Through a glass, darkly? HepaRG and HepG2 cells as models of human phase I drug metabolism.

The pharmacokinetic and safety assessment of drug candidates is becoming increasingly dependent upon in vitro models of hepatic metabolism and toxicity. Predominant among these is the HepG2 cell line, although HepaRG is becoming increasingly popular because of its perceived closer resemblance to human hepatocytes. We review the functionality of these cell lines in terms of Phase I protein expression, basal cytochrome P450-dependent activity, and utility in P450 induction studies. Our analysis indicates that HepG2 cells are severely compromised: proteomic studies show that they express few key proteins in common with hepatocytes and they lack drug-metabolizing capacity. Differentiated HepaRGs are more hepatocyte-like than HepG2s, but they also have limitations, and it is difficult to assess their utility because of the enormous variability in data reported, possibly arising from the complex differentiation protocols required to obtain hepatocyte-like cells. This is exacerbated by the use of DMSO in the induction protocol, together with proprietary supplements whose composition is a commercial secret. We conclude that, while currently available data on the utility of HepaRG generates a confusing picture, this line does have potential utility in drug metabolism studies. However, to allow studies to be compared directly a standardized, reproducible differentiation protocol is essential and the cell line's functionality in terms of known mechanisms of P450 regulation must be demonstrated. We, therefore, support the development of regulatory guidelines for the use of HepaRGs in induction studies as a first step in generating a database of consistent, reliable data.

The European Registered Toxicologist (ERT): Current status and prospects for advancement.

Following its inception in 1994, the certification of European Registered Toxicologists (ERT) by EUROTOX has been recognized as ensuring professional competence as well as scientific integrity and credibility. Criteria and procedures for registration are contained in the ERT "Guidelines for Registration 2012". The register of ERT currently has over 1900 members. In order to continue the harmonisation of requirements and processes between national registering bodies as a prerequisite for official recognition of the ERT title as a standard, and to take account of recent developments in toxicology, an update of the ERT Guidelines has been prepared in a series of workshops by the EUROTOX subcommittees for education and registration, in consultation with representatives of national toxicology societies and registers. The update includes details of topics and learning outcomes for theoretical training, and how these can be assessed. The importance of continuing professional development as the cornerstone of re-registration is emphasised. To help with the process of harmonisation, it is necessary to collate and share best practices of registration conditions and procedures across Europe. Importantly, this information can also be used to audit compliance with the EUROTOX standards. As recognition of professionals in toxicology, including specialist qualifications, is becoming more important than ever, we believe that this can best be achieved based on the steps for harmonisation outlined here together with the proposed new Guidelines.

Cytochrome b5 is a major determinant of human cytochrome P450 CYP2D6 and CYP3A4 activity in vivo.

The cytochrome P450-dependent mono-oxygenase system is responsible for the metabolism and disposition of chemopreventive agents, chemical toxins and carcinogens, and >80% of therapeutic drugs. Cytochrome P450 (P450) activity is regulated transcriptionally and by the rate of electron transfer from P450 reductase. In vitro studies have demonstrated that cytochrome b5 (Cyb5) also modulates P450 function. We recently showed that hepatic deletion of Cyb5 in the mouse (HBN) markedly alters in vivo drug pharmacokinetics; a key outstanding question is whether Cyb5 modulates the activity of the major human P450s in drug disposition in vivo. To address this, we crossed mice humanized for CYP2D6 or CYP3A4 with mice carrying a hepatic Cyb5 deletion. In vitro triazolam 4-hydroxylation (probe reaction for CYP3A4) was reduced by >50% in hepatic microsomes from CYP3A4-HBN mice compared with controls. Similar reductions in debrisoquine 4-hydroxylation and metoprolol α-hydroxylation were observed using CYP2D6-HBN microsomes, indicating a significant role for Cyb5 in the activity of both enzymes. This effect was confirmed by the concentration-dependent restoration of CYP3A4-mediated triazolam turnover and CYP2D6-mediated bufuralol and debrisoquine turnover on addition of Escherichia coli membranes containing recombinant Cyb5. In vivo, the peak plasma concentration and area under the concentration time curve from 0 to 8 hours (AUC0-8 h) of triazolam were increased 4- and 5.7-fold, respectively, in CYP3A4-HBN mice. Similarly, the pharmacokinetics of bufuralol and debrisoquine were significantly altered in CYP2D6-HBN mice, the AUC0-8 h being increased ∼1.5-fold and clearance decreased by 40-60%. These data demonstrate that Cyb5 can be a major determinant of CYP3A4 and CYP2D6 activity in vivo, with a potential impact on the metabolism, efficacy, and side effects of numerous therapeutic drugs.

