natgillin/translations-raw huggingface dataset:
DHT is significantly more potent as an androgen than T and is also considered an end-product hormone.
DHT je znatno potentniji kao androgen u usporedbi s testosteronom te se smatra i krajnjim hormonom.
The H295R assay does not measure DHT (see paragraph 10).
Testom H295R ne mjeri se DHT (vidjeti stavak 10.).
The enzyme in the steroidogenic pathway which converts androgenic chemicals into oestrogenic chemicals is aromatase (CYP19).
Aromataza (CYP19) jest enzim na steroidogenskom putu kojim se androgenske kemikalije pretvaraju u estrogenske.
CYP19 converts T into 17β-oestradiol (E2) and androstenedione into oestrone.
Njome se testosteron pretvara u 17β-estradiol (E2), a androstendion u estron.
E2 and T are considered end-product hormones of the steroidogenic pathway.
Hormoni E2 i T smatraju se krajnjim hormonima steroidogenskog puta.
The specificity of the lyase activity of CYP17 differs for the intermediate substrates among species.
Specifičnost djelovanja liaze aromataze CYP17 razlikuje se za supstrate posrednike među vrstama.
In the human, the enzyme favours substrates of the Δ5-hydroxysteroid pathway (pregnenolone), whereas substrates in the Δ4-ketosteroid pathway (progesterone) are favoured in the rat (19).
U ljudi enzim favorizira supstrate puta Δ5-hidroksisteroida (pregnenolon), dok su u štakora poželjniji supstrati na putu Δ4-ketosteroida (progesteron) (19).
Such differences in the CYP17 lyase activity may explain some species-dependent differences in response to chemicals that alter steroidogenesis in vivo (6).
Takvim razlikama u aktivnosti liaze aromataze CYP17 mogu se objasniti neke razlike u reakciji na kemikalije kojima se mijenja steroidogeneza in vivo prema vrstama (6).
The H295 cells have been shown to most closely reflect human adult adrenal enzyme expression and steroid production pattern (20), but are known to express enzymes for both the Δ5-hydroxysteroid and Δ4-ketosteroid pathways for androgen synthesis (7) (11) (13) (15).
Dokazano je da su stanice H295 najsličnije adrenalnoj ekspresiji enzima i uzorku proizvodnje steroida u odraslih ljudi (20), ali je poznato da one izražavaju enzime za putove Δ5-hidroksisteroida i Δ4-ketosteroida za sintezu androgena (7) (11) (13) (15).
Steroidogenic pathway in H295R cells.
Steroidogenski put u stanicama H295R
Enzymes are in italics, hormones are bolded and arrows indicate the direction of synthesis.
Enzimi su u kurzivu, hormoni su podebljani, a strelice ukazuju na smjer sinteze.
Gray background indicates corticosteroid pathways/products.
Siva pozadina predstavlja putove/proizvode kortikosteroida.
Sex steroid pathways/products are circled.
Putovi/proizvodi spolnih steroida su zaokruženi.
CYP = cytochrome P450;
CYP = citokrom P450;
HSD = hydroxysteroid dehydrogenase;
HSD = hidroksisteroid-dehidrogenaza;
DHEA = dehydroepiandrosterone.
DHEA = dehidroepiandrosteron.
The human H295R adreno-carcinoma cell line is a useful in vitro model for the investigation of effects on steroid hormone synthesis (2) (7) (8) (9) (10).
Stanična linija humanog adrenokarcinoma (H295R) koristan je model in vitro za ispitivanje učinaka na sintezu steroidnih hormona (2) (7) (8) (9) (10).
The H295R cell line expresses genes that encode for all the key enzymes for steroidogenesis noted above (11) (15) (Figure 1).
Stanična linija H295R izražava gene koji kodiraju sve gore navedene ključne enzime za steroidogenezu (11) (15) (slika 1.).
This is a unique property because in vivo expression of these genes is tissue and developmental stage-specific with typically no one tissue or one developmental stage expressing all of the genes involved in steroidogenesis (2).
To je jedinstveno obilježje jer je ekspresija tih gena in vivo specifična za tkivo i razvojnu fazu, uz u pravilu više tkiva i razvojnih faza koji izražavaju sve gene uključene u steroidogenezu (2).
