Anti-Aspergillus activities of olorofim at sub-MIC levels during early-stage growth.

Olorofim, the first member of the novel class of antifungal drugs, the orotomides, shows promising anti-Aspergillus activity and is currently in phase III clinical development. Using high-throughput microscopy, we monitored olorofim's antifungal potential at sub-minimum inhibitory concentration (MIC) levels with a focus on early-stage growth. Unlike voriconazole, olorofim showed significant growth inhibitory activities against three main pathogenic Aspergillus species, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger, at concentrations >100,000-fold below its MIC. IMPORTANCE: Among antifungal compounds in clinical development for systemic disease, the orotomide olorofim is one of only two that target a completely new mechanism of action. Olorofim is highly potent against pathogenic Aspergillus species including cryptic species that frequently show increased resistance to current agents. In this study, our primary focus was on evaluating in detail the inhibitory activity of voriconazole and olorofim against different pathogenic Aspergillus species employing high-throughput microscopy. Compared to standardized, less-sensitive visual assessment-based methods, microscopy-assisted growth monitoring allowed us to detect sub-MIC drug concentration ranges with significant inhibitory activity at early-stage growth. This revealed that olorofim exerts growth inhibition at concentrations that are several magnitudes below those of voriconazole.

Aspergillus fumigatus strains that evolve resistance to the agrochemical fungicide ipflufenoquin in vitro are also resistant to olorofim.

Widespread use of azole antifungals in agriculture has been linked to resistance in the pathogenic fungus Aspergillus fumigatus. We show that exposure of A. fumigatus to the agrochemical fungicide, ipflufenoquin, in vitro can select for strains that are resistant to olorofim, a first-in-class clinical antifungal with the same mechanism of action. Resistance is caused by non-synonymous mutations within the target of ipflufenoquin/olorofim activity, dihydroorotate dehydrogenase (DHODH), and these variants have no overt growth defects.

Characterisation of putative class 1A DHODH-like proteins from Mucorales and dematiaceous mould species.

Olorofim is a new antifungal in clinical development which has a novel mechanism of action against dihydroorotate dehydrogenase (DHODH). DHODH form a ubiquitous family of enzymes in the de novo pyrimidine biosynthetic pathway and are split into class 1A, class 1B and class 2. Olorofim specifically targets the fungal class 2 DHODH present in a range of pathogenic moulds. The nature and number of DHODH present in many fungal species have not been addressed for large clades of this kingdom. Mucorales species do not respond to olorofim; previous work suggests they have only class 1A DHODH and so lack the class 2 target that olorofim inhibits. The dematiaceous moulds have mixed susceptibility to olorofim, yet previous analyses imply that they have class 2 DHODH. As this is at odds with their intermediate susceptibility to olorofim, we hypothesised that these pathogens may maintain a second class of DHODH, facilitating pyrimidine biosynthesis in the presence of olorofim. The aim of this study was to investigate the DHODH repertoire of clinically relevant species of Mucorales and dematiaceous moulds to further characterise these pathogens and understand variations in olorofim susceptibility. Using bioinformatic analysis, S. cerevisiae complementation and biochemical assays of recombinant protein, we provide the first evidence that two representative members of the Mucorales have only class 1A DHODH, substantiating a lack of olorofim susceptibility. In contrast, bioinformatic analyses initially suggested that seven dematiaceous species appeared to harbour both class 1A-like and class 2-like DHODH genes. However, further experimental investigation of the putative class 1A-like genes through yeast complementation and biochemical assays characterised them as dihydrouracil oxidases rather than DHODHs. These data demonstrate variation in dematiaceous mould olorofim susceptibility is not due to a secondary DHODH and builds on the growing picture of fungal dihydrouracil oxidases as an example of horizontal gene transfer.

Resistance profiling of Aspergillus fumigatus to olorofim indicates absence of intrinsic resistance and unveils the molecular mechanisms of acquired olorofim resistance.