Evidence that the capacity of nongenotoxic carcinogens to induce oxidative stress is subject to marked variability.

Many drugs and environmental chemicals which are not directly mutagenic have the capacity to increase the incidence of tumors in the liver and other tissues. For this reason, such compounds are known as nongenotoxic carcinogens. The mechanisms underlying their effects remain unclear; however, their capacity to induce oxidative stress is considered to be a critical step in the carcinogenic process, although the evidence that this is actually the case remains equivocal and sparse. We have exploited a novel heme oxygenase-1 reporter mouse to evaluate the capacity of nongenotoxic carcinogens with different mechanisms of action to induce oxidative stress in the liver in vivo. When these compounds were administered at doses reported to cause liver tumors, marked differences in activation of the reporter were observed. 1,4-Dichlorobenzene and nafenopin were strong inducers of oxidative stress, whereas phenobarbital, piperonyl butoxide, cyproterone acetate, and WY14,643 were, at best, only very weak inducers. In the case of phenobarbital and thioacetamide, the number of LacZ-positive hepatocytes increased with time, and for the latter also with dose. The data obtained demonstrate that although some nongenotoxic carcinogens can induce oxidative stress, it is not a dominant feature of the response to these compounds. Therefore in contrast to the current models, these data suggest that oxidative stress is not a key determinant in the mechanism of nongenotoxic carcinogenesis but may contribute to the effects in a compound-specific manner.

Differences between murine arylamine N-acetyltransferase type 1 and human arylamine N-acetyltransferase type 2 defined by substrate specificity and inhibitor binding.

BACKGROUND: The mouse has three arylamine N-acetyltransferase genes, (MOUSE)Nat1, (MOUSE)Nat2 and (MOUSE)Nat3. These are believed to correspond to (HUMAN)NAT1, (HUMAN)NAT2 and NATP in humans. (MOUSE)Nat3 encodes an enzyme with poor activity and human NATP is a pseudogene. (MOUSE)Nat2 is orthologous to (HUMAN)NAT1 and their corresponding proteins are functionally similar, but the relationship between (MOUSE)Nat1 and (HUMAN)NAT2 is less clear-cut. METHODS: To determine whether the (MOUSE)NAT1 and (HUMAN)NAT2 enzymes are functionally equivalent, we expressed and purified (MOUSE)NAT1*1 and analysed its substrate specificity using a panel of arylamines and hydrazines. To understand how specific residues contribute to substrate selectivity, three site-directed mutants of (MOUSE)NAT2*1 were prepared: these were (MOUSE)NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L. All three exhibited diminished activity towards "(MOUSE)NAT2-specific" arylamines but were more active against hydrazines than (MOUSE)NAT1*1. The inhibitory and colorimetric properties of a selective naphthoquinone inhibitor of (HUMAN)NAT1 and (MOUSE)NAT2 were investigated. RESULTS: Comparing (MOUSE)NAT1*1 with other mammalian NAT enzymes demonstrated that the substrate profiles of (MOUSE)NAT1 and (HUMAN)NAT2 are less similar than previously believed. Three key residues (F125, R127 and Y129) in (HUMAN)NAT1*4 and (MOUSE)NAT2*1 were required for enzyme inhibition and the associated colour change on naphthoquinone binding. In silico modelling of selective ligands into the appropriate NAT active sites further implicated these residues in substrate and inhibitor specificity in mouse and human NAT isoenzymes. CONCLUSIONS: Three non-catalytic residues within (HUMAN)NAT1*4 (F125, R127 and Y129) contribute both to substrate recognition and inhibitor binding by participating in distinctive intermolecular interactions and maintaining the steric conformation of the catalytic pocket. These active site residues contribute to the definition of substrate and inhibitor selectivity, an understanding of which is essential for facilitating the design of second generation (HUMAN)NAT1-selective inhibitors for diagnostic, prognostic and therapeutic purposes. In particular, since the expression of (HUMAN)NAT1 is related to the development and progression of oestrogen-receptor-positive breast cancer, these structure-based tools will facilitate the ongoing design of candidate compounds for use in (HUMAN)NAT1-positive breast tumours.

