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Functional xenobiotic metabolism and efflux transporters in trout hepatocyte spheroid cultures.

Uchea C, Owen SF, Chipman JK - Toxicol Res (Camb) (2015)

Bottom Line: Significantly greater levels of expression of genes involved in xenobiotic metabolism and efflux were measured in spheroids (which have been shown to remain viable in excess of 30 days), compared to hepatocytes cultured using conventional suspension and monolayer culture techniques.Functionality of efflux transporters in spheroids was also demonstrated using fluorescent markers and specific inhibitors.In conclusion, the more physiologically relevant architecture in spheroid cultures provides a high functional integrity in relation to xenobiotic metabolism and efflux.

View Article: PubMed Central - PubMed

Affiliation: University of Birmingham , School of Biosciences , Birmingham , B15 2TT , UK ; AstraZeneca , Alderley Park , Macclesfield , Cheshire , SK10 4TF , UK . Email: Stewart.Owen@AstraZeneca.com.

ABSTRACT

Prediction of xenobiotic fate in fish is important for the regulatory assessment of chemicals under current legislation. Trout hepatocyte spheroids are a promising in vitro model for this assessment. In this investigation, the gene expression and function for xenobiotic metabolism and cellular efflux were characterised. Using fluorescence, transport and real time PCR analysis, the expression and functionality of a variety of genes related to xenobiotic metabolism and drug efflux were assessed in a range of trout hepatocyte culture preparations. Significantly greater levels of expression of genes involved in xenobiotic metabolism and efflux were measured in spheroids (which have been shown to remain viable in excess of 30 days), compared to hepatocytes cultured using conventional suspension and monolayer culture techniques. A transient decline in the expression of genes related to both xenobiotic metabolism and transport was determined during spheroid development, with a subsequent recovery in older spheroids. The most mature spheroids also exhibited an expression profile most comparable to that reported in vivo. Functionality of efflux transporters in spheroids was also demonstrated using fluorescent markers and specific inhibitors. In conclusion, the more physiologically relevant architecture in spheroid cultures provides a high functional integrity in relation to xenobiotic metabolism and efflux. Together with the enhanced gene expression and longevity of the model, hepatocytes in spheroid culture may prove to be an accurate alternative model to study the mechanisms of these processes in fish liver and provide an assay to determine the bioaccumulation potential of environmental contaminants.

No MeSH data available.


Related in: MedlinePlus

Relative expression levels of genes related to xenobiotic metabolism in freshly isolated hepatocytes, hepatocytes in monolayer culture (24 and 48 hours post isolation) and hepatocyte spheroids. The bar above the heat map indicates the scale of Ct values measured in cell samples with red representing the lowest extreme Ct value, therefore the greatest level of expression; blue representing the highest extreme Ct value, therefore the lowest level of expression; and black representing the median level of expression.
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fig5: Relative expression levels of genes related to xenobiotic metabolism in freshly isolated hepatocytes, hepatocytes in monolayer culture (24 and 48 hours post isolation) and hepatocyte spheroids. The bar above the heat map indicates the scale of Ct values measured in cell samples with red representing the lowest extreme Ct value, therefore the greatest level of expression; blue representing the highest extreme Ct value, therefore the lowest level of expression; and black representing the median level of expression.

Mentions: The transcript levels of CYP1A, CYP2K1, CYP2M1, CYP3A27 and UGT were also assessed and compared in the same hepatocyte preparations as used in transporter assessments. A number of gene specific changes in expression levels in different hepatocyte preparations were identified (Fig. 5). A decline in the expression of UGT, CYP2K1 and CYP2M1 was evident in early monolayer culture, followed by a recovery in late monolayer culture and early stages of spheroid culture. Expression levels of CYP1A and CYP3A27 gradually increased in this timeframe; with a sharp decline in CYP1A expression evident in spheroids 5 days post isolation. Levels of expression of the genes of interest appeared to be relatively more stable in more mature spheroids (Fig. 5).