Olorofim (F901318) is a new antifungal currently under clinical development that shows both in vitro and in vivo activity against a number of filamentous fungi including Aspergillus fumigatus. In this study, we screened A. fumigatus isolates for intrinsic olorofim-resistant A. fumigatus and evaluated the ability of A. fumigatus to acquire an olorofim-resistant phenotype. No intrinsic resistance was found in 975 clinical A. fumigatus isolates. However, we found that isolates with increased olorofim MICs (> 8 mg/L) could be selected using a high number of conidia and olorofim exposure under laboratory conditions. Assessment of the frequency of acquired olorofim resistance development of A. fumigatus was shown to be higher than for voriconazole but lower than for itraconazole. Sequencing the PyrE gene of isogenic isolates with olorofim MICs of >8 mg/L identified various amino acid substitutions with a hotspot at locus G119. Olorofim was shown to have reduced affinity to mutated target protein dihydroorotate dehydrogenase (DHODH) and the effect of these mutations was proven by introducing the mutations directly in A. fumigatus. We then investigated whether G119 mutations were associated with a fitness cost in A. fumigatus. These experiments showed a small but significant reduction in growth rate for strains with a G119V substitution, while strains with a G119C substitution did not exhibit a reduction in growth rate. These in vitro findings were confirmed in an in vivo pathogenicity model.

The Dynamic Influence of Olorofim (F901318) on the Cell Morphology and Organization of Living Cells of Aspergillus fumigatus.

The first characterized antifungal in the orotomide class is olorofim. It targets the de novo pyrimidine biosynthesis pathway by inhibiting dihydroorotate dehydrogenase (DHODH). The pyrimidines uracil, thymine and cytosine are the building blocks of DNA and RNA; thus, inhibition of their synthesis is likely to have multiple effects, including affecting cell cycle regulation and protein synthesis. Additionally, uridine-5'-triphosphate (UTP) is required for the formation of uridine-diphosphate glucose (UDP-glucose), which is an important precursor for several cell wall components. In this study, the dynamic effects of olorofim treatment on the morphology and organization of Aspergillus fumigatus hyphae were analyzed microscopically using confocal live-cell imaging. Treatment with olorofim led to increased chitin content in the cell wall, increased septation, enlargement of vacuoles and inhibition of mitosis. Furthermore, vesicle-like structures, which could not be stained or visualized with a range of membrane- or vacuole-selective dyes, were found in treated hyphae. A colocalization study of DHODH and MitoTracker Red FM confirmed for the first time that A. fumigatus DHODH is localized in the mitochondria. Overall, olorofim treatment was found to significantly influence the dynamic structure and organization of A. fumigatus hyphae.

Madurella mycetomatis, the main causative agent of eumycetoma, is highly susceptible to olorofim.

OBJECTIVES: Eumycetoma is currently treated with a combination of itraconazole therapy and surgery, with limited success. Recently, olorofim, the lead candidate of the orotomides, a novel class of antifungal agents, entered a Phase II trial for the treatment of invasive fungal infections. Here we determined the activity of olorofim against Madurella mycetomatis, the main causative agent of eumycetoma. METHODS: Activity of olorofim against M. mycetomatis was determined by in silico comparison of the target gene, dihydroorotate dehydrogenase (DHODH), and in vitro susceptibility testing. We also investigated the in vitro interaction between olorofim and itraconazole against M. mycetomatis. RESULTS: M. mycetomatis and Aspergillus fumigatus share six out of seven predicted binding residues in their DHODH DNA sequence, predicting susceptibility to olorofim. Olorofim demonstrated excellent potency against M. mycetomatis in vivo with MICs ranging from 0.004 to 0.125 mg/L and an MIC90 of 0.063 mg/L. Olorofim MICs were mostly one dilution step lower than the itraconazole MICs. In vitro interaction studies demonstrated that olorofim and itraconazole work indifferently when combined. CONCLUSIONS: We demonstrated olorofim has potent in vitro activity against M. mycetomatis and should be further evaluated in vivo as a treatment option for this disease.

Fluconazole Monotherapy Is a Suboptimal Option for Initial Treatment of Cryptococcal Meningitis Because of Emergence of Resistance.