H295R cells have physiological characteristics of zonally undifferentiated human foetal adrenal cells (11).
Stanice H295R odlikuju se fiziološkim karakteristikama zonski nediferenciranih humanih fetalnih adrenalnih stanica (11).
The cells represent a unique in vitro system in that they have the ability to produce all of the steroid hormones found in the adult adrenal cortex and the gonads, allowing testing for effects on both corticosteroid synthesis and the production of sex steroid hormones such as androgens and oestrogens, although the assay was validated only to detect T and E2.
Stanice predstavljaju jedinstveni sustav in vitro s obzirom na to da se odlikuju sposobnošću proizvodnje svih steroidnih hormona koji se nalaze u odraslome adrenalnom korteksu i gonadama, čime se omogućava ispitivanje učinaka na sintezi kortikosteroida i proizvodnju spolnih steroidnih hormona poput androgena i estrogena, iako je test validiran samo za otkrivanje hormona T i E2.
Changes recorded by the test system in the form of alteration in the production of T and E2 can be the result of a multitude of different interactions of test chemicals with steroidogenic functions that are expressed by the H295R cells.
Promjene zabilježene ispitnim sustavom u obliku promjene u proizvodnji hormona T i E2 mogu biti posljedica brojnih interakcija ispitivanih kemikalija i steroidogenih funkcija izraženih stanicama H295R.
These include modulation of the expression, synthesis or function of enzymes involved in the production, transformation, or elimination of steroid hormones (12) (13) (14).
One uključuju modulaciju ekspresije, sintezu ili funkciju enzima uključenih i proizvodnju, transformaciju ili eliminaciju steroidnih hormona (12) (13) (14).
Inhibition of hormone production can be due to direct competitive binding to an enzyme in the pathway, impact on co-factors such as NADPH (Nicotinamide Adenine Dinucleotide Phosphate) and cAMP (cyclic Adenosine Monophosphate), and/or increase in steroid metabolism or suppression of gene expression of certain enzymes in the steroidogenesis pathway.
Razlozi inhibicije proizvodnje hormona mogu biti izravno konkurentsko povezivanje s enzimom na putu, učinak na sučimbenike poput NADPH-a (nikotinamid adenin dinukleotid fosfat) i cAMP-a (ciklički adenozin monofosfat) i/ili porast metabolizma steroida ili suzbijanje ekspresije gena određenih enzima na putu steroidogeneze.
While inhibition can be a function of both direct or indirect processes involved with hormone production, induction is typically of an indirect nature, such as by affecting co-factors such as NADPH and cAMP (as in the case of forskolin), decreasing steroid metabolism (13), and or up-regulating steroidogenic gene expression.
Iako inhibicija može biti funkcija izravnih ili neizravnih postupaka uključenih u proizvodnju hormona, indukcija je u pravilu neizravnog karaktera, poput utjecaja na sučimbenike poput NADPH-a i cAMP-a (kao u slučaju forskolina), smanjenja metabolizma steroida (13) i/ili pospješivanja steroidogenske ekspresije gena.
The H295R assay has several advantages:
Test H295R ima nekoliko prednosti:
It allows for the detection of both increases and decreases in the production of both T and E2;
njime se omogućava otkrivanje povećanja i smanjenja proizvodnje hormona T i E2,
It permits the direct assessment of the potential impact of a chemical on cell viability/cytotoxicity.
njime se dopušta izravna procjena potencijalnog učinka kemikalije na vitalnost/citotoksičnost stanice.
This is an important feature as it allows for the discrimination between effects that are due to cytotoxicity from those due to the direct interaction of chemicals with steroidogenic pathways, which is not possible in tissue explants systems that consist of multiple cell types of varying sensitivities and functionalities;
To je važno obilježje jer se njime omogućava razlikovanje učinaka koji su posljedica citotoksičnosti i onih koji su posljedica izravne interakcije kemikalija i steroidogenskih putova, što nije moguće u sustavima u kojima se upotrebljavaju eksplantati tkiva koji se sastoje od brojnih vrsta stanica različitih osjetljivosti i funkcionalnosti,
It does not require the use of animals;
za njegovo provođenje nisu potrebne životinje,
The H295R cell line is commercially available.
stanična linija H295R komercijalno je dostupna.
