Ubururu bwa metilene mu bushakashatsi ku mafi ya zebrafish: inyongeramusaruro mu bworozi n’ikintu gishobora kuyobya igerageza

Amazi arimo amagi ya zebrafish akenshi aba afite ibara ry’ubururu ryerurutse, kandi hafi nta n’umwe wandika impamvu. Iryo bara rituruka ku bururu bwa metilene, bwongerwamo kurwanya uduhumyo, kandi amabwiriza menshi y’igerageza abufata nk’ikintu gisanzwe kititabwaho. Kubufata gutyo si byo hano. Ahubwo bukwiye gufatwa nk’ikintu kigira uruhare mu igerageza ariko kitanditswe: kiba kiri mu bikoresho by’amatsinda ahabwa ubuvuzi bw’igerageza ariko ntikibe mu by’amatsinda agereranywa na yo, kikagira ibikorwa bya farumakoloji ku ngano ikoreshwa, kandi kigashobora guhindura ibipimo nyirizina igerageza ripima.

Iryo tandukaniro ni ryo rigenderwaho muri byose bikurikira. Uburyo busanzwe bwo kurwanya uduhumyo mu bworozi ni ikibazo kimwe. Kumenya niba iyo nyongeramusaruro nta ngaruka igira ku isuzuma ryawe ni ikindi kibazo. Ubushakashatsi bwa vuba kuri zebrafish busubiza icya mbere mu buryo butanga icyizere, naho icya kabiri bukagisubiza mu buryo butera impungenge.

Icyo amabwiriza asaba mu by’ukuri

Nta gipimo kimwe gisanzwe gihuriweho, kandi ibyo biri mu bigize ikibazo. Amabwiriza ya Canadian Council on Animal Care yemera 0.5 mg/L, cyangwa 0.00005%, mu gisukika gitwarwamo amagi n’ibyana kugira ngo agabanye imikurire y’uduhumyo. Amabwiriza y’i Burayi yerekeye inyamaswa zo muri laboratwari asobanura insoro zibikwa mu gisukika akenshi kirimo icyo gipimo cya 0.00005%. Uburyo bwa Cold Spring Harbor bwo gutegura E3 busaba inshuro ebyiri z’icyo gipimo, ni ukuvuga 1 mg/L cyangwa 0.0001%, nk’ikigize urwo ruvange. Icyakora, hari ikintu kigaragara kitavugwa ahandi: amabwiriza ya OECD yo gusuzuma uburozi ku nsoro z’amafi asaba amazi yongeye gutegurwa afite ibigize bizwi yo kuvangamo ikinyabutabire gipimwa, ariko nta nama yo gukoresha ubururu bwa metilene iri mu nyandiko yayo yo mu 2013 cyangwa mu ivugurura ryakurikiyeho. Bityo, laboratwari imwe yororera insoro za zebrafish mu mazi arimo ubururu bwa metilene kuva mu masaha ya mbere y’ubuzima, indi igakora isuzuma ry’insoro rigengwa n’amabwiriza mu mazi atarimo iryo bara, kandi zombi zikita ibisukika zikoresha ibisanzwe. Ugereranya ibyavuye muri laboratwari zitandukanye, cyangwa uhuza amakuru y’amatsinda agereranywa yo mu bushakashatsi bwabanje, na we ahura n’uko kudahuza.

Kugira ngo ibyo bipimo byumvikane neza, 0.00005% ni hafi 1.56 micromolar naho 0.0001% ni hafi 3.13 micromolar. Iyo ni imibare mito, kandi biroroshye gutekereza ko ari mito cyane ku buryo nta ngaruka yagira. Ubushakashatsi bukurikira busuzuma neza icyo gitekerezo, bukoresheje ibyo bipimo nyirizina.

Abasomyi bamenyereye korora amafi aho kumenyera laboratwari bahura n’iyo molekile mu bundi buryo, busobanurwa mu gitabo cy’ubururu bwa metilene mu byogero by’amafi. Ubutabire ni bumwe; ibisobanuro by’uko iryo bara ritwara elegitoroni mu turemangingo byerekana impamvu ikinyabutabire kigira ibikorwa bya redox gikomeza kugaragara mu nyandiko zivuga kuri metabolizime. Ibikurikira byibanda ku mazi arimo amagi ya zebrafish n’ubururu bwa metilene.