Cryptococcal meningitis is a lethal disease with few therapeutic options. Induction therapy with fluconazole has been consistently demonstrated to be associated with suboptimal microbiological and clinical outcomes. Exposure to fluconazole causes dynamic changes in antifungal susceptibility, which are associated with the development of aneuploidy. The implications of this phenomenon for pharmacodynamics of fluconazole for cryptococcal meningitis are poorly understood. The pharmacodynamics of fluconazole were studied using a hollow-fiber infection model (HFIM) and a well-characterized murine model of cryptococcal meningoencephalitis. The relationship between drug exposure and both antifungal killing and the emergence of resistance was quantified. The same relationships were further evaluated in a recently described group of patients with cryptococcal meningitis undergoing induction therapy with fluconazole at 800 to 1,200 mg/day. The pattern of emergence of fluconazole resistance followed an "inverted U." Resistance amplification was maximal and suppressed at ratios of the area under the concentration-time curve for the free, unbound fraction of the drug to the MIC (fAUC:MIC) of 34.5 to 138 and 305.6, respectively. Emergence of resistance was observed in vivo with an fAUC:MIC of 231.4. Aneuploidy with duplication of chromosome 1 was demonstrated to be the underlying mechanism in both experimental models. The pharmacokinetic (PK)-pharmacodynamic model accurately described the PK, antifungal killing, and emergence of resistance. Monte Carlo simulations from the clinical pharmacokinetic-pharmacodynamic model showed that only 12.8% of simulated patients receiving fluconazole at 1,200 mg/day achieved sterilization of the cerebrospinal fluid (CSF) after 2 weeks and that 83.4% had a persistent subpopulation that was resistant to fluconazole. Fluconazole is primarily ineffective due to the emergence of resistance. Treatment with 1,200 mg/day leads to the killing of a susceptible subpopulation but is compromised by the emergence of resistance.IMPORTANCE Cryptococcal meningitis is a lethal disease with few treatment options. The incidence remains high and intricately linked with the HIV/AIDS epidemic. In many parts of the world, fluconazole is the only agent that is available for the initial treatment of cryptococcal meningitis despite considerable evidence that it is associated with suboptimal microbiological and clinical outcomes. Fluconazole has a fungistatic mode of action: it predominantly inhibits growth rather than causing fungal killing. Our work shows that the pattern of fluconazole activity is caused by the emergence of resistance in Cryptococcus not detected by standard susceptibility tests, with chromosomal duplication/aneuploidy as the main mechanism. Resistance emergence is related to drug exposure and occurs with the use of clinically relevant regimens. Hence, fluconazole (and potentially other agents that target 14-alpha-demethylase) is compromised by an intrinsic property that limits its effectiveness. However, this resistance may be potentially overcome by dosage escalation or the use of combination therapy.

Effect of the Novel Antifungal Drug F901318 (Olorofim) on Growth and Viability of Aspergillus fumigatus.

F901318 (olorofim) is a novel antifungal drug that is highly active against Aspergillus species. Belonging to a new class of antifungals called the orotomides, F901318 targets dihydroorotate dehydrogenase (DHODH) in the de novo pyrimidine biosynthesis pathway. In this study, the antifungal effects of F901318 against Aspergillus fumigatus were investigated. Live cell imaging revealed that, at a concentration of 0.1 µg/ml, F901318 completely inhibited germination, but conidia continued to expand by isotropic growth for >120 h. When this low F901318 concentration was applied to germlings or vegetative hyphae, their elongation was completely inhibited within 10 h. Staining with the fluorescent viability dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC) showed that prolonged exposure to F901318 (>24 h) led to vegetative hyphal swelling and a decrease in hyphal viability through cell lysis. The time-dependent killing of F901318 was further confirmed by measuring the fungal biomass and growth rate in liquid culture. The ability of hyphal growth to recover in drug-free medium after 24 h of exposure to F901318 was strongly impaired compared to that of the untreated control. A longer treatment of 48 h further improved the antifungal effect of F901318. Together, the results of this study indicate that F901318 initially has a fungistatic effect on Aspergillus isolates by inhibiting germination and growth, but prolonged exposure is fungicidal through hyphal swelling followed by cell lysis.

A Nonredundant Phosphopantetheinyl Transferase, PptA, Is a Novel Antifungal Target That Directs Secondary Metabolite, Siderophore, and Lysine Biosynthesis in Aspergillus fumigatus and Is Critical for Pathogenicity.

