Znanstvena istraživanja u 21. stoljeću - etički aspekti uporabe laboratorijskih životinja i alternativni modeli

U toksikološkim istraživanjima uz uporabu klasičnih (in vivo) istraživanja, primjenjuju se alternativni test sustavi. Korištenje laboratorijskih životinja, embrija, humanog i animalnog tkiva, kultura stanica i fetalnog seruma u istraživanjima smatra se etički problematičnim te se ograničava zakonima, pravilnicima i praksom. Razmatranjem načina kojima bi se neetičnost mogla izbjeći, došlo je do razvoja “3R” načela (akronim za tri pristupa koja bi se trebala provoditi pri istraživanjima na laboratorijskim životinjama), a to su: smanjenje/racionalizacija uporabe laboratorijskih životinja (engl. Reduction), načelo njihove zamjene (engl. Replacement) i poboljšanje uvjeta uzgoja, smještaja i skrbi za životinje (engl. Refinement). Većina je alternativnih testova toksičnosti još uvijek u postupku validacije. Pojedini in vitro testovi za istraživanja embriotoksičnosti (etički posebno osjetljivo područje) koja su priznala nadležna regulatorna tijela, su EST (engl. Embryonic Stem cell Test), WEC (engl. Whole- Embryo Culture) i MM (engl. MicroMass) test. Standardizacija protokola i uvođenje novih in vitro modela predstavlja važan segment napretka u toksikološkim istraživanjima. Znanstvena budućnost tu vidi mogućnost razvoja i implementacije načela etičnosti u istraživanja primjenjujući sustave koji će promišljeno i bez korištenja živih organizama dijelom nadomjestiti metode u biomedicini, veterinarskoj medicini, biotehnologiji i užem smislu - toksikologiji i farmakologiji.

Ionizing Radiation Effects In Vitro Study; Using The Rat Whole Embryo Culture Model

Background: Ionizing radiation poses a threat to the early embryo possibly leading to prenatal death, growth retardation, organ malformation, or mental retardation. It is important, assessment of any adverse effects of radiation upon the embryo. This study aimed to evaluate the outcomes of embryos irradiated with 1Gy doses in vitro and investigate hematopoiesis in the yolk sac of the irradiated embryos. Materials and methods: In the study, the experimental group of rats was be exposed to total body ionizing radiation on days 8.5th of gestation. All embryos in the control and radiation group cultured from gestation day 9.5 to 11.5 were alive at the end of the culture period. After 48 hours culture period, the embryos from each group were harvested and analyzed morphologically. Histological evaluation of the vWF+ cell number was performed in vivo. Results: The results showed that the embryonic growth and development during organogenesis decreased in the radiation exposed embryos when compared to control embryos. Additionally, the immunofluorescent examination showed that the vWF+ cell number reduced in the yolk sac of embryos exposed to ionizing radiation. Conclusion: Consequently, these findings support the conclusion that 1 Gy ionizing irradiation may increase prenatal death, intrauterine growth restriction on embryonic development when ionizing irradiation decreases the vWF+ cell number in the yolk sac compared to control embryos. This research related to radiation was the first study using the in vitro embryo culture technique; thus, future studies that will be performed by using different doses of radiation will contribute to the literature.

Inhibition of Acetyl-CoA Carboxylase Causes Malformations in Rats and Rabbits: Comparison of Mammalian Findings and Alternative Assays

Acetyl-CoA carboxylase (ACC) is an enzyme within the de novo lipogenesis (DNL) pathway and plays a role in regulating lipid metabolism. Pharmacologic ACC inhibition has been an area of interest for multiple potential indications including oncology, acne vulgaris, metabolic diseases such as type 2 diabetes mellitus, and nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. A critical role for ACC in de novo synthesis of long-chain fatty acids during fetal development has been demonstrated in studies in mice lacking Acc1, where the absence of Acc1 results in early embryonic lethality. Following positive predictions of developmental toxicity in the alternative in vitro assays (positive in murine embryonic stem cell [mESC] assay and rat whole embryo culture, but negative in zebrafish), developmental toxicity (growth retardation and dysmorphogenesis associated with disrupted midline fusion) was observed with the oral administration of the dual ACC1 and 2 inhibitors, PF-05175157, in Sprague Dawley rats and New Zealand White rabbits. The results of these studies are presented here to make comparisons across the assays, as well as mechanistic insights from the mESC assay demonstrating high ACC expression in the mESC and that ACC-induced developmental toxicity can be rescued with palmitic acid providing supportive evidence for DNL pathway inhibition as the underlying mechanism. Ultimately, while the battery of alternative approaches and weight-of-evidence case were useful for hazard identification, the embryo-fetal development studies were necessary to inform the risk assessment on the adverse fetal response, as malformations and/or embryo-fetal lethality were limited to doses that caused near-complete inhibition of DNL.

Teratogenic Potential of Traditionally Formulated and Nano-Encapsulated Vitamin A in Two Vertebrate Models, Rattus norvegicus and Xenopus Laevis

Nano-encapsulation is applied for the preparation of functional food to preserve micronutrients degradation and to ameliorate their absorption. Being nano-encapsulation already related to increased vitamin A embryotoxicity, we aimed to evaluate the effect of traditionally formulated (BULK-A) and nano-encapsulated vitamin A (NANO-A) in two different vertebrate models: rat post implantation Whole Embryo Culture (WEC) and Frog Embryo Teratogenesis Assay-Xenopus (FETAX). After benchmark-dose modelling, WEC results showed that NANO-A was 7 times more effective than BULK-A, while FETAX results indicated that X. laevis development was affected only by NANO-A. The relative potency of WEC was 14 times the potency of FETAX, suggesting a minor role of preformed vitamin A in X. laevis development in respect to mammal embryogenesis. Results from this work prompt the necessity to monitor the use of food supplemented with NANO A, since even low doses can elicit teratogenic effects on vertebrate embryos due to its increased bioavailability.