Functional xenobiotic metabolism and efflux transporters in trout hepatocyte spheroid cultures.

Uchea C, Owen SF, Chipman JK - Toxicol Res (Camb) (2015)

Relative expression levels of genes related to xenobiotic metabolism in freshly isolated hepatocytes, hepatocytes in monolayer culture (24 and 48 hours post isolation) and hepatocyte spheroids. The bar above the heat map indicates the scale of Ct values measured in cell samples with red representing the lowest extreme Ct value, therefore the greatest level of expression; blue representing the highest extreme Ct value, therefore the lowest level of expression; and black representing the median level of expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384106&req=5

fig5: Relative expression levels of genes related to xenobiotic metabolism in freshly isolated hepatocytes, hepatocytes in monolayer culture (24 and 48 hours post isolation) and hepatocyte spheroids. The bar above the heat map indicates the scale of Ct values measured in cell samples with red representing the lowest extreme Ct value, therefore the greatest level of expression; blue representing the highest extreme Ct value, therefore the lowest level of expression; and black representing the median level of expression.
Mentions: The transcript levels of CYP1A, CYP2K1, CYP2M1, CYP3A27 and UGT were also assessed and compared in the same hepatocyte preparations as used in transporter assessments. A number of gene specific changes in expression levels in different hepatocyte preparations were identified (Fig. 5). A decline in the expression of UGT, CYP2K1 and CYP2M1 was evident in early monolayer culture, followed by a recovery in late monolayer culture and early stages of spheroid culture. Expression levels of CYP1A and CYP3A27 gradually increased in this timeframe; with a sharp decline in CYP1A expression evident in spheroids 5 days post isolation. Levels of expression of the genes of interest appeared to be relatively more stable in more mature spheroids (Fig. 5).

Bottom Line: Significantly greater levels of expression of genes involved in xenobiotic metabolism and efflux were measured in spheroids (which have been shown to remain viable in excess of 30 days), compared to hepatocytes cultured using conventional suspension and monolayer culture techniques.Functionality of efflux transporters in spheroids was also demonstrated using fluorescent markers and specific inhibitors.In conclusion, the more physiologically relevant architecture in spheroid cultures provides a high functional integrity in relation to xenobiotic metabolism and efflux.

View Article: PubMed Central - PubMed

Affiliation: University of Birmingham , School of Biosciences , Birmingham , B15 2TT , UK ; AstraZeneca , Alderley Park , Macclesfield , Cheshire , SK10 4TF , UK . Email: Stewart.Owen@AstraZeneca.com.

ABSTRACT

Prediction of xenobiotic fate in fish is important for the regulatory assessment of chemicals under current legislation. Trout hepatocyte spheroids are a promising in vitro model for this assessment. In this investigation, the gene expression and function for xenobiotic metabolism and cellular efflux were characterised. Using fluorescence, transport and real time PCR analysis, the expression and functionality of a variety of genes related to xenobiotic metabolism and drug efflux were assessed in a range of trout hepatocyte culture preparations. Significantly greater levels of expression of genes involved in xenobiotic metabolism and efflux were measured in spheroids (which have been shown to remain viable in excess of 30 days), compared to hepatocytes cultured using conventional suspension and monolayer culture techniques. A transient decline in the expression of genes related to both xenobiotic metabolism and transport was determined during spheroid development, with a subsequent recovery in older spheroids. The most mature spheroids also exhibited an expression profile most comparable to that reported in vivo. Functionality of efflux transporters in spheroids was also demonstrated using fluorescent markers and specific inhibitors. In conclusion, the more physiologically relevant architecture in spheroid cultures provides a high functional integrity in relation to xenobiotic metabolism and efflux. Together with the enhanced gene expression and longevity of the model, hepatocytes in spheroid culture may prove to be an accurate alternative model to study the mechanisms of these processes in fish liver and provide an assay to determine the bioaccumulation potential of environmental contaminants.

No MeSH data available.


Related in: MedlinePlus