Secondary metabolites are key mediators of virulence for many pathogens. Aspergillus fumigatus produces a vast array of these bioactive molecules, the biosynthesis of which is catalyzed by nonribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs). Both NRPSs and PKSs harbor carrier domains that are primed for acceptance of secondary metabolic building blocks by a phosphopantetheinyl transferase (P-pant). The A. fumigatus P-pant PptA has been shown to prime the putative NRPS Pes1 in vitro and has an independent role in lysine biosynthesis; however, its role in global secondary metabolism and its impact on virulence has not been described. Here, we demonstrate that PptA has a nonredundant role in the generation of the vast majority of detectable secondary metabolites in A. fumigatus, including the immunomodulator gliotoxin, the siderophores triacetylfusarinine C (TAFC) and ferricrocin (FC), and dihydroxy naphthalene (DHN)-melanin. We show that both the lysine and iron requirements of a pptA null strain exceed those freely available in mammalian tissues and that loss of PptA renders A. fumigatus avirulent in both insect and murine infection models. Since PptA lacks similarity to its mammalian orthologue, we assert that the combined role of this enzyme in both primary and secondary metabolism, encompassing multiple virulence determinants makes it a very promising antifungal drug target candidate. We further exemplify this point with a high-throughput fluorescence polarization assay that we developed to identify chemical inhibitors of PptA function that have antifungal activity.IMPORTANCE Fungal diseases are estimated to kill between 1.5 and 2 million people each year, which exceeds the global mortality estimates for either tuberculosis or malaria. Only four classes of antifungal agents are available to treat invasive fungal infections, and all suffer pharmacological shortcomings, including toxicity, drug-drug interactions, and poor bioavailability. There is an urgent need to develop a new class of drugs that operate via a novel mechanism of action. We have identified a potential drug target, PptA, in the fungal pathogen Aspergillus fumigatus PptA is required to synthesize the immunotoxic compound gliotoxin, DHN-melanin, which A. fumigatus employs to evade detection by host cells, the amino acid lysine, and the siderophores TAFC and FC, which A. fumigatus uses to scavenge iron. We show that strains lacking the PptA enzyme are unable to establish an infection, and we present a method which we use to identify novel antifungal drugs that inactivate PptA.

F901318 represents a novel class of antifungal drug that inhibits dihydroorotate dehydrogenase.

There is an important medical need for new antifungal agents with novel mechanisms of action to treat the increasing number of patients with life-threatening systemic fungal disease and to overcome the growing problem of resistance to current therapies. F901318, the leading representative of a novel class of drug, the orotomides, is an antifungal drug in clinical development that demonstrates excellent potency against a broad range of dimorphic and filamentous fungi. In vitro susceptibility testing of F901318 against more than 100 strains from the four main pathogenic Aspergillus spp. revealed minimal inhibitory concentrations of ≤0.06 µg/mL-greater potency than the leading antifungal classes. An investigation into the mechanism of action of F901318 found that it acts via inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) in a fungal-specific manner. Homology modeling of Aspergillus fumigatus DHODH has identified a predicted binding mode of the inhibitor and important interacting amino acid residues. In a murine pulmonary model of aspergillosis, F901318 displays in vivo efficacy against a strain of A. fumigatus sensitive to the azole class of antifungals and a strain displaying an azole-resistant phenotype. F901318 is currently in late Phase 1 clinical trials, offering hope that the antifungal armamentarium can be expanded to include a class of agent with a mechanism of action distinct from currently marketed antifungals.

Characterisation of the Candida albicans Phosphopantetheinyl Transferase Ppt2 as a Potential Antifungal Drug Target.

Antifungal drugs acting via new mechanisms of action are urgently needed to combat the increasing numbers of severe fungal infections caused by pathogens such as Candida albicans. The phosphopantetheinyl transferase of Aspergillus fumigatus, encoded by the essential gene pptB, has previously been identified as a potential antifungal target. This study investigated the function of its orthologue in C. albicans, PPT2/C1_09480W by placing one allele under the control of the regulatable MET3 promoter, and deleting the remaining allele. The phenotypes of this conditional null mutant showed that, as in A. fumigatus, the gene PPT2 is essential for growth in C. albicans, thus fulfilling one aspect of an efficient antifungal target. The catalytic activity of Ppt2 as a phosphopantetheinyl transferase and the acyl carrier protein Acp1 as a substrate were demonstrated in a fluorescence transfer assay, using recombinant Ppt2 and Acp1 produced and purified from E.coli. A fluorescence polarisation assay amenable to high-throughput screening was also developed. Therefore we have identified Ppt2 as a broad-spectrum novel antifungal target and developed tools to identify inhibitors as potentially new antifungal compounds.

