LA EXTRAFISCALIDAD DE LOS IMPUESTOS EN MÉXICO

Thymocytes at 1x10^ cells/ml were pre-incubated for Ih with ferric citrate 50pM (from a 18mM stock in O.IM HCl), washed x 2 with medium, then either CP20 (150 pM)

ro .2 'w o Q . o Q . < 8 0 1 6 0 - 4 0 - 20 - P B S C P 2 0 TPEN

Figure 5.6a: Effect of zinc chelation on thymocyte apoptosis

T h y m o c y t e s w e r e i n c u b a te d w ith e it h er c o n tr o l, P B S or C P 2 0 , 3 0 0 p M I B E or the z in c c h e la t o r T P E N , 5 0 ) i M for 2 4 h at 3 7 ‘’C /5% CO^. T h e c e l l s w e re spun in a p e lle t, f i x e d in 7 0 % e t h a n o l and sta ine d w ith PI for a p o p t o s is m e a su r em e n t by ( l o w c y to m e tr y . T h e data s h o w n are the m e a n ± S D o f 3 in d e p en d e n t e x p e r im e n t s d o n e in duplicate. 8 0 7 0 - <0 6 0 - CO CO o Q . o a . < 5 0 - 4 0 - .01 1 1 1 0 1 0 0 1 0 0 0 C o n e . T P E N pM

Figure 5.6b: Effect of TPEN concentration on thymocyte apoptosis

T h y m o c y t e s w e r e i n c u b a te d w ith i n c r e a s i n g c o n c e n t r a t i o n s o f T P E N , 0 . l - 5 0 ) i M fo r 2 4 h at 37 " C /5 % C 0 2 . T h e c e l l s w e r e su n and f i x e d in 70% e th a n o l and s t a i n e d w ith PI for a p o p t o s i s m e a s u r e m e n t b y f l o w c y to m e tr y . T h e data s h o w n are the m e a n ± S D o f 3 in d e p e n d e n t e x p e r i m e n t s do n e in d u p licate .

(A O D . O a < 80 - | 9 T P E N 60 - 40 - _{P B S} 20 - 0 1 0 2 0 3 0 T i m e Mrs

Figure 5.6c: Effect of incubation time on zinc chelation by TPEN

T h y m o c y t e s w e r e inc ub ate d for b e t w e e n 3 0 m i n s and 24h w ith T P E N (50)liM) at 37"C/5% CO,. T h e

c e l l s w e r e spun in a p e lle t and fix ed in 70% ethanol and stained with PI for a p o p t o s i s m e a s u r e m e n t by f l o w c y t o m e t r y . T h e data s h o w n are the m e a n ± S D o f 3 in d e p e n d e n t e x p e r i m e n t s d o n e in du plica te. 0) u (O < o 0) o S Q . C N 200 -1 100 - C t r l C P 2 0 T P E N

Figure 5.6d: Effect of TPEN on zinquin fluorescence

T h y m o c y t e s w e r e in c u b a te d w ith T P E N , 5 0 | i M for 2 4 h at 3 7 " C / 5 % C 0 2 . T h e c e l l s w e r e w a s h e d x3 prior to the a d d itio n o f z in qu in , 2 5 | i M . Z in q u in h u o r e s c e n c e w a s m e a s u r ed by sp e c t r o llu o r im e t r y . T h e data s h o w n are the m ean ± S D o f 3 ind ep en dent e x p e rim e n ts d o n e in du plicate.

or DFO (50|iM ) added then TPEN (50jiM) added and incubated for 24h. Samples were then analysed for apoptosis as described previously using flow cytometry (s e c tio n 2.4.1) or assessed for intracellular zinc levels using zinquin incorporation (section 2.5)

R esu lts and d iscu ssio n .