The principle limitations of the assay are as follows:
Načelna su ograničenja testa kako slijedi:
Its metabolic capability is unknown but probably quite limited;
nepoznat je njegov metabolički kapacitet, ali je vjerojatno vrlo ograničen.
therefore, chemicals that need to be metabolically activated will probably be missed in this assay.
Stoga će kemikalije koje treba metabolički aktivirati vjerojatno biti izostavljene iz ovog testa.
Being derived from adrenal tissue, the H295R possesses the enzymes capable of producing the gluco-, and mineral-corticoids as well as the sex hormones;
s obzirom na to da nastaje iz adrenalnog tkiva, H295R se dolikuje enzimima sposobnima za proizvodnju glukokortikoida i mineralnih kortikoida, kao i spolnih hormona.
therefore, effects on the production of gluco-, and mineral corticoids could influence the levels of T and E2 observed in the assay.
Stoga bi učinci na proizvodnju glukokortikoida i mineralnih kortikoida mogli utjecati na razine hormona T i E2 uočene u testu,
It does not measure DHT and, therefore, would not be expected to detect chemicals that inhibit 5α-reductase in which case the Hershberger assay (16) can be used.
njime se ne mjeri DHT i zato se ne očekuje da se njime otkriju kemikalije koje inhibiraju 5α-reduktazu, za što se može upotrebljavati Hershbergerov test (16),
The H295R assay will not detect chemicals that interfere with steroidogenesis by affecting the hypothalamic-pituitary-gonadal axis (HPG) axis as this can only be studied in intact animals.
testom H295R neće se otkriti kemikalije koje ometaju steroidogenezu tako da utječu na os hipotalamus-hipofiza-gonada jer se nju može ispitivati samo na netaknutim životinjama.
The purpose of the assay is the detection of chemicals that affect T and E2 production.
Namjena je testa otkrivanje kemikalija koje utječu na proizvodnju hormona T i E2.
T is also an intermediate in the pathway to produce E2.
Testosteron je i hormon posrednik na putu proizvodnje hormona E2.
The assay can detect chemicals that typically inhibit or induce the enzymes of the steroidogenesis pathway.
Testom se mogu otkriti kemikalije kojima se u pravilu inhibiraju ili induciraju enzimi puta steroidogeneze.
The assay is usually performed under standard cell culture conditions in 24-well culture plates.
Test se obično provodi u standardnim uvjetima stanične kulture na pločicama za uzgoj kulture s 24 jažice.
Alternatively, other plate sizes can be used for conducting the assay;
Alternativno, za provođenje testa mogu se upotrebljavati i druge veličine pločica.
however, seeding and experimental conditions should be adjusted accordingly to maintain adherence to the performance criteria.
Međutim, uvjete razmnožavanja i pokusa treba odgovarajuće prilagoditi kako bi se kriteriji učinka i dalje primjenjivali.
After an acclimation period of 24 h in multi-well plates, cells are exposed for 48 h to seven concentrations of the test chemical in at least triplicate.
Nakon razdoblja aklimatizacije u trajanju od 24 sata na pločicama s višestrukim jažicama, stranice se 48 sati izlaže sedam koncentracija ispitivane kemikalije u najmanje tri primjerka.
Solvent and a known inhibitor and inducer of hormone production are run at a fixed concentration as negative and positive controls.
Otapalo i poznati inhibitor i induktor proizvodnje hormona dodaju se u fiksnoj koncentraciji kao negativne i pozitivne kontrole.
At the end of the exposure period, the medium is removed from each well.
Na kraju razdoblja izlaganja iz svake se jažice uklanja medij.
Cell viability in each well is analysed immediately after removal of medium.
Vitalnost stanica u svakoj jažici analizira se neposredno nakon uklanjanja medija.
Concentrations of hormones in the medium can be measured using a variety of methods including commercially available hormone measurement kits and/or instrumental techniques such as liquid chromatography-mass spectrometry (LC-MS).