The Aspergillus fumigatus dihydroxyacid dehydratase Ilv3A/IlvC is required for full virulence.

Dihydroxyacid dehydratase (DHAD) is a key enzyme in the branched-chain amino acid biosynthetic pathway that exists in a variety of organisms, including fungi, plants and bacteria, but not humans. In this study we identified four putative DHAD genes from the filamentous fungus Aspergillus fumigatus by homology to Saccharomyces cerevisiae ILV3. Two of these genes, AFUA_2G14210 and AFUA_1G03550, initially designated AfIlv3A and AfIlv3B for this study, clustered in the same group as S. cerevisiae ILV3 following phylogenetic analysis. To investigate the functions of these genes, AfIlv3A and AfIlv3B were knocked out in A. fumigatus. Deletion of AfIlv3B gave no apparent phenotype whereas the Δilv3A strain required supplementation with isoleucine and valine for growth. Thus, AfIlv3A is required for branched-chain amino acid synthesis in A. fumigatus. A recombinant AfIlv3A protein derived from AFUA_2G14210 was shown to have DHAD activity in an in vitro assay, confirming that AfIlv3A is a DHAD. In addition we show that mutants lacking AfIlv3A and ilv3B exhibit reduced levels of virulence in murine infection models, emphasising the importance of branched-chain amino acid biosynthesis in fungal infections, and hence the potential of targeting this pathway with antifungal agents. Here we propose that AfIlv3A/AFUA_2G2410 be named ilvC.

Functional analysis of a mitochondrial phosphopantetheinyl transferase (PPTase) gene pptB in Aspergillus fumigatus.

The mitochondrial phosphopantetheinyl transferase gene pptB of the opportunistic pathogen Aspergillus fumigatus has been identified and characterised. Unlike pptA, which is required for lysine biosynthesis, secondary metabolism, and iron assimilation, pptB is essential for viability. PptB is located in the mitochondria. In vitro expression of pptA and pptB has shown that PptB is specific for the mitochondrial acyl carrier protein AcpA.

The transposon impala is activated by low temperatures: use of a controlled transposition system to identify genes critical for viability of Aspergillus fumigatus.

Genes that are essential for viability represent potential targets for the development of anti-infective agents. However, relatively few have been determined in the filamentous fungal pathogen Aspergillus fumigatus. A novel solution employing parasexual genetics coupled with transposon mutagenesis using the Fusarium oxysporum transposon impala had previously enabled the identification of 20 essential genes from A. fumigatus; however, further use of this system required a better understanding of the mode of action of the transposon itself. Examination of a range of conditions indicated that impala is activated by prolonged exposure to low temperatures. This newly identified property was then harnessed to identify 96 loci that are critical for viability in A. fumigatus, including genes required for RNA metabolism, organelle organization, protein transport, ribosome biogenesis, and transcription, as well as a number of noncoding RNAs. A number of these genes represent potential targets for much-needed novel antifungal drugs.

Gene ontology mapping as an unbiased method for identifying molecular pathways and processes affected by toxicant exposure: application to acute effects caused by the rodent non-genotoxic carcinogen diethylhexylphthalate.

Toxicogenomics has the potential to reveal the molecular pathways and cellular processes that mediate the adverse responses to a toxicant. However, the initial output of a toxicogenomic experiment often consists of large lists of genes whose expression is altered after toxicant exposure. To interpret gene expression changes in the context of underlying biological pathways and processes, new bioinformatics methods must be developed. We have used global gene expression profiling combined with an evaluation of Gene Ontology (GO) and pathway mapping tools as unbiased methods for identifying the molecular pathways and processes affected upon toxicant exposure. We chose to use the acute effects caused by the non-genotoxic carcinogen and peroxisome proliferator (PP) diethylhexylphthalate (DEHP) in the mouse liver as a model system. Consistent with what is known about the mode of action of DEHP, our GO analysis of transcript profiling data revealed a striking overrepresentation of genes associated with the peroxisomal cellular component, together with genes involved in carboxylic acid and lipid metabolism. Furthermore we reveal gene expression changes associated with additional biological functions, including complement activation, hemostasis, the endoplasmic reticulum overload response, and circadian rhythm. Together, these data reveal potential new pathways of PP action and shed new light on the mechanisms by which non-genotoxic carcinogens control hepatocyte hypertrophy and proliferation. We demonstrate that GO mapping can identify, in an unbiased manner, both known and novel DEHP-induced molecular changes in the mouse liver and is therefore a powerful approach for elucidating modes of toxicity based on toxicogenomic data.

