Ziad Abdel-Razzak

Oxidative stress plays a major role in chlorpromazine-induced cholestasis in human HepaRG cells.

Drugs induce cholestasis by diverse and still poorly understood mechanisms in humans. Early hepatic effects of chlorpromazine (CPZ), a neuroleptic drug known for years to induce intrahepatic cholestasis, were investigated using the differentiated human hepatoma HepaRG cells. Generation of reactive oxygen species (ROS) was detected as early as 15 minutes after CPZ treatment and was associated with an altered mitochondrial membrane potential and disruption of the pericanalicular distribution of F‐actin. Inhibition of [3H]‐taurocholic acid efflux was observed after 30 minutes and was mostly prevented by N‐acetyl cysteine (NAC) cotreatment, indicating a major role of oxidative stress in CPZ‐induced bile acid (BA) accumulation. Moreover, 24‐hour treatment with CPZ decreased messenger RNA (mRNA) expression of the two main canalicular bile transporters, bile salt export pump (BSEP) and multidrug resistance protein 3 (MDR3). Additional CPZ effects included inhibition of Na+‐dependent taurocholic cotransporting polypeptide (NTCP) expression and activity, multidrug resistance‐associated protein 4 (MRP4) overexpression and CYP8B1 inhibition that are involved in BA uptake, basolateral transport, and BA synthesis, respectively. These latter events likely represent hepatoprotective responses which aim to reduce intrahepatic accumulation of toxic BA. Compared to CPZ effects, overloading of HepaRG cells with high concentrations of cholic and chenodeoxycholic acids induced a delayed oxidative stress and, similarly, after 24 hours it down‐regulated BSEP and MDR3 in parallel to a decrease of NTCP and CYP8B1 and an increase of MRP4. By contrast, low BA concentrations up‐regulated BSEP and MDR3 in the absence of oxidative stress. Conclusion: These data provide evidence that, among other mechanisms, oxidative stress plays a major role as both a primary causal and an aggravating factor in the early CPZ‐induced intrahepatic cholestasis in human hepatocytes. (HEPATOLOGY 2013)

Comparative localization and functional activity of the main hepatobiliary transporters in HepaRG cells and primary human hepatocytes

The role of hepatobiliary transporters in drug-induced liver injury remains poorly understood. Various in vivo and in vitro biological approaches are currently used for studying hepatic transporters; however, appropriate localization and functional activity of these transporters are essential for normal biliary flow and drug transport. Human hepatocytes (HHs) are considered as the most suitable in vitro cell model but erratic availability and inter-donor functional variations limit their use. In this work, we aimed to compare localization of influx and efflux transporters and their functional activity in differentiated human HepaRG hepatocytes with fresh HHs in conventional (CCHH) and sandwich (SCHH) cultures. All tested influx and efflux transporters were correctly localized to canalicular [bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), multidrug resistance protein 1 (MDR1), and MDR3] or basolateral [Na(+)-taurocholate co-transporting polypeptide (NTCP) and MRP3] membrane domains and were functional in all models. Contrary to other transporters, NTCP and BSEP were less abundant and active in HepaRG cells, cellular uptake of taurocholate was 2.2- and 1.4-fold and bile excretion index 2.8- and 2.6-fold lower, than in SCHHs and CCHHs, respectively. However, when taurocholate canalicular efflux was evaluated in standard and divalent cation-free conditions in buffers or cell lysates, the difference between the three models did not exceed 9.3%. Interestingly, cell imaging showed higher bile canaliculi contraction/relaxation activity in HepaRG hepatocytes and larger bile canaliculi networks in SCHHs. Altogether, our results bring new insights in mechanisms involved in bile acids accumulation and excretion in HHs and suggest that HepaRG cells represent a suitable model for studying hepatobiliary transporters and drug-induced cholestasis.