From arylamine N-acetyltransferase to folate-dependent acetyl CoA hydrolase: impact of folic acid on the activity of (HUMAN)NAT1 and its homologue (MOUSE)NAT2.

Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme's active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer.

Modeling human cytochrome P450 2D6 metabolism and drug-drug interaction by a novel panel of knockout and humanized mouse lines.

The highly polymorphic human cytochrome P450 2D6 enzyme is involved in the metabolism of up to 25% of all marketed drugs and accounts for significant individual differences in response to CYP2D6 substrates. Because of the differences in the multiplicity and substrate specificity of CYP2D family members among species, it is difficult to predict pathways of human CYP2D6-dependent drug metabolism on the basis of animal studies. To create animal models that reflect the human situation more closely and that allow an in vivo assessment of the consequences of differential CYP2D6 drug metabolism, we have developed a novel straightforward approach to delete the entire murine Cyp2d gene cluster and replace it with allelic variants of human CYP2D6. By using this approach, we have generated mouse lines expressing the two frequent human protein isoforms CYP2D6.1 and CYP2D6.2 and an as yet undescribed variant of this enzyme, as well as a Cyp2d cluster knockout mouse. We demonstrate that the various transgenic mouse lines cover a wide spectrum of different human CYP2D6 metabolizer phenotypes. The novel humanization strategy described here provides a robust approach for the expression of different CYP2D6 allelic variants in transgenic mice and thus can help to evaluate potential CYP2D6-dependent interindividual differences in drug response in the context of personalized medicine.

Drug transporters: gatekeepers controlling access of xenobiotics to the cellular interior.

In this paper, we evaluate methodologies and null mouse models used to study drug transporter function in vitro and in vivo. P-glycoprotein and MRP null mice have been used to examine many aspects of xenobiotic distribution and bioavailability. Their advantage over conventional models is that they allow the exclusion of transporters from a particular process; however, they cannot be used to study the activity of the transporter that has been deleted. Use of humanized mice permits a logical progression from phenomena in wild-type mice via the effects of removing the mouse transporter to the consequences of replacing it with its human counterpart.

Dermal penetration and metabolism of p-aminophenol and p-phenylenediamine: application of the EpiDerm human reconstructed epidermis model.

To address the provision of the 7th Amendment to the EU Cosmetics Directive banning the use of in vivo genotoxicity assays for testing cosmetic ingredients in 2009, the 3D EpiDerm reconstructed human skin micronucleus assay has been developed. To further characterise the EpiDerm tissue for potential use in genotoxicity testing, we have evaluated the dermal penetration and metabolism of two hair dye ingredients, p-aminophenol (PAP) and p-phenylenediamine (PPD) in this reconstructed epidermis model. When EpiDerm tissue was topically exposed to PAP or PPD for 30 min (typical for a hair dye exposure), the majority (80->90%) of PAP or PPD was excluded from skin tissue and removed by rinsing. After a 23.5h recovery period, the PAP fraction that did penetrate was completely N-acetylated to acetaminophen (APAP). Similarly, 30 min topical application of PPD resulted in the formation of the N-mono- and N,N'-diacetylated metabolites of PPD. These results are consistent with published data on the dermal metabolism of these compounds from other in vitro systems as well as from in vivo studies. When tissue was exposed topically (PAP) or via the culture media (PPD) for 24h, there was good batch-to-batch and donor-to-donor reproducibility in the penetration and metabolism of PAP and PPD. Overall, the results demonstrate that these two aromatic amines are biotransformed in 3D EpiDerm tissue via N-acetylation. Characterising the metabolic capability of EpiDerm tissue is important for the evaluation of this model for use in genotoxicity testing.

