Flanagan et al. (2001)
reported a total loss of viability of HepG2 at 48 hr exposure to 1.24 μM OA, producing changes prior to cell death that reflect cellular responses to toxic challenge such as alterations of the actin cytoskeleton. The studies presented here demonstrated toxic effects of OA at 50 nM. At OA concentrations below 25 nM for 48 hr, HepG2 cells proliferated at 50–80% the rate of controls, but at higher concentrations more negative effects of OA such as pronounced cell rounding and detachment were observed. These effects culminated in nearly complete cell detachment from the substrate at concentrations above approximately 50 nM OA in a process that included some apoptosis, as previously shown for OA (Valdiglesias et al 2011c
; Fujiki and Suganuma 1993
; Rossini et al. 2001
). The similarity in slopes of the concentration-response relationships for detachment and proliferation suggest that cessation of proliferation and cell detachment are linked in a continuum of damage with increasing OA concentration. In addition, greater DNA laddering in detached fractions suggested greater apoptosis in detached cells.
The effects of OA on HepG2 cells appear to be independent of its metabolism via induction of phase 1 enzymes such as the CYP which is the route by which many carcinogens act (Perkinson 1996
). CYP3A transforms several procarcinogens to their carcinogenic form (Medina-Diaz and Elizondo 2005
; Perera 2010
). Although Hashizume et al. (2009)
demonstrated an increased tolerance to OA in HepG2 cells transformed to express CYP1A2. Guo et al. (2010)
noted that among 9 human CYP tested including CYP1A2, only CYP3A4 and 3A5 metabolize OA. Therefore it is likely that OA did not induce CYP expression in HepG2 cells and OA toxicity in HepG2 cells may be attributed entirely to OA itself and not to its metabolites. Several lines of evidence lead us to this conclusion.
HepG2 cells exhibited low or undetectable levels of CYP3A4 or 3A5 activity in 2 studies (Yoshitomi et al. 2001
; Omasa et al. 2005
), with no CYP mRNA transcript levels detected in the former study and the latter study demonstrating only 0.6 pmol/min/mg testosterone 6β hydroxylation by HepG2 cells. With the single exception of o, p
-DDT (Medina-Diaz and Elizondo 2005
), there is little evidence that most CYPs are more than minimally induced in HepG2 cells by the introduction of xenobiotics (Yoshitomi et al. 2001
; Wilkening et al. 2003
). OA was shown to be converted to 4 metabolites by human cytochrome P450 3A4 and 3A5 (Guo et al. 2010
). However, incubation of OA with HepG2 cells failed to produce any of these metabolites. Testosterone metabolism, which is dependent on CYP3A4, was also not observed. Finally, mRNA expression of all members of the (CYP3A) family was low.
Valdiglesias and colleagues (2011a
) studied OA toxicity in the range 0.005–1 μM in different preparations including HepG2 cells to demonstrate differences in OA effects in different human cell types. Caspase 3-dependent apoptosis occurred in HepG2 cells exposed to OA for 3 hr without evidence of necrosis (Valdiglesias et al 2011c
). A marker of γH2AX phosphorylation denoting DNA damage in chromatin domains near DNA double strand breaks (Andrievski and Wilkins 2009
) was highest in HepG2 of the 3 cell types tested (Valdiglesias et al 2011b
). In the comet assay, OA showed no marked oxidative damage to DNA in HepG2 cells (Valdiglesias et al 2011a
). OA-induced DNA damage in HepG2 cells over a 48 hr exposure period was suggested by both apoptosis and necrosis in the detached cell fraction. DNA damage induced by CYP-dependent conversion of OA to its metabolites may not have been detected in these experiments due to absence of CYP expression in HepG2 cells. The lack of CYP expression is considered a shortcoming of the HepG2 cell line for genotoxicological testing studies. Guillouzo et al. (2007)
developed a human liver-derived cell line, HepaRG, which is inducible for many CYP. It is possible that HepaRG might distinguish DNA damage induced by OA metabolites from the cell death induced by OA as documented here.
Several studies on gene expression in humans have demonstrated diverse actions of OA on cell processes. Chin et al (2000)
used differential display in human glioma model T89G cells to show OA-induced upregulation of transcription factors, oxidative metabolism proteins, phosphorylation substrates, and stress response genes after 2.5 hr exposures. Valdiglesias et al (2012)
used subtractive hybridization in the human neuroblastoma cell line SHSY5Y to show OA induced up and down gene regulation depending on the time of exposure from 2–48 hr with the affected genes playing roles in translation, transcription, signal transduction, metabolism, cell cycle control and apoptosis, transport, and cytoskeletal processes. The study of OA-mediated concentration effects adds to these insights. More genes were significantly different from the controls at OA concentrations of 37–200 nM than at low or high concentrations, with twice as many genes having curvilinear rather than linear responses. Expression of many genes peaked at intermediate concentrations. Most of these gene expression responses involved cell cycle and secretion or extracellular matrix genes. The array data showed specific cell cycle genes were downregulated at moderate OA concentrations consistent with the significant decrease in cell proliferation observed, while dozens more were upregulated at these same OA concentrations. Cell cycle inhibition at G0/G1 in HepG2 cells during short exposures to OA, with concomitant decreased expression of cyclins A2 and B1 and increased expression of cyclin D has been observed (Rubiolo et al. 2011
; Valdiglesias et al. 2011c
The upregulation of many array cell cycle genes may reflect the phosphorylation state of retinoblastoma protein, pRb, in the presence of OA, rather than a physiological response. pRb is a gatekeeper of cell cycle gene transcription that binds to the promoter region of cell cycle genes and whose activity is controlled by its phosphorylation state. pRB is active and in the promoter-bound form when dephosphorylated. When phosphorylated, it detaches from the promoter, allowing gene transcription. Since pRb is normally dephosphorylated by a serine/theronine phosphatase such as PP-1 or PP-2A (Magenta et al. 2008
; Krucher et al. 2006
; Grana 2008
), in the presence of the PP-1 and PP-2A inhibitor OA, pRb would remain phosphorylated and inactivated, and cell cycle gene transcription would proceed. RBBP6 interacts with both pRb and p53 (Chibi et al. 2008
), and promotes degradation of the latter, a sequence hypothesized to increase cell proliferation (Motadi et al. 2011
). Thus OA may have induced inappropriate inactivation of both pRb and RBBP6, although it was possible to track only RBBP6 in our array, stimulating cell cycle gene transcription even though the cells were unable to divide due to damage and activation of apoptosis gene pathways. The observed gene expression patterns allude to both changes in morphology and the loss of viability in HepG2 cells.
In conclusion, the deleterious effects of OA on HepG2 cells reflect OA-induced toxicity and not that of its metabolites. While, as in past studies, many genes were affected by OA, cell cycle genes were significantly elevated at low OA while some apoptotic gene pathways were upregulated at moderate and high OA concentrations. Overall, the concentration dependent effects of OA on gene expression may explain the divergent effects of OA at low concentrations stimulating genes involved in the cell cycle while and at high concentrations stimulating apoptosis.