Impact of Inflammation on Chlorpromazine-Induced Cytotoxicity and Cholestatic Features in HepaRG Cells

Several factors are thought to be implicated in the occurrence of idiosyncratic adverse drug reactions. The present work aimed to question as to whether inflammation is a determinant factor in hepatic lesions induced by chlorpromazine (CPZ) using the human HepaRG cell line. An inflammation state was induced by a 24-hour exposure to proinflammatory cytokines interleukin-6 (IL-6) and IL-1β; then the cells were simultaneously treated with CPZ and/or cytokine for 24 hours or daily for 5 days. The inflammatory response was assessed by induction of C-reactive protein and IL-8 transcripts and proteins as well as inhibition of CPZ metabolism and down-regulation of cytochrome 3A4 (CYP3A4) and CYP1A2 transcripts, two major cytochrome P450 (P450) enzymes involved in its metabolism. Most effects of cotreatments with cytokines and CPZ were amplified or only observed after five daily treatments; they mainly included increased cytotoxicity and overexpression of oxidative stress-related genes, decreased Na+-taurocholate cotransporting polypeptide mRNA levels and activity, a key transporter involved in bile acids uptake, and deregulation of several other transporters. However, CPZ-induced inhibition of taurocholic acid efflux and pericanalicular F-actin distribution were not affected. In addition, a time-dependent induction of phospholipidosis was noticed in CPZ-treated cells, without obvious influence of the inflammatory stress. In summary, our results show that an inflammatory state induced by proinflammatory cytokines increased cytotoxicity and enhanced some cholestatic features induced by the idiosyncratic drug CPZ in HepaRG cells. These changes, together with inhibition of P450 activities, could have important consequences if extrapolated to the in vivo situation.

Differential sensitivity of metabolically competent and non-competent HepaRG cells to apoptosis induced by diclofenac combined or not with TNF-alpha

The role of reactive metabolites and inflammatory stress has been largely evoked in idiosyncratic hepatotoxicity of diclofenac (DCF); however mechanisms remain poorly understood. We aimed to evaluate the influence of liver cell phenotype on the hepatotoxicity of DCF combined or not with TNF-α using differentiated and undifferentiated HepaRG cells, and for comparison, HepG2 cells. Our results demonstrate that after a 24h-treatment metabolizing HepaRG cells were less sensitive to DCF than their undifferentiated non-metabolizing counterparts as shown by lower oxidative and endoplasmic reticulum stress responses and lower activation of caspase 9. Differentiated HepaRG cells were also less sensitive than HepG2 cells. Their lower sensitivity to DCF was related to their high content in glutathione transferases. DCF-induced apoptotic effects were potentiated by TNF-α only in death receptor-expressing differentiated HepaRG and HepG2 cells and were associated with marked activation of caspase 8. TNF-α co-treatment did not aggravate DCF-induced cholestatic features. Altogether, our results demonstrate that (i) lower sensitivity to DCF of differentiated HepaRG cells compared to their non-metabolically active counterparts was related to their high detoxifying capacity, giving support to the higher sensitivity of nonhepatic tissues than liver to this drug; (ii) TNF-α-potentiation of DCF cytotoxicity occurred only in death receptor-expressing cells.

Alteration of human hepatic drug transporter activity and expression by cigarette smoke condensate

Smoking is well-known to impair pharmacokinetics, through inducing expression of drug metabolizing enzymes. In the present study, we demonstrated that cigarette smoke condensate (CSC) also alters activity and expression of hepatic drug transporters, which are now recognized as major actors of hepatobiliary elimination of drugs. CSC thus directly inhibited activities of sinusoidal transporters such as OATP1B1, OATP1B3, OCT1 and NTCP as well as those of canalicular transporters like P-glycoprotein, MRP2, BCRP and MATE1, in hepatic transporters-overexpressing cells. CSC similarly counteracted constitutive OATP, NTCP and OCT1 activities in human highly-differentiated hepatic HepaRG cells. In parallel, CSC induced expression of BCRP at both mRNA and protein level in HepaRG cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B1, OATP2B1, OAT2, NTCP, OCT1 and BSEP, and enhanced that of MRP4. Such changes in transporter gene expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin, a reference activator of the aryl hydrocarbon receptor (AhR) pathway, and were counteracted, for some of them, by siRNA-mediated AhR silencing. This suggests that CSC alters hepatic drug transporter levels via activation of the AhR cascade. Importantly, drug transporter expression regulations as well as some transporter activity inhibitions occurred for a range of CSC concentrations similar to those required for inducing drug metabolizing enzymes and may therefore be hypothesized to be relevant for smokers. Taken together, these data established human hepatic transporters as targets of cigarette smoke, which could contribute to known alteration of pharmacokinetics and some liver adverse effects caused by smoking.

