Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry

John T. Heinonen, Jaspreet S. Sidhu, Maureen T. Reilly, Federico M. Farin, Curtis John Omiecinski, David L. Eaton, Terrance J. Kavanagh

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and β-naphthoflavone (βNF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-μm thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O- dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from βNF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur.

Original languageEnglish (US)
Pages (from-to)536-543
Number of pages8
JournalEnvironmental health perspectives
Volume104
Issue number5
DOIs
StatePublished - Jan 1 1996

Fingerprint

Cytochrome P-450 CYP2B1
Cytochrome P-450 Enzyme System
Lasers
Fluorescence
Liver
Cytochrome P-450 CYP1A1
Aptitude
Isoenzymes
Dealkylation
Individuality
Perfusion
resorufin
Kidney
Lung
Messenger RNA
Enzymes

All Science Journal Classification (ASJC) codes

  • Public Health, Environmental and Occupational Health
  • Health, Toxicology and Mutagenesis

Cite this

Heinonen, J. T., Sidhu, J. S., Reilly, M. T., Farin, F. M., Omiecinski, C. J., Eaton, D. L., & Kavanagh, T. J. (1996). Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry. Environmental health perspectives, 104(5), 536-543. https://doi.org/10.1289/ehp.96104536
Heinonen, John T. ; Sidhu, Jaspreet S. ; Reilly, Maureen T. ; Farin, Federico M. ; Omiecinski, Curtis John ; Eaton, David L. ; Kavanagh, Terrance J. / Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry. In: Environmental health perspectives. 1996 ; Vol. 104, No. 5. pp. 536-543.
@article{105405efb7304c4a9e2e240185107420,
title = "Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry",
abstract = "Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and β-naphthoflavone (βNF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-μm thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O- dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from βNF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur.",
author = "Heinonen, {John T.} and Sidhu, {Jaspreet S.} and Reilly, {Maureen T.} and Farin, {Federico M.} and Omiecinski, {Curtis John} and Eaton, {David L.} and Kavanagh, {Terrance J.}",
year = "1996",
month = "1",
day = "1",
doi = "10.1289/ehp.96104536",
language = "English (US)",
volume = "104",
pages = "536--543",
journal = "Environmental Health Perspectives",
issn = "0091-6765",
publisher = "Public Health Services, US Dept of Health and Human Services",
number = "5",

}

Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry. / Heinonen, John T.; Sidhu, Jaspreet S.; Reilly, Maureen T.; Farin, Federico M.; Omiecinski, Curtis John; Eaton, David L.; Kavanagh, Terrance J.

In: Environmental health perspectives, Vol. 104, No. 5, 01.01.1996, p. 536-543.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry

AU - Heinonen, John T.

AU - Sidhu, Jaspreet S.

AU - Reilly, Maureen T.

AU - Farin, Federico M.

AU - Omiecinski, Curtis John

AU - Eaton, David L.

AU - Kavanagh, Terrance J.

PY - 1996/1/1

Y1 - 1996/1/1

N2 - Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and β-naphthoflavone (βNF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-μm thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O- dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from βNF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur.

AB - Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and β-naphthoflavone (βNF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-μm thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O- dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from βNF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur.

UR - http://www.scopus.com/inward/record.url?scp=0029891444&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029891444&partnerID=8YFLogxK

U2 - 10.1289/ehp.96104536

DO - 10.1289/ehp.96104536

M3 - Article

C2 - 8743442

AN - SCOPUS:0029891444

VL - 104

SP - 536

EP - 543

JO - Environmental Health Perspectives

JF - Environmental Health Perspectives

SN - 0091-6765

IS - 5

ER -