Kinetic Interference Upon Embryo Development

Biomphalaria glabrata (molúsco, gastrópodo), em diferentes estágios de desenvolvimento foi tratado com D2O a 1% e 0,1 v/v em H2O (~5 vezes a abundância nat u ral). Além de uma alta mortalidade em relação ao controle, observa-se uma velocidade de desenvolvimento extremamente baixa. Surpreendentemente, este an i mal pode ser mantido por ~1/3 da sua vida em dimensões reduzidas. O re torno às condições normais leva a uma retomada do desenvolvimento atingindo o estado de adulto após um intervalo de tempo muito maior do que o observado no controle. O real papel dos isótopos na natureza é questionado.

Biomphalaria glabrata (mol lusc, gas tro pod), in dif fer ent de vel op ment stages were treated with D 2O at 1% and 0.1 v/v in H 2O (~5 times nat u ral abun dance).Be sides a high mor tal ity rate in re la tion to the con trols an amaz ing slow rate of de vel op ment was ob served.Sur pris ingl y, this an i mal may be kept for ~1/3 of its life in re duced di men sions.Re covering is how ever pos si ble.Upon re turn to nor mal con di tions they re sume de vel op ment reach ing adult stage in a much lo nger pe riod of time as com pared to the con trols.The ac tual role of iso topes in na ture is dis cussed Key words: Biomphalaria glabrata, deu te rium , en vi ron ment, iso tope, tox i col ogy

In tro duc tion
Iso tope sub sti tu tion has been a quite com mon technique to in ves ti gate chem i cal re ac tions.More re cently [1][2][3][4] how ever, this same tech nique has shown a spe cial in ter est for stud ies con cern ing ac tiv ity of drugs 4 , com plex mech anisms such as taste of liv ing be ings, growth and trans forming ac tiv i ties of mi cro or gan isms, such as Strep to coc cus pneumoniae , cell sur vival 3 and tem per a ture shift, ef fect over some dis ease such us Fanconi's ane mia, and its ef fect upon hu man car ci noma.
As it is well known, iso tope ef fect upon a chem i cal reac tion 9 is quite un pre dict able spe cially when we con sider chem i cal re ac tions among high mo lec u lar weight compounds are con sid ered.Ob vi ously, more un pre dict able effects can be ex pected when deal ing with liv ing or gan isms where the mod i fied mol e cules take part of an enor mous num ber of re ac tions in con cert.In this pa per we deal with the ef fect of iso tope sub sti tu tion upon the pro cess of growth of an an i mal from its em bryo phase.For the purpose we have cho sen as our sub ject Biomphalaria glabrata (mol lusc, gas tro pod) in dif fer ent de vel op men tal stages (Phase I, close to 130 blastomers; II , Vel iger , 5 days af ter first cleav age; III , al ready formed em bryo , ready to hatch out, and IV , al ready ecloded, two days old).

Ma te rials and Method
Biomphalaria glabrata , from the State of Minas Gerais, Brazil was used in the pres ent re search.Adults were raised in lab o ra tory for ten years.Biomphalaria glabrata is a si mul ta neous her maph ro dite and re pro duce ei ther by selfing or cross-fertilization.In the pres ent research em bryos and adults were used.The adults were raised ac cord ing to our lab o ra tory rou tine 5,6,7 and the embryos were kept in Petri dishes and fil tered wa ter, at room tem per a ture.

No te
Em bryos at dif fer ent stages of de vel op ment were treated in wa ter con tain ing 0.1% and 1% D 2 O. Con trols were kept in plain wa ter.The ex per i ment were per formed us ing adults, and em bryos which were clas si fied ac cord ing Camey 5 as fol lows: phase I, 130 blastomers, one day af ter the eggmass was laid; phase II , Vel iger, five days af ter the eggmass was laid; phase III , em bryos ten days old em bryos and fi nally, phase IV , pos-hatching, young snails one or two days old.In all cases con trols were used un der the same con di tions.Ex per i ments were car ried out with mu tant albino and wild type.
The eggmasses of phases I-III were kept in mini Petri dishes (2 x 2 cm), con tain ing two eggmasses each.For each plate the num ber of eggs and hatch ing ra tio were ver i fied.
In all ex per i ments the sub jects were treated with two differents so lu tions of D 2 O (Aldrich, 99% D 2 O ), in wa ter, at con ve nient con cen tra tions of 0.1 and 1% (v/v).The an imals in phase IV , post-hatching num bered 18 young snails were kept only at 0.1% (v/v) D 2 O in H 2 O.All of them were kept in the same dish con tain ing D 2 O at 0.1% in wa ter (v/v), fed with dry ground let tuce leaves.