N-acetylcysteine potentiates diclofenac toxicity in Saccharomyces cerevisiae: stronger potentiation in ABC transporter mutant strains

Abstract Diclofenac (DCF) adverse reactions involve diverse mechanisms in different models. We recently demonstrated that DCF-induced toxicity in HepaRG decreases as they express DCF-metabolizing enzymes. DCF metabolism promotes toxicity in Saccharomyces cerevisiae expressing heterologous cytochromes-P450. N-Acetylcysteine (NAC) is used to treat diverse medical conditions due to its multiple properties (antioxidant, metal chelator, thiol-disulfide disruption). The latter property accounts for its mucolytic effects and broadens its potential molecular targets to signal transduction proteins, ABC transporters and others. Interaction of NAC with DCF effects depends on the experimental model. This study aims to investigate NAC/DCF interaction and the involvement of ABC transporters in wild type and mutant Saccharomyces cerevisiae. DCF inhibited yeast growth in a dose- and time-dependent manner and the cells started adapting to DCF 24-h post-treatment. NAC potentiated DCF-induced toxicity if added prior or parallel to DCF. Pretreatment with NAC increased its potentiation effect and compromised cells adaption to DCF. Post-treatment with NAC potentiated DCF toxicity without compromising adaptation. Moreover, mutant strains in ABC transporters Pdr5, Yor1, Bpt1 or Pdr15, were more sensitive to DCF; while mutant strains in Pdr5, Vmr1 or Pdr12 were more sensitive to NAC/DCF interaction. DCF ± NAC elicited on the mutant strain in Yap1, an oxidative stress-related protein, the same effects as on the wild type. Therefore, oxidative stress does not seem to be key actor in DCF toxicity in our model. Our hypothesis is that NAC potentiation effect is at least due to its ability to disrupt disulfide bridge in proteins required to overcome DCF toxicity in yeast.

Inhibition of organic anion transporter (OAT) activity by cigarette smoke condensate

Cigarette smoke condensate (CSC) has previously been shown to impair activity and expression of hepatic drug transporters. In the present study, we provided evidence that CSC also hinders activity of organic anion transporters (OATs), notably expressed at the kidney level. CSC thus cis-inhibited OAT substrate uptake in OAT1- and OAT3-transfected HEK293 cells, in a concentration-dependent manner (IC50=72.1μg/mL for OAT1 inhibition and IC50=27.3μg/mL for OAT3 inhibition). By contrast, OAT4 as well as the renal organic cation transporter (OCT) 2 were less sensitive to the inhibitory effect of CSC (IC50=351.5μg/mL and IC50=226.2μg/mL, for inhibition of OAT4 and OCT2, respectively). OAT3 activity was further demonstrated to be blocked by some single chemicals present in cigarette smoke such as the heterocyclic amines AαC (IC50=11.3μM) and PhIP (IC50=1.9μM), whereas other major cigarette smoke components used at 100μM, like nicotine, the nitrosamine NNK and the polycyclic aromatic hydrocarbons benzo(a)pyrene and phenanthrene, were without effect. AαC and PhIP however failed to trans-stimulate activity of OAT3, suggesting that they were not substrates for this transporter. Taken together, these data establish OAT1 and OAT3 transporters as targets of cigarette smoke chemicals, which may contribute to smoking-associated pharmacokinetics alterations.