W hen thymocytes were pre-incubated with ferric citrate prior to the addition o f chelator, no significant increase in apoptosis after 24h was observed when compared to control. In contrast when ferric citrate and TPEN, or ferric citrate iron chelator and TPEN , was added to thymocytes there is a significant amount of apoptosis (55.8±3.2% and 68.5±5.3% respectively) compared to control, PBS (39.5±4.1% ), P<0.001 (F ig u re 5 .7 a ) .

Using zinquin to assess the levels o f intracellular zinc, it is shown (F igure 5 .7 b )

that by adding ferric citrate, alone or in combination with an iron chelator, there is no fall in intracellular zinc levels, confirming previous preliminary experiments in sectio n 5 . 2

which showed that the addition of iron to zinquin had no effect on zinquin fluorescence. H owever ferric citrate together with TPEN or ferric citrate with chelator and TPEN, caused a significant decrease in the amount of intracellular zinc, p<0.01 (Figure 5.7b).

Therefore it is clear from these experiments that by pre-loading thymocytes with iron, the fall in intracellular zinc that was previously observed when similar concentrations o f iron chelators were subsequently added to the thymocytes (section 5 .2 ), is abrogated together with the effect on apoptosis. These findings are consistent with the inhibition o f iron chelator induced apoptosis, being due to the excess added iron being scavenged by the iron chelator intracellularly in preference to zinc, thereby decreasing intracellular zinc chelation and thus apoptosis. Therefore iron overload could protect against the thymocyte apoptotic induction by iron chelators by indirect inhibition o f zinc chelation (due to excess preferentially chelatable iron) rather than inhibition o f a primary iron induced apoptotic mechanism.

From these experiments, the possibility that iron chelation is the direct mechanism o f apoptotic induction by iron chelators cannot be absolutely excluded. However to examine the relative contributions of zinc and iron chelation more closely, further experiments were designed to examine the interaction o f zinc and iron chelator combinations on apoptosis.

<N <0 <0 (fl O Q. O CL <

F igure 5.7a: Effect of iron addition to chelator-induced thymocyte apoptosis

Thym ocytes were pre-incubated for 2h with ferric sulphate, lOOpM prior to the addition of the iron chelators, CP20 and DFO, lOOpM IBE or the zinc chelator, TPEN, 50p.M. The cells were spun and fixed in 70% cold ethanol and stained with PI for apoptosis measurement by flow cytometry. The data shown are the mean ± SD of 4 independent experiments done in triplicate.

200 - w 0) o E a. "F N 100 -

CP20+Fe TPEN+Fe CP20+Fe+TPEN

Figure 5.7b: Effect of addition of iron on zinquin fluorescence

Thymocytes were incubated with either PBS, CP20 (300)iM IBE) TPEN (50)iM), Ferric-sulphate (lOOjiM), CP20+Fe, TPEN+Fe or CP20+Fe+TPEN for 24h at 3 7"C/5%C0 2. The cells were washed x3 prior to the addition of zinquin, 25pM. Zinquin fluorescence was measured by spectrofluorimetry. The data shown are the mean ± of 4 independent experiments done in duplicate.

5 . 5 . 3 In teraction s o f zinc and iron chelator com bin ations.

R a tio n a le .

In order to examine such potential interactions, experiments were designed to construct a series o f isobolograms using the zinc chelator T P E N together with the iron chelators CP20 or DFO. Isobolograms are a simple way of visualising drug interactions and show lines of equivalent effect, i.e. show the relative concentration o f pairs o f agents that in combination induce the same magnitude of effect (50% o f maximal response was chosen). Synergistic interaction is shown by a line o f points curving underneath the diagonal zero line to produce a concave-up Isobologram. This type o f interaction indicates that the combined effect of two drugs at given concentrations is greater than the sum o f each drug individually, in other words by combining two drugs you obtain a better than expected effect. In contrast, a line if points curving above the zero interaction line, to produce a concave-down isobole, indicates that the two drugs antagonise each other and may not suitable to be used in combination therapy. Finally, a line o f points along the zero interaction line indicates that the combination is merely additive (Berenbaum, 1989).