine and putrescine pools have been depleted. Such inhibition of DNA synthesis and cell replication can be reversed by addition of spermidine suggesting that spermidine is needed for these processes [29-31,34]. It is possible that part of the inhibition

of DNA synthesis caused by a-difluoromethylornithine in regenerating liver is mediated via its effect on spermidine accumulation. However, as shown in Tables I, II and IV, the effect of the drug on spermidine content in the liver at 28 h after operation is quite small and is not statistically significant in the experiment shown in Table IV. Exposure to a-difluoromethylornithine produced a much greater depletion of putrescine which was reduced to at most 2 n m o l / g tissue in rats receiving 400 m g / k g doses of the inhibitor. Such reduction in hepatic putrescine was partially reversed by administration of putrescine although putrescine

185 content was not restored to the level seen in the partially hepatectomized rats not exposed to the inhibitor. However, the administration of putrescine completely reversed the inhibition of DNA synthesis produced by the drug. This suggests that putrescine, itself, may be needed for DNA synthesis in this system and it can be seen in Tables I, II and IV that DNA synthesis was only inhibited when putrescine was reduced to 2 n m o l / g or less. This requirement for putrescine may be indirect in that the diamine could be needed for some cellular event crucial for DNA synthesis or it could be needed for DNA replication. Several other recent papers have also suggested that putrescine may be essential for proliferation of cells in rodent tissues including epidermis [35], sarcoma cells [36] and liver [7,37]. The present results are consistent with a role for putrescine in the stimulation of DNA synthesis following partial hepatectomy, but show clearly that only a small fraction of the rise in putrescine normally seen after the operation is essential. It should also be noted that the reversal of inhibition of DNA synthesis by putrescine was not seen when the dose of putrescine was increased to 500 /~mol/kg (Table II). Toxic effects of repeated high doses of putrescine and other diamines have been reported previously [8,11,38] and have repeatedly been observed in our laboratory when a-difluoromethylornithine-treated animals were exposed to putrescine (unpublished data). The results obtained in the experiment shown in Table IV demonstrate that polyamine synthesis is needed for a normal rate of liver regeneration as measured by DNA synthesis. The large dose of a-difluoromethylornithine used in this experiment produced no obvious toxicity and did not inhibit other enzymes in the liver such as S-adenosylmethionine decarboxylase (unpublished data). Its inhibitory effect on spermidine accumulation and DNA synthesis could be completely abolished by administration of putrescine, the product of the reaction inhibited by the drug. Therefore, liver regeneration may be slowed by exposure to inhibitors of polyamine synthesis and our results are consistent with a recent abstract [39] which notes that the gain in liver weight after partial hepatectomy was reduced in rats given 2% adifluoromethylornithine in drinking water for 2

weeks. However, as discussed above, such treatment with the drug is known to affect the normal weight gain in growing rats. Also, the most pronounced changes in liver polyamine synthesis occur within the first 48 h after partial hepatectomy. During this period, fluid intake by the rats recovering from surgery is significantly reduced (Table III) and in order to maintain a concentration of the inhibitor sufficient to inhibit polyamine production injection of the drug is needed. Finally, it is interesting that even in rats treated with doses of a-difluoromethylornithine that reduce hepatic putrescine to the limit of detection there was still a substantial rise in spermidine in response to partial hepatectomy. The putrescine formed by the vestigial ornithine decarboxylase activity is clearly very efficiently converted into spermidine. Under these circumstances, ornithine decarboxylase is the rate-limiting enzyme in spermidine production, whereas in the partially hepatectomized rats not treated with a-difluoromethylornithine, S-adenosylmethionine decarboxylase activity is more likely to be the ratelimiting factor. It also appears that the total normal induction of ornithine decarboxylase is not needed for polyamine production in the early stages of liver regeneration. Part of the stimulation may be nonspecific and due to enhanced rate of protein synthesis or reduced rate of protein degradation. Such changes would bring about a particularly marked increase in the amount of a protein such as ornithine decarboxylase which turns over very rapidly [40]. It is also possible that the increased synthesis of putrescine is needed for cellular functions other than DNA synthesis.

Acknowledgements This reseach was supported by grant CA-I 8138 from the N.I.H. and by an Established Investigatorship to A.E.P. from the American Heart Association and its Pennsylvania Affiliate. H.P. is a recipient of a research fellowship from the Research Council for Natural Sciences of the Academy of Finland and of a travel grant from the League of Finnish American Societies Scholarship Foundation (thanks to Scandinavia grant). We also want to thank Mrs. Bonnie Merlino for help in the preparation of this manuscript.

186

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24 25

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30 31 32 32

34 35 36 37 38 39 40

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