Cryopreserved Hepatocytes from Rainbow Trout (Oncorhynchus mykiss): A Validation Study to Support Their Application in Bioaccumulation Assessment

Abstract

Determination of biotransformation rates of xenobiotics in freshly isolated trout hepatocytes has been demonstrated to significantly improve the performance of bioaccumulation assessment models. In order to promote this in vitro approach, trout hepatocytes need to be cryopreserved to facilitate their availability while ensuring their metabolic competency. In the present study, we obtained basal level metabolic enzyme activities for cytochrome P450 (CYP) 1A, CYP3A, glutathione-S-transferase, and uridine 5'-diphospho-glucuronosyltransferase from trout hepatocytes cryopreserved for various periods of time up to three months and compared their values with those obtained from freshly isolated hepatocytes. Similarly, we compared intrinsic clearance (CL(int)) values determined in cryopreserved trout hepatocytes to those determined in freshly isolated hepatocytes for reference compounds molinate, michler's ketone, 4-nonylphenol, 2,4-ditert-butylphenol, benzo(a)pyrene, and pyrene. Our results show that cryopreserved trout hepatocytes maintained greater than 75% of their basal level enzyme activities and greater than 72% of xenobiotic biotransformation capabilities, regardless of the length of cryostorage. As a result, bioconcentration factors of the reference compounds were adequately predicted based on the CL(int) values. We simulated the condition for shipping cryopreserved trout hepatocytes and demonstrated that 24 h dry ice storage did not negatively affect the rates of xenobiotic biotransformation. We conclude that cryopreserved trout hepatocytes are suitable for biotransformation rate determination of xenobiotics in vitro, and therefore, are an acceptable alternative to freshly isolated trout hepatocytes in the application in bioaccumulation assessment.

Evaluation of the sensitivity, responsiveness and reproducibility of primary rainbow trout hepatocyte vitellogenin expression as a screening assay for estrogen mimics

“…chemical depletion, well format, wall sorption) in which partition-driven administration dosing may alleviate some of the problems (Booij et al, 2011;Kwon et al, 2009;Riedl and Altenburger, 2007;Schreiber et al, 2008;Smith et al, 2009). Development of continuous liver cell lines or use of cryopreserved primary hepatocytes, which retain properties of freshly isolated hepatocytes (Markell et al, 2014;Mingoia et al, 2010), may additionally reduce inter-lab and inter-assay variance to a level that accommodates suitable high-throughput screening formats (Bols et al, 2005;Tollefsen, 2012, 2011;Tollefsen et al, 2003Tollefsen et al, , 2008b.…”

Comparison of trout hepatocytes and liver S9 fractions as in vitro models for predicting hepatic clearance in fish

“…Most research in this area has been performed using isolated hepatocytes and liver S9 fractions from rainbow trout [6,9,11,12,14,15]. This primary focus on a single species reflects the availability of established methods for collecting this biological material from trout, as well as the existence of physiological and anatomical information required to develop necessary extrapolation factors.…”

Essai n° 319A : Détermination de la clairance intrinsèque in vitro sur des hépatocytes de truite arc en ciel (rt-hep) cryoconservés

“…Une viabilité élevée (> 80 %) est généralement obtenue après décongélation des RT-HEP cryoconservés, alors que le rendement (taux de récupération, c'est-à-dire nombre de RT-HEP viables obtenus après décongélation rapporté au nombre de RT-HEP viables initialement cryoconservés) est généralement de 25-45 % (Fay et al, 2014a(Fay et al, , 2014bMarkell et al, 2014 ;Mingoia et al, 2010). 1.…”

“…Plusieurs équipes de © OCDE 2018 recherche ont montré que l'intégration de données de biotransformation établies par des méthodes in vitro à des modèles à un compartiment applicables aux poissons améliore notablement les performance de ces modèles ; les niveaux d'accumulation prédits sont alors beaucoup plus proches des valeurs mesurées que les prédictions obtenues en ne tenant pas compte du métabolisme (Cowan-Ellsberry et al, 2008 ;Dyer et al, 2008Dyer et al, , 2009Han et al, 2007 ;Laue et al, 2014 ;Mingoia et al, 2010 ;Fay et al, 2014aFay et al, , 2014b. D'autres considérations relatives à la prédiction des BCF figurent dans le Document d'orientation RT-HEP et RT-S9 (OCDE, 2018a).…”

“…La truite arc-en-ciel constitue une source d'hépatocytes relativement facile à utiliser, et il a été établi que les RT-HEP se prêtent bien à la cryoconservation, avec une perte minimale de capacité de métabolisation des xénobiotiques (Mingoia et al, 2010 ;Fay et al, 2014a). Il est donc possible de congeler les RT-HEP dans un laboratoire et de les distribuer à d'autres laboratoires en vue d'un usage ultérieur, et d'utiliser le même lot pour plusieurs essais séparés dans le temps.…”