Koncentracije hormona u mediju moguće je izmjeriti raznim metodama, uključujući komercijalno dostupne komplete za mjerenje hormona i/ili instrumentalnim tehnikama poput masene spektrometrije s tekućinskom kromatografijom (LC-MS).
Data are expressed as fold change relative to the solvent control and the Lowest-Observed-Effect-Concentration (LOEC).
Podaci su izraženi kao promjena omjera u odnosu na kontrolu otapala i najnižu koncentraciju s vidljivim učinkom (LOEC).
If the assay is negative, the highest concentration tested is reported as the No-Observed-Effect-Concentration (NOEC).
Ako je test negativan, najviša ispitivana koncentracija navodi se kao koncentracija bez vidljivog učinka (NOEC).
Conclusions regarding the ability of a chemical to affect steroidogenesis should be based on at least two independent test runs.
Zaključke o sposobnosti kemikalije da utječe na steroidogenezu treba temeljiti na najmanje dva neovisna ispitivanja.
The first test run may function as a range finding run with subsequent adjustment of concentrations for runs 2 and 3, if applicable, if solubility or cytotoxicity problems are encountered or the activity of the chemical seems to be at the end of the range of concentrations tested.
Prvo ispitivanje može služiti za utvrđivanje raspona, uz naknadnu prilagodbu koncentracija za drugo i treće ispitivanje ako dođe do problema radi topljivosti ili citotoksičnosti ili ako se čini da je djelovanje kemikalije pri kraju raspona ispitivanih koncentracija.
CULTURE PROCEDURE
POSTUPAK U POGLEDU KULTURA
Cell Line
Stanična linija
The NCI-H295R cells are commercially available from the American Type Culture Collections (ATCC) upon signing a Material Transfer Agreement (MTA) [14].
Stanice NCI-H295R mogu se kupiti od centra American Type Culture Collections (ATCC) nakon potpisa ugovora o prijenosu materijala [14].
Due to changes in the E2 producing capacity of the cells with increasing age/passages (2), cells should be cultured following a specific protocol before they are used and the number of passages since the cells were defrosted as well as the passage number at which the cells were frozen and placed in liquid nitrogen storage should be noted.
Zbog promjena kapaciteta za proizvodnju hormona E2 stanica zbog starenja/prolaza (2), stranice prije upotrebe treba kultivirati u skladu s posebnim protokolom te treba zabilježiti broj prolaza od odmrzavanja stanica, kao i broj prolaza kod zamrzavanja stanica i njihova smještaja u spremnik s tekućim dušikom.
The first number indicates the actual cell passage number and the second number describes the passage number at which the cells were frozen and placed in storage.
Prvi broj odnosi se na stvarni broj staničnih prolaza, dok se drugi odnosi na broj prolaza kod zamrzavanja stanica i njihova smještaja u spremnik.
For example, cells that were frozen after passage five and defrosted and then were split three times (4 passages counting the freshly thawed cells as passage 1) after they were cultured again would be labelled passage 4.5.
Primjerice, stanice koje su smrznute nakon petog prolaza i odmrznute te potom podijeljene tri puta (četiri prolaza, pri čemu su svježe odmrznute stanice prvi prolaz) nakon ponovne kultivacije imale bi oznaku prolaz 4,5.
An example of a numbering scheme is illustrated in Appendix I to the validation report (4).
Primjer sheme za označivanje brojevima opisuje se u Dodatku 1. izvješću o validaciji (4).
Stock medium is used as the base for the supplemented and freezing mediums.
Osnovni se medij upotrebljava kao osnova za dopunjene medije i medije za zamrzavanje.
Supplemented medium is a necessary component for culturing cells.
Dopunjeni je medij nužna komponenta za kultivaciju stanica.
Freezing medium is specifically designed to allow for impact-free freezing of cells for long-term storage.
Medij za zamrzavanje posebno je namijenjen zamrzavanju stranica bez posljedica radi dugotrajnog čuvanja.
Prior to use, Nu-serum (or a comparable serum of equal properties that has been demonstrated to produce data that meets the test performance and Quality Control (QC) requirements), which is a constituent of supplemented media, should be analysed for background T and E2 concentrations.
