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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

Expression levels of genes related to ABC efflux transporters 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 (n = 5 fish).
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fig1: Expression levels of genes related to ABC efflux transporters 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 (n = 5 fish).

Mentions: Differences in transcript levels of the transporters of interest (MDR1, BSEP, MRP2 and BCRP) were seen in the different hepatocyte cultures assessed (freshly isolated suspensions, monolayer cultures 24 and 48 hours post isolation and spheroid cultures 5, 7, 10, 15 and 25 days post isolation) (Fig. 1). Expression levels of BSEP and BCRP in monolayer culture were lower than those measured in freshly isolated hepatocytes and, following a recovery in early spheroid culture, an increase in expression was evident in mature spheroids.


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

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

Expression levels of genes related to ABC efflux transporters 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 (n = 5 fish).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Expression levels of genes related to ABC efflux transporters 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 (n = 5 fish).
Mentions: Differences in transcript levels of the transporters of interest (MDR1, BSEP, MRP2 and BCRP) were seen in the different hepatocyte cultures assessed (freshly isolated suspensions, monolayer cultures 24 and 48 hours post isolation and spheroid cultures 5, 7, 10, 15 and 25 days post isolation) (Fig. 1). Expression levels of BSEP and BCRP in monolayer culture were lower than those measured in freshly isolated hepatocytes and, following a recovery in early spheroid culture, an increase in expression was evident in mature spheroids.

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