The primary substrate binding site in the b' domain of ERp57 is adapted for endoplasmic reticulum lectin association.

ERp57 is a member of the protein disulfide isomerase (PDI) family that is located in the endoplasmic reticulum (ER) and characterized by its specificity for glycoproteins. Substrate selection by ERp57 is dependent upon its formation of discrete complexes with two ER resident lectins, soluble calreticulin and membrane-bound calnexin. It is these two lectins that directly associate with glycoproteins bearing correctly trimmed oligosaccharide side chains. Thus, ERp57 is presented with a preselected set of substrates upon which it can act, and the specific binding of calreticulin and calnexin to ERp57 is pivotal to the functions of the resulting complexes. To gain further insights into the formation of these ERp57-ER lectin complexes, we have investigated the regions of ERp57 that are specifically required for its binding to calreticulin. Using a quantitative pull-down assay to investigate the binding of ERp57/PDI chimeras to calreticulin, we define the b and b' domains of ERp57 as the minimal elements that are sufficient for complex formation. This analysis further identifies a novel role for the distinctive C-terminal extension of ERp57 in reconstituting complex formation to wild type levels. Using our understanding of substrate binding to the b' domain of PDI as a paradigm, we show that alterations to specific residues in the b' domain of ERp57 dramatically reduce or completely abolish its binding to calreticulin. On the basis of these data, we propose a model where the region of ERp57 equivalent to the primary substrate binding site of archetypal PDI is occupied by calreticulin and suggest that the ER lectins act as adaptor molecules that define the substrate specificity of ERp57.

Tail-anchored and signal-anchored proteins utilize overlapping pathways during membrane insertion.

Tail-anchored proteins are a distinct class of membrane proteins that are characterized by a C-terminal membrane insertion sequence and a capacity for post-translational integration. Although it is now clear that tail-anchored proteins are inserted into the membrane at the endoplasmic reticulum (ER), the molecular basis for their integration is poorly understood. We have used a cross-linking approach to identify ER components that may be involved in the membrane insertion of tail-anchored proteins. We find that several newly synthesized tail-anchored proteins are transiently associated with a defined subset of cellular components. Among these, we identify several ER proteins, including subunits of the Sec61 translocon, Sec62p, Sec63p, and the 25-kDa subunit of the signal peptidase complex. When we analyze the cotranslational membrane insertion of a comparable signal-anchored protein we find the nascent polypeptide associated with a similar set of ER components. We conclude that the pathways for the integration of tail-anchored and signal-anchored membrane proteins at the ER exhibit a substantial degree of overlap, and we propose that this reflects similarities between co- and post-translational membrane insertion.

Receptor-mediated hepatocarcinogenesis: role of hepatocyte proliferation and apoptosis.

The rodent liver is a target organ for the action of several non-genotoxic carcinogens. These include dioxins, polychlorinated biphenyls, phenobarbital, peroxisome proliferators and organochlorine pesticides. These chemicals disrupt the homeostasis of the liver by perturbing hepatocyte cell death and proliferation, causing hyperplasia leading to tumour formation. Significant progress has been made towards elucidating the mechanisms of action of these toxicants since the discovery of receptors that bind specific classes of xenobiotics. Dioxins and polychlorinated biphenyls bind to the aryl hydrocarbon receptor, phenobarbital binds to the constitutive androstane receptor and peroxisome proliferators act via the their activated receptor alpha. These three receptors have ligand-dependent transcription activities and therefore mediate changes in gene expression in response to toxicant exposure. The development of transgenic mouse strains where the genes for these receptors are disrupted has demonstrated that receptor activity is essential for the toxicity of these carcinogens. This implies that changes in the expression of key target genes control proliferation and apoptosis in the xenobiotic-induced hepatocyte phenotype.