Prije upotrebe treba analizirati nu-serum (ili usporedivi serum jednakih svojstava za koji je dokazano da daje podatke u skladu sa zahtjevima u pogledu provođenja ispitivanja i kontrole kvalitete), koji je sastavni dio dopunjenih medija, radi osnovnih koncentracija hormona T i E2.
The preparation of these solutions is described in Appendix II to the validation report (4).
Priprema tih otopina opisuje se u Dodatku 1. izvješću o validaciji (4).
After initiation of an H295R cell culture from an original ATCC batch, cells should be grown for five passages (i.e.the cells are split 4 times).
Nakon inicijacije stanične kulture H295R iz prvotne ATCC-ove serije, stanice treba uzgajati za pet prolaza (tj. stanice se dijele 4 puta).
Passage five cells are then frozen in liquid nitrogen for storage.
Stanice petog prolaza potom se zamrzavaju u spremniku s tekućim dušikom.
Prior to freezing the cells, a sample of the previous passage four cells is run in a QC plate (See paragraph 36 and 37) to verify whether the basal production of hormones and the response to positive control chemicals meet the assay quality control criteria as defined in Table 5.
Prije zamrzavanja stanica uzorak prethodnih stanica četvrtog prolaza stavlja se na pločicu za kontrolu kvalitete (vidjeti stavke 36. i 37.) kako bi se potvrdilo jesu li osnovna proizvodnja hormona i reakcija na pozitivne kontrolne kemikalije u skladu s kriterijima kontrole testa kako su definirani u tablici 5.
H295R cells need to be cultured, frozen and stored in liquid nitrogen to make sure that there are always cells of the appropriate passage/age available for culture and use.
Stanice H295R treba kultivirati, zamrznuti i pohraniti u tekućem dušiku kako bi se osiguralo da su one uvijek stanice odgovarajućeg prolaza/starosti za kultivaciju i upotrebu.
The maximum number of passages after taking a new [15] or frozen [16] batch of cells into culture that is acceptable for use in the H295R assay should not exceed 10.
Najveći broj prolaza nakon uzimanja nove [15] ili zamrznute [16] serije stanica u kulturu koji je prihvatljiv za upotrebu u testu H295R ne smije premašiti deset.
For example, acceptable passages for cultures of cells from a batch frozen at passage 5 would be 4.5 through 10.5.
Primjerice, prihvatljivi bi prolazi za stanične kulture iz serije zamrznute kod petog prolaza bili 4,5 do 10,5.
For cells started from these frozen batches, the procedure described in paragraph 19 should be followed.
Za stanice započete iz tih zamrznutih serija treba slijediti postupak iz stavka 19.
These cells should be cultured for at least four (4) additional passages (passage 4.5) prior to their use in testing.Starting Cells from the Frozen Stock
Te stanice treba kultivirati za najmanje četiri (4) dodatna prolaza (prolaz 4,5) prije njihove upotrebe u ispitivanju.Započinjanje stanica iz zamrznute zalihe
The procedure for starting the cells from frozen stock is to be used when a new batch of cells is removed from liquid nitrogen storage for the purpose of culture and testing.
Postupak za započinjanje stanica iz zamrznute zalihe treba upotrebljavati kad se nova serija stanica vadi iz spremnika s tekućim dušikom radi kultivacije i ispitivanja.
Details for this procedure are set forth in Appendix III to the validation report (4).
Pojedinosti o ovom postupku navedene su u Dodatku III. izvješću o validaciji (4).
Cells are removed from liquid nitrogen storage, thawed rapidly, placed in supplemented medium in a centrifuge tube, centrifuged at room temperature, re-suspended in supplemented medium, and transferred to a culture flask.
Stanice se vade iz spremnika s tekućim dušikom, ubrzano odmrzavaju, smještaju u dopunjeni medij u kivetu za centrifugiranje, centrifugiraju na sobnoj temperaturi, ponovno suspendiraju u dopunjenom mediju te premještaju u tikvicu s kulturom.
The medium should be changed the following day.
Medij treba promijeniti sljedećeg dana.
The H295R cells are cultivated in an incubator at 37 °C with 5 % CO2 in air atmosphere and the medium is renewed 2-3 times per week.
