PMID- 36799572
OWN - NLM
STAT- MEDLINE
DCOM- 20230222
LR  - 20240202
IS  - 2691-1299 (Electronic)
IS  - 2691-1299 (Linking)
VI  - 3
IP  - 2
DP  - 2023 Feb
TI  - Evaluating the Impact of Physiologically Relevant Oxygen Tensions on Drug 
      Metabolism in 3D Hepatocyte Cultures in Paper Scaffolds.
PG  - e662
LID - 10.1002/cpz1.662 [doi]
AB  - Oxygen is an essential regulator of cellular function and phenotype. Despite its 
      importance, the incorporation of physiologically relevant oxygen tensions is 
      often overlooked in experimental setups. Ambient oxygen tensions (pO(2)  approximately 152 
      mmHg) are significantly higher than those in the alveolar-capillary barrier of 
      the lung, which is the most oxygen-rich interface in the body (pO(2)  approximately 104 mmHg). 
      The discrepancy between standard culture practices and physiologically relevant 
      oxygen tensions is more pronounced when considering the hepatocyte-lined 
      sinusoids of the liver, whose pO(2) values range from 65 mm Hg in the periportal 
      region to 30 mm Hg in the perivenous region. Our previous work highlights the 
      need to transition from standard culture conditions to more physiologically 
      relevant microenvironments when predicting hepatocyte responses to drug 
      candidates or potential toxins. This protocol details an experimental pipeline 
      for quantifying differences in transcript levels, protein levels, and activity of 
      the cytochrome P450 1A (CYP1A) enzyme family in hepatocytes maintained in a 
      three-dimensional environment at ambient and physiologically relevant oxygen 
      tensions. We quantify changes in transcript with qRT-PCR, protein expression with 
      western blots, and activity with the ethoxyresorufin-O-deethylase (EROD) assay. 
      This approach can be adapted to any drug-metabolizing enzyme. (c) 2023 Wiley 
      Periodicals LLC. Basic Protocol 1: Preparing tissue-like environments to evaluate 
      HepG2 cells in paper-based cell culture platform at physiological oxygen levels 
      Basic Protocol 2: Evaluating CYP1A activity of hepatocytes grown in the paper 
      scaffolds using the EROD assay Basic Protocol 3: Evaluating CYP1A transcript 
      levels of hepatocytes grown in the paper scaffolds using RT-qPCR Basic Protocol 
      4: Evaluating CYP1A protein levels of hepatocytes grown in the paper scaffolds 
      using western blotting.
CI  - (c) 2023 Wiley Periodicals LLC.
FAU - Sitte, Zachary R
AU  - Sitte ZR
AD  - Department of Chemistry, University of North Carolina at Chapel Hill, Chapel 
      Hill, North Carolina.
FAU - DiProspero, Thomas J
AU  - DiProspero TJ
AD  - Department of Chemistry, University of North Carolina at Chapel Hill, Chapel 
      Hill, North Carolina.
FAU - Lockett, Matthew R
AU  - Lockett MR
AD  - Department of Chemistry, University of North Carolina at Chapel Hill, Chapel 
      Hill, North Carolina.
AD  - Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel 
      Hill, Chapel Hill, North Carolina.
LA  - eng
GR  - P30 ES010126/ES/NIEHS NIH HHS/United States
GR  - R35 GM128697/GM/NIGMS NIH HHS/United States
GR  - R35GM128697/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PL  - United States
TA  - Curr Protoc
JT  - Current protocols
JID - 101773894
RN  - EC 1.14.14.1 (Cytochrome P-450 CYP1A1)
RN  - 9035-51-2 (Cytochrome P-450 Enzyme System)
RN  - S88TT14065 (Oxygen)
SB  - IM
EIN - Curr Protoc. 2023 Apr;3(4):e756. PMID: 37014791
MH  - *Cytochrome P-450 CYP1A1/genetics/metabolism
MH  - *Hepatocytes/metabolism
MH  - Cytochrome P-450 Enzyme System/metabolism
MH  - Liver/metabolism
MH  - Oxygen/metabolism
PMC - PMC10249672
MID - NIHMS1861001
OTO - NOTNLM
OT  - 3D cell culture
OT  - RT-qPCR
OT  - cytochrome P450
OT  - hepatocyte
OT  - oxygen
OT  - western blot
COIS- CONFLICT OF INTEREST STATEMENT: The authors have no conflict of interest to 
      declare.
EDAT- 2023/02/18 06:00
MHDA- 2023/02/22 06:00
PMCR- 2024/02/01
CRDT- 2023/02/17 08:53
PHST- 2023/02/17 08:53 [entrez]
PHST- 2023/02/18 06:00 [pubmed]
PHST- 2023/02/22 06:00 [medline]
PHST- 2024/02/01 00:00 [pmc-release]
AID - 10.1002/cpz1.662 [doi]
PST - ppublish
SO  - Curr Protoc. 2023 Feb;3(2):e662. doi: 10.1002/cpz1.662.