Evaluation of the HC-04 Cell Line as an In Vitro Model for Mechanistic Assessment of Changes in Hepatic Cytochrome P450 3A during Adenovirus Infection

Abstract

HC-04 cells were evaluated as an in vitro model for mechanistic study of changes in the function of hepatic CYP3A during virus infection. Similar to in vivo observations, infection with a first generation recombinant adenovirus significantly inhibited CYP3A4 catalytic activity in an isoform-specific manner. Virus (MOI 100) significantly reduced expression of the retinoid X receptor (RXR) by 30% 96 hours after infection. Cytoplasmic concentrations of the pregnane X receptor (PXR) were reduced by 50%, whereas the amount of the constitutive androstane receptor (CAR) in the nuclear fraction doubled with respect to uninfected controls. Hepatocyte nuclear factor 4a (HNF-4a) and peroxisome proliferator-activated receptor g coactivator 1a (PGC-1a) were also reduced by ∼70% during infection. Virus suppressed CYP3A4 activity in the presence of the PXR agonist rifampicin and did not affect CYP3A4 activity in the presence of the CAR agonist CITCO [6-(4-chlorophenyl) imidazosuggesting that virus-induced modification of PXR may be responsible for observed changes in hepatic CYP3A4. The HC-04 cell line is easy to maintain, and CYP3A4 in these cells was responsive to known inducers and suppressors. Dexamethasone (200 mM) and phenobarbital (500 mM) increased activity by 230 and 124%, whereas ketoconazole (10 mM) and lipopolysaccharide (LPS) (10 mg/ml) reduced activity by 90 and 92%, respectively. This suggests that HC-04 cells can be a valuable tool for mechanistic study of drug metabolism during infection and for routine toxicological screening of novel compounds prior to use in the clinic.

Integrin Receptors Play a Key Role in the Regulation of Hepatic CYP3A

“…CYP3A11 mRNA levels were unaffected by AdDRGD throughout the study period. The observation that CYP3A activity was suppressed to a greater extent than protein and gene expression after infection with AdlacZ is not unique to these studies because it has previously been seen in vitro and could be attributable to post-translational modifications of the CYP3A enzyme (Wonganan et al, 2014).…”

“…On a transcriptional level, hepatic CYP3A is constitutively expressed in the liver, but several nuclear receptors, including PXR, CAR, and RXR, are primarily responsible for regulating CYP3A gene expression (Croyle, 2009). However, it remains to be known which is predominantly influenced by virus infection and is ultimately responsible for our observed changes in CYP3A (Callahan et al, 2008a;Wonganan et al, 2014). In an effort to determine how integrin engagement changes each of these key regulators of CYP3A in vivo, gene and protein expression was determined 24 and 48 hours after infection with each virus (Fig.…”

“…A secondary goal of these studies was to identify specific integrin receptor subunits responsible for changes in CYP3A. In vitro assessment of CYP3A activity and expression after knockdown of various a-and b-subunits of integrin receptors in a novel human-derived hepatocyte cell line (Wonganan et al, 2014) allowed us to prioritize the role that each plays in the regulation of CYP3A and several transcription factors known to moderate this enzyme. To our knowledge, this is the first report describing a role for these ubiquitous receptors in the regulation of drug metabolism in vivo and in human hepatocytes.…”

The Role of CYP3A in Health and Disease

“…Some clinical research indicates that inhibitors of IL-6 enhance drug metabolism via CYP3A4, implying that IL-6 is an important regulator of this enzyme [ 396 ]. It has been demonstrated that CYP3A4 activity is suppressed in adenovirus-infected primary hepatocytes and that adenovirus-induced modification of PXR expression may be responsible for the alterations of CYP3A4 activity in the liver [ 397 ].…”

The Role of Cytochrome P450 Enzymes in COVID-19 Pathogenesis and Therapy

“…CYP1A1 activity was suppressed by 75% in coxsackievirus B3 infected mice (Funseth et al, 2002). CYP3A4 activity was suppressed in primary hepatocytes infected with adenovirus, and adenovirus-induced modification of PXR may be responsible for changes in hepatic CYP3A4 activity (Wonganan et al, 2014). CYP2A5 and CYP3A expression increased in hepatitis B virus (HBV)transgenic mice (Kirby et al, 1994), whilst CYP2D6 expression decreased in hepatitis C virus (HCV) infected mice (Kikuchi et al, 2010).…”