Metabolizime: icyerekezo cy’ingaruka gihinduka bitewe n’icyiciro

Ubushakashatsi butanga amakuru menshi kurusha ubundi bwashyize zebrafish zikura bisanzwe mu mazi arimo iryo bara kuva ku munsi wa mbere w’ubuzima kugeza ku minsi 5 nyuma y’ifatanywa ry’intanga, maze bupima metabolizime ikoresha ogisijeni kuri buri cyiciro, nk’uko raporo yatangajwe y’amagerageza kuri metabolizime y’insoro ibisobanura. Uko ingaruka zakurikiranye gukwiye gusobanurwa neza kuko zihindura icyerekezo.

Ku munsi 1 nyuma y’ifatanywa ry’intanga, ikoreshwa rya ogisijeni mu mimerere y’ibanze ryariyongereye mu nsoro zashyizwe muri iryo bara, ku bipimo byombi bikoreshwa mu bworozi. Ku minsi 2, itandukaniro ry’umuriro w’amashanyarazi ku gahu ka mitokondiriya mu gice cyo ku buso bw’ibyana ryariyongereye ku bipimo bya 0.00005%, 0.0001%, na 0.0003%. Ku minsi 4 kugeza kuri 5, nyuma yo kuva mu magi, icyerekezo cyari cyahindutse: ikoreshwa ry’ingufu rishingiye kuri ogisijeni ryaragabanutse, haba ku byari byarakomeje kuba mu mazi arimo iryo bara kuva ku ifatanywa ry’intanga cyangwa ku byarishyizwemo igihe gito gusa, kuva ku munsi 3 kugeza ku munsi 4. Muri icyo gihe kandi, inyandiko za RNA z’ibice bigize amatsinda y’imikorere y’ubuhumekero bwa mitokondiriya zaragabanutse ku munsi 1 no ku munsi 4, kimwe n’inyandiko za RNA zijyanye n’ikorwa ry’imisemburo ya steroyide n’ikorwa rya glukoze mu bindi bintu bitari karubohayidire.

Ibindi bintu bibiri byagaragaye bituma ishusho irushaho gusobanuka. Ku gipimo kinini muri ibyo bikoreshwa mu bworozi, kuva mu magi byabaye mbere gato y’igihe: ku minsi 2 insoro zashyizwe muri iryo bara zari ziri imbere, ariko ku munsi 3 izo mu itsinda rigereranywa zari zamaze kuzigeraho. Kandi uburyo umubiri witabira imimerere itera umunaniro bwasaga n’aho bukora neza: igihe ibyana bifite iminsi 5 byashyirwaga mu mazi azunguruka abiteza umunaniro, kortizole yo mu mubiri wose yariyongereye nk’uko bisanzwe mu matsinda yombi. Bityo, kuri ibyo bipimo, iryo bara si uburozi bubangamira imikurire muri rusange, kandi ntirigabanya buri gisubizo cy’imikorere y’umubiri. Rihindura metabolizime y’ingufu bitewe n’icyiciro, rikayongera mbere yo kuva mu magi rikayigabanya nyuma yaho.

Imyitwarire: impamvu ubushakashatsi bubiri butavuga kimwe, n’impamvu bwombi bushobora kuba ari ukuri

Inyandiko z’ubushakashatsi ku myitwarire zisa n’izivuguruzanya kugeza igihe uburyo bwo gushyira ibinyabuzima muri iryo bara bugereranyijwe. Ubushakashatsi bumwe ku mutekano mu mikurire no mu myitwarire bwashyize insoro mu mazi arimo iryo bara kuva hafi y’amasaha 6 nyuma y’ifatanywa ry’intanga, ku bipimo bigera kuri 10 micromolar, bugasimbuza ibisukika buri munsi kugeza ku munsi 4. Nyuma yaho, bwashyize ibyana mu gisukika kitarimo iryo bara ku minsi 5 na 6, maze ku munsi 6 bupima ingendo zabyo mu isuzuma rihinduranya urumuri n’umwijima. Ibyavuye mu bushakashatsi: nta ngaruka zifite ubusobanuro mu ibarurishamibare ku rupfu, ku kuva mu magi cyangwa ku miterere y’umubiri, kandi nta ngaruka z’iryo bara ku ngendo. Byari bifite ishingiro ko abanditsi batanga 10 micromolar nk’igipimo ntarengwa mu mimerere y’ubushakashatsi bwabo.