Re sults and Con clu sions
Al though all ex per i ments were car ried out with al bino and wild type (pig mented), we will pres ent the re sults consid er ing only the wild type since the re sponse to the tests were iden ti cal for both strains.The re sults that fol low are sum ma rized in Ta ble 1.
As it can be seen from our data, the con tent of D 2 O in the en vi ron ment is crit i cal for dif fer ent phases.From them we no tice that youn ger an i mals (phases I and II) are more re sis tant than the older ones, as far as the D 2 O ef fects are con cerned.Al though a sim i lar be hav ior was ob served when those an i mals were tested with nic o tine sul fate 6 and cop per sul fate 7 that was not the case with caf feine 8 ; in this case youn ger an i mals were more sus cep ti ble to its ef fect than the older ones.
Be sides the above men tioned ef fect of D 2 O upon the an i mals, em bryos in phase I and II , sur vived but their devel op ment was con sid er ably slowed down.In these cases egg hatch ing out was pos si ble only me chan i cally.An i mals of the other phase even tu ally died as the re sult of the treatment.
It is in ter est ing to no tice that, fol low ing our treat ment, we were able to keep em bryos en cap su lated for 26 days, liv ing nor mally, when we de cided to ecclode the eggs mechan i cally.Normally an em bryo stay en cap su lated for no more than 10 days be fore to ecclode.The an i mals thus mechan i cally ob tained man aged to sur vive; how ever, all them had an ex tremely slow de vel op ment; their di men sions were kept sur pris ingly diminute re gard ing weight and size as com pared to the nor mal un der con trol.We did not ob -serve any anom aly in their shell in spite of their di men sions of about ~1/4 to ~1/6 of the con trols at the same age.
When those an i mals thus de scribed were sub mit ted to nor mal feed ing con di tions in clud ing wa ter with nat u ral iso tope abun dance of deu te rium, they ap prox i mately recover reg u lar di men sions.Af ter 9 months their shell di ame ter reached ~15 mm as com pared to the con trols with ~20 mm on the av er age.This re cov er ing pro cess showed it self fea si ble even when the an i mals were kept for about 1/3-1/4 of his life be fore re turn ing to nor mal con di tion.Finally, after re cov er ing the an i mal ini ti ated nor mal re pro duc tion.
For al most half a cen tury sta ble-isotope has found usage 1 in hu man met a bolic stud ies with out doc u mented signif i cant ad verse ef fect.Side-effects with acute deu te rium dos ing are re ported tran si tory with no dem on strated ev idence of a per ma nent del e te ri ous ac tion.The thresh old of D tox ic ity has been de fined in an i mals and is far in ex cess of con cen tra tions used in hu man stud ies.For that rea son it is thought that D may have ad di tional ben e fi cial phar ma colog i cal ap pli ca tions.In fact, re cently there are re ports concern ing the ef fect of D 2 O re tard ing the growth of hu man car ci no mas 2 in oc u lated in mice af ter in ges tion of wa ter con tain ing ~30 atom % D 2O.Also, PtK2 cells 3 grown in pres ence of D 2 O re mained non-proliferating; how ever, they were able to di vide af ter re cov ery in a non-deuterated me dium.On the other hand, when they were in cu bated for 8 weeks in 75% D 2 O mi totic ac tiv ity was not re sumed, as re ported re cently.Be sides, it is re ported that mice in gest ing 30 to 50% D 2O have de veloped a dose-dependent de pres sion 4 of formed pe riph eral blood el e ments in 4 to 9 days.In this case, de spite pancytopenia in the pe riph eral blood, it was ob served that bone mar row cellularity and mor phol ogy re mained normal.Al though platelet and neutrophil con cen tra tions may re turn to nor mal within 48 to 72 h upon sub sti tu tion of D 2 O with tap wa ter, a pro longed deuteration has proven toxic in an i mal mod els.
The above men tioned stud ies how ever deal with a rather high con tent of D 2O.Con se quently fol lows the idea that much lower % of deu te rium in a me dium might turn out to be harm less.Ac cord ing to our stud ies, differents phases of an an i mal de vel op ment can be have in a quite dis tinct man ner.An em bryo may be more re sis tant than an adult, when we con sider the ef fect of deuteration.As a con sequence, un pre dict able ef fects upon their de vel op ment are ob served.
Nat u ral abun dance of deu te rium is ~ 0.02% rel a tive to hy dro gen; how ever, work ing with val ues only five times higher, it is pos si ble to re tard the hatch ing out of an em bryo for a rather long pe riod of time.Fur ther, the an i mal thus grown may be kept for al most 1/4-1/3 of its live in suprisingly re duced di men sions and later it is pos si ble to re cover its de vel op ment to an al most nor mal size with out ap par ent anom a lies and with out af fect its re pro duc tion.
For those rea sons we are then tempted to ask what is indeed the role iso topes are play ing upon the liv ing or ganisms on Earth.Is deu te rium a sub tle mech a nism al low ing na ture to fine tune ki net ics pro cesses of life?