Stanice H295R kultiviraju se u inkubatoru na 37 °C s 5 %-tnim CO2 u zraku, a medij se obnavlja 2 do 3 puta tjedno.
When the cells are approximately 85-90 % confluent, they should be split.
Kad su stanice približno 85 – 90 % konfluentne, treba ih podijeliti.
Splitting of the cells is necessary to ensure the health and growth of the cells and to maintain cells for performing bioassays.
Dijeljenje stanica nužno je radi osiguranja zdravlja i rasta stanica te čuvanja stanica za provođenje biotestova.
The cells are rinsed three times with phosphate-buffered saline (PBS, without Ca2+ Mg2+.) and freed from the culture flask by the addition of an appropriate detachment enzyme, e.g. trypsin, in PBS (without Ca2+ Mg2+).
Stanice se tri puta ispiru fiziološkom otopinom s fosfatnim pulferom (bez Ca2+ Mg2+) i oslobađaju iz tikvice s kulturom dodavanjem odgovarajućeg enzima za odvajanje, npr. tripsina u fiziološku otopinu s fosfatnim pulferom (bez Ca2+ Mg2+).
Immediately after the cells detach from the culture flask, the enzyme action should be stopped with the addition of supplemented medium at a ratio of 3× the volume used for the enzyme treatment.
Neposredno nakon odvajanja stanica od tikvice s kulturom treba zaustaviti djelovanje enzima dodavanjem dopunjenog medija u omjeru trostrukog volumena upotrijebljenog za tretman enzimom.
Cells are placed into a centrifuge tube, centrifuged at room temperature, the supernatant is removed and the pellet of cells is re-suspended in supplemented medium.
Stanice se stavljaju u kivetu za centrifugiranje, centrifugiraju se na sobnoj temperaturi, uklanja se supernatant te se stanični talog ponovno suspendira u dopunjenom mediju.
The appropriate amount of cell solution is placed in the new culture flask.
Odgovarajuća količina otopine stanica stavlja se u novu tikvicu s kulturom.
The amount of cell solution should be adjusted so that the cells are confluent within 5-7 days.
Količinu otopine stanica treba prilagoditi tako da su stanice konfluentne u roku od 5 do 7 dana.
The recommended sub-cultivation ratio is 1:3 to 1:4.
Preporučeni je omjer potkultivacije 1:3 do 1:4.
The plate should be carefully labelled.
Pločicu treba pažljivo označiti.
The cells are now ready to be used in the assay and excess cells should be frozen in liquid nitrogen as described in paragraph 20.
Stanice su sada spremne za upotrebu u testu, a višak stanica treba zamrznuti u tekućem dušiku kako je opisano u stavku 20.
Freezing H295R Cells (preparing cells for liquid nitrogen storage)
Zamrzavanje stanica H295R (priprema stanica za čuvanje u tekućem dušiku)
To prepare H295R cells for freezing, the procedure described above for splitting cells should be followed until the step for re-suspending the pellet of cells in the bottom of the centrifuge tube.
Kod pripreme stanica H295R za zamrzavanje treba slijediti gore opisani postupak za dijeljenje stanica do koraka za ponovnu suspenziju staničnog taloga s dna kivete za centrifugiranje.
Here, the pellet of cells is re-suspended in freezing medium.
Ovdje se stanični talog ponovno suspendira u mediju za zamrzavanje.
The solution is transferred to a cryogenic vial, labelled appropriately, and frozen at – 80 °C for 24 hours after which the cryogenic vial is transferred to liquid nitrogen for storage.
Otopina se premješta u kriogenu posudu, odgovarajuće označava te 24 sata zamrzava na – 80 °C, nakon čega se kriogena posuda stavlja u spremnik s tekućim dušikom.
Plating and Pre-incubation of Cells for Testing
Nanošenje na pločice i preinkubacija stanica za ispitivanje
The number of 24-well plates, prepared as outlined in paragraph 19, that will be needed depends on the number of chemicals to be tested and the confluency of the cells in the culture dishes.
Broj potrebnih pločica s 24 jažice pripremljenih kako se navodi u stavku 19. ovisi o broju kemikalija koje treba ispitati i konfluenciji stanica u posudama s kulturom.