Itsinda rya kabiri, ririmo bamwe mu banditsi b’ubushakashatsi kuri metabolizime, ryakoresheje iryo bara nk’uko rikoreshwa mu bworozi nyirizina: ryari mu mazi kuva ku masaha 2 kugeza kuri 3 nyuma y’ifatanywa ry’intanga, igice cy’ayo mazi kigasimbuzwa buri munsi, kandi imyitwarire igapimwa ibinyabuzima bikiri muri iryo bara cyangwa bimaze igihe gito birivuyemo. Kuri bya bipimo bisanzwe, ibyana byagaragaje ingendo zagabanutse mu isuzuma ry’amasaha 24, kandi ibyo byashoboye kongera kugaragara mu mirongo y’inkomoko itandukanye no muri laboratwari zitandukanye. Ubushakashatsi ku ngaruka ziyobya ibipimo by’imyitwarire bwahuje izo ngaruka n’urwego rw’ibikorwa bya serotonine, bihuye n’uko iryo bara rizwiho kubuza imikorere ya monoamine oxidase. Ikigaragaza cyane kurushaho ni uko igisukika cy’insoro kirimo ubururu bwa metilene cyahishe impinduka z’ingendo zari zitezwe ku muti wa SSRI witwa fluoxetine. Bityo, isuzuma ryo gushakisha ingaruka rikorewe mu mazi y’ubururu rishobora kutabona ingaruka z’umuti zari kugaragara mu mazi atarimo iryo bara.

Nta kuvuguruzanya nyakuri kurimo. Ikinyabutabire gishobora kutagaragaza ingaruka zisigaye zipimika nyuma y’iminsi ibiri kivanyweho mu isuzuma rimwe, ariko kigakomeza kugira ibikorwa igihe kikiri mu rindi. Icyo bivuze mu bikorwa ni uko igisubizo kitagaragaje ingaruka gishyigikira igipimo ntarengwa, aho kwemeza ko nta ngaruka na mba zibaho. Naho igisubizo cyagaragaje ingaruka gisobanura ibyago bikwiye kwitabwaho: ibinyabuzima kuba bikiri mu mazi arimo iryo bara mu gihe cyo gupima, mu buryo nyirizina laboratwari nyinshi zikoresha.

Ibara rya redox mu isuzuma rya redox

Hari undi mutego ukwiye igika cyawo kuko wangiza igipimo ubwacyo aho kugira ingaruka ku nyamaswa. Isuzuma ry’ikoreshwa ry’ingufu rikoresha Alamar Blue ubwaryo rishingiye ku butabire bwa redox: resazurin yakira elegitoroni igahinduka resorufin itanga urumuri rwa floresansi. Ubururu bwa metilene na bwo bugira ubutabire bwa redox. Mu matsinda agereranywa atarimo uturemangingo, ibipimo byombi bikoreshwa mu bworozi byagabanyije gato urumuri rwa floresansi rwa Alamar Blue nyuma y’amasaha 24, ariko iryo gabanuka rifite ubusobanuro mu ibarurishamibare. Mu magerageza ku nyamaswa, insoro zari zashyizwe muri iryo bara zozwe nibura inshuro eshatu mu gisukika kitarimo iryo bara mbere y’isuzuma, kandi ingaruka ku mikorere y’umubiri mu byana byari byaravuye mu magi zarutaga ukubangamira igipimo kwagaragaye ahatari uturemangingo. Bityo, ibyo byombi ni ukuri: inyamaswa zozwe zigumana ingaruka nyakuri kuri metabolizime, kandi iryo bara ritakuweho no koza ryongeraho iyobya ry’igipimo rituruka ku butabire bwaryo butaziguye. Amatsinda agereranywa agomba gutandukanya ibyo byombi, bitabaye ibyo nta mubare n’umwe uba ufite icyo usobanura. Amabwiriza rusange yo gusobanura ibyavuye mu isuzuma rishingiye ku mabara afite akamaro kihariye hano.

Uko ibyiciro by’imikurire bikurikirana

Imbonerahamwe y’umwimerere yateguriwe iyi nyandiko. Ibipimo ni bibiri bisanzwe bikoreshwa mu bworozi, 0.00005% na 0.0001%, keretse aho havuzwe ibindi.

Icy’ingenzi cyo kwibuka ni ukwiyongera kwabanje n’igabanuka ryakurikiyeho mu bipimo bya metabolizime, hamwe n’ingaruka ku myitwarire zigaragara ibinyabuzima bikiri mu bara ariko zitagaragara nyuma yo kurikuraho no kubireka bikaryisohoramo, mu isuzuma rimwe ryabipimye.

Urutonde rwo kugenzura itegurwa ry’amatsinda agereranywa

Urutonde rwa kabiri rw’umwimerere. Rukoreshe igihe amabwiriza yo korora zebrafish ukoresheje ubururu bwa metilene ahuriye n’igerageza.