Inhibition of organic cation transporter (OCT) activities by carcinogenic heterocyclic aromatic amines

Carcinogenic heterocyclic aromatic amines (HAAs) interact with some drug transporters, like the efflux pump BCRP and the organic anion transporters OAT1 and OAT3. The present study was designed to determine whether they can also target activities of the organic cation transporters (OCTs), using mainly OCT1-, OCT2- and OCT3-overexpressing HEK293 cells. Fifteen HAAs were demonstrated to differently alter OCT activities; with a cut-off of at least 50% reduction of transporter activity by 100 μM HAAs, 5/15 HAAs, including Trp-P-1 and Trp-P-2, inhibited activities of OCT1, OCT2 and OCT3, whereas 7/15 HAAs, including PhIP and MeIQx, blocked those of OCT2 and OCT3, 1/15 HAAs reduced those of OCT1 and OCT2 and 2/15 HAAs, including AαC, only that of OCT2. IC50 values of Trp-P-1 and Trp-P-2 towards OCT activities were found to be in the 2-6 μM range, likely not relevant for human exposure to HAAs through smoking or the diet. Trp-P-1 and Trp-P-2 additionally failed to trans-stimulate OCT1 and OCT2 activities and exhibited similar accumulation in OCT1/2-transduced HEK293 cells and control HEK293-MOCK cells. These data demonstrate that HAAs, notably Trp-P-1 and Trp-P-2, interact with OCT1/2, without however being transported, thus likely discarding a major role for OCT1/2 in HAA systemic toxicokinetics.

Archaeal SF1 and SF2 Helicases: Unwinding in the Extreme

Abstract Archaea, the third domain of life, are significant microorganism models in understanding fundamental aspects of molecular biology. Since the archaeal informational system shares many eukaryal features, structure–function studies using Archaea as models have largely contributed to our understanding of many eukaryotic cellular processes. Helicases of superfamilies 1 (SF1) and 2 (SF2) have been shown to be of major importance in RNA and DNA metabolism in Eukarya and in Bacteria. In Archaea, the cellular functions of these enzymes remain dispersed and only few members were characterized. In this chapter, we review our knowledge on the archaeal SF1 and SF2 helicases. We focus on phylogenomic studies that revealed archaeal helicase families and give insights into their respective biochemical and structural properties. Finally, we raise the question of the mode of actions of these helicases in archaeal DNA and RNA metabolism.

Cigarette smoke condensate alters Saccharomyces cerevisiae efflux transporter mRNA and activity and increases caffeine toxicity

In animals, cigarette smoke may alter pharmacokinetics by altering activity and expression of ABC drug transporters. We previously demonstrated that cigarette smoke condensate (CSC) impairs activity and expression of several hepatic ABC drug transporters which mediate toxicant efflux. However, CSC effects on efflux transporters are still unknown in Saccharomyces cerevisiae which resists diverse chemical stresses, by inducing pleiotropic drug resistance (PDR) genes among others. The yeast ABC transporters are functionally and structurally homologous to the mammalian ones. In this study, Saccharomyces cerevisiae exposure to CSC for 15 min caused a dose-dependent inhibition of rhodamine 123 efflux, whereas a longer exposure (3 h) induced mRNA expression of the ABC PDR efflux pumps Pdr5, Snq2, Pdr 10 and Pdr15, and of Tpo1, a member of the major facilitator superfamily (MFS). CSC also increased toxicity of caffeine, which is handled by two PDR transporters, Pdr5 and Snq2. Taken together, these data demonstrated that yeast efflux transporters are targets of cigarette smoke chemicals, and that Saccharomyces cerevisiae may cope with CSC-induced stress, including the initial efflux inhibition, by induction of the mRNA of several plasma membrane PDR and MFS efflux transporters. Saccharomyces cerevisiae is therefore a valid model to investigate pollutant effects on ABC and MFS transporters.