Preliminary data on the efficacy of the Incredible Years Basic Parent Programme in New Zealand.

OBJECTIVE: The aim of the present study was to conduct a preliminary examination of the efficacy and cultural acceptability of the Incredible Years Basic Parent Programme (IYBPP) using data provided by the New Zealand Ministry of Education. METHODS: Data were gathered on a series of 214 parents attending IYBPP for at least nine sessions. These data included (i) pre-test and post-test T scores on the Eyberg intensity and problem scales; (ii) pre-test and post-test scores on the parent version of the child Social Competence Scale; and (iii) parent satisfaction ratings. RESULTS: Pre-test-post-test comparisons indicated significant improvements in behaviour and social competence scores (p<0.001). Effect sizes ranged from 0.50 to 0.77. Effects were similar for Maori and non-Maori subjects. Parental satisfaction with the programme was high, with Maori and non-Maori parents reporting similar levels of satisfaction. CONCLUSIONS: These preliminary data are consistent with the view that IYBPP is an effective and culturally appropriate programme. There is a need for a more comprehensive evaluation using pilot research to assess the fidelity of programme delivery and randomized trials to assess programme efficacy.

Update on the pharmacogenetics of NATs: structural considerations.

The arylamine N-acetyltransferase (NAT) genes encode enzymes that catalyze the N-acetylation of aromatic amines and hydrazines and the O-acetylation of heterocyclic amines. These genes, which play a key role in cellular homeostasis as well as in gene-environment interactions, are subject to marked pharmacogenetic variation, and different combinations of SNPs in the human NAT genes lead to different acetylation phenotypes. Our understanding of the consequences of pharmacogenetic variability in NATs has recently been enhanced by structural studies showing that effects on protein folding, aggregation and turnover, as well as direct changes in active site topology, are involved. These developments pave the way for a better understanding of the role played by NATs in maintaining cellular homeostasis. In addition, the NATs represent a model for studying fundamental processes associated with protein folding and pharmacogenomic effects mediated by inheritance in human populations across a polymorphic region of the genome.

PXR and CAR: nuclear receptors which play a pivotal role in drug disposition and chemical toxicity.

Xenobiotic metabolism and detoxification is regulated by receptors (e.g., PXR, CAR) whose characterization has contributed significantly to our understanding of drug responses in humans. Technologies facilitating the screening of compounds for receptor interactions provide valuable tools applicable in drug development. Most use in vitro systems or mice humanized for receptors in vivo. In vitro assays are limited by the reporter systems and cell lines chosen and are uninformative about effects in vivo. Humanized mouse models provide novel, exciting ways of understanding the functions of these genes. This article evaluates these technologies and current knowledge on PXR/CAR-mediated regulation of gene expression.

Lack of evidence for metabolism of p-phenylenediamine by human hepatic cytochrome P450 enzymes.

p-Phenylenediamine (PPD) is a widely used ingredient in permanent hair dyes; however, little has been published on its metabolism, especially with respect to hepatic cytochrome P450 (CYP)-mediated oxidation. This is regarded as a key step in the activation of carcinogenic arylamines that ultimately leads to the development of bladder cancer. Most epidemiology studies show no significant association between personal use of hair dyes and bladder cancer, but one recent study reported an increased risk of bladder cancer in women who were frequent users of permanent hair dyes. The aim of the present study was to use intact human hepatocytes, human liver microsomes, and heterologously expressed human CYPs to determine whether PPD is metabolised by hepatic CYPs to form an N-hydroxylamine. p-Phenylenediamine was N-acetylated by human hepatocytes to form N-acetylated metabolites, but there was no evidence for the formation of mono-oxygenated metabolites or for enzyme-mediated covalent binding of 14C-PPD to microsomal protein. In contrast, 2-aminofluorene underwent CYP-mediated metabolism to > or = 4 different hydroxylated metabolites. The lack of evidence for hepatic CYP-mediated metabolism of PPD is inconsistent with the hypothesis that this compound plays a causal role in the development of bladder cancer via a mode of action involving hepatic metabolism to an N-hydroxyarylamine.