1. Andika iryo bara nk’ikinyabutabire gikoreshwa mu igerageza, aho kuryita amazi gusa. Garagaza igipimo mu ijanisha no muri micromolar, igihe cyo gutangira no guhagarika mu masaha cyangwa iminsi nyuma y’ifatanywa ry’intanga, gahunda yo gusimbuza amazi, n’igihe cyo kuvanaho ibara mbere ya buri gipimo.
2. Huza amatsinda agereranywa n’ikibazo gisuzumwa. Ubushakashatsi ku kinyabutabire gipimwa bukeneye amatsinda agereranywa atarimo iryo bara; ubushakashatsi ku bworozi bukeneye kugereranya amatsinda atarimo iryo bara n’ayarimo, bukandika ibyavuye mu kurwanya uduhumyo, kuko nta na bumwe muri bwa bushakashatsi bwavuzwe bwapimye ubushobozi bwo kurwanya uduhumyo.
3. Oza mbere y’isuzuma rya redox, hanyuma ugenzure. Oza nibura inshuro eshatu mu gisukika kitarimo iryo bara, kandi ukore iruhande rw’isuzuma itsinda rigereranywa ritarimo uturemangingo ariko ririmo iryo bara n’ikinyabutabire cy’isuzuma, kuko iryo bara rigabanya mu buryo butaziguye ikimenyetso cya Alamar Blue.
4. Fata ibipimo by’imyitwarire n’ibya monoamine nk’ibifite ibyago byinshi byo kuyobywa. Ubushakashatsi ku ngendo, ku kwikanga, ku myitwarire isa n’iy’impungenge, ku miti igabanya kwiheba no ku bibuza imikorere ya MAO bukwiye kwirinda iryo bara mu gihe cyo gupima, cyangwa bukagaragaza ko nta ngaruka rigira kuri buri gipimo kigamijwe; kuvanaho iryo bara mu gihe cy’umunsi umwe kugeza ku minsi ibiri byakuyeho ingaruka imwe ku ngendo, ariko ibyo ntibirahamya ko ari ko bigenda mu bundi buryo bw’igerageza.
5. Fata ibipimo bya metabolizime n’ibya mitokondiriya nk’ibifite ibyago byinshi byo kuyobywa kuri buri cyiciro. Ikoreshwa rya ogisijeni, ikoreshwa ry’ingufu, itandukaniro ry’umuriro w’amashanyarazi ku gahu n’inyandiko za RNA z’uruhererekane rw’ubuhumekero birahinduka ku bipimo bikoreshwa mu bworozi, mu byerekezo binyuranye mbere na nyuma yo kuva mu magi.
6. Ntukemeze ko nta ngaruka zibaho mu buryo bumwe bw’igerageza ushingiye ku bundi. Igisubizo kitagaragaje ingaruka nyuma yo kuvanaho ibara ntigikoreshwa ku gihe ibinyabuzima bikiririmo; kutabona ingaruka ku miterere y’umubiri ntibyemeza ko nta ngaruka kuri metabolizime cyangwa ku mikoranire n’umuti; igisubizo cyo mu murongo umwe w’inkomoko, ku bushyuhe bumwe cyangwa mu gihe kimwe cy’isuzuma ntigikoreshwa ku bindi.
7. Vuga amabwiriza wakurikije n’andi ashobora gukoreshwa. Garagaza neza ibipimo bya CCAC, iby’i Burayi cyangwa ibya Cold Spring Harbor, kandi werekane aho isuzuma ry’insoro rya OECD ritavuga iryo bara, kugira ngo umusomyi yumve imimerere inyamaswa zawe zarimo.

Nta na kimwe muri ibi kirwanya gukoresha iryo bara kugira ngo amagi akomeze kubaho. Ahubwo birwanya kureka icyemezo cyo mu bworozi kikagira uruhare mu makuru y’ubushakashatsi kitasuzumwe. Ubururu buri mu gikoresho ni molekile ifite ibikorwa bya farumakoloji, kandi amagerageza ashobora kuyobywa cyane ni ya yandi nyirizina apima metabolizime n’imyitwarire ijyanye n’imikorere y’imyakura ku bwinshi kandi vuba, ari na yo asigaye ahitamo gukoresha zebrafish zikiri nto. Bwandike, ugenzure ingaruka zabwo, ubukureho mbere yo gupima, kandi ugenzure isuzuma bushobora kuba bugiramo uruhare rutagaragara.