Disinfection by-products are produced in water disinfected with chlorine-based products. One such group is trihalomethanes, and chloroform is the most abundant trihalomethane in swimming pool areas. Chloroform can be absorbed by inhalation, ingestion, and dermal absorption, and is classified as possibly carcinogenic.

To investigate if chloroform concentrations in air and water affect the chloroform concentration in urine samples of exposed swimming pool workers.

Workers from 5 adventure indoor swimming pools carried personal chloroform air samplers and provided up to 4 urine samples during one workday. Chloroform concentrations were analyzed with a linear mixed model analysis to investigate a possible correlation between air and urine concentrations.

The geometric mean chloroform concentration was 11 μg/m3 in air and 0.009 µg/g creatinine in urine among individuals with ≤2 h at work, 0.023 µg/g creatinine among those with >2-5 working hours, and 0.026 µg/g creatinine in the group with >5-10 working hours. A risk of higher levels of chloroform in urine was associated with longer hours at work (≤2 h versus >5-10 h, odds ratio [OR] 2.04, 95% confidence interval [CI] 1.25-3.34), personal chloroform concentrations in air (≤17.00 µg/m3 versus >28.00 µg/m3, OR 9.23, 95% CI 3.68-23.13) and working at least half the working day near the swimming pools (OR 3.16, 95% CI 1.33-7.55). Executing work tasks in the swimming pool water was not associated with higher chloroform concentrations in urine compared to only working on land (OR 0.82, 95% CI 0.27-2.45).

There is an accumulation of chloroform concentrations in urine during a workday and a correlation between personal air and urine concentrations of chloroform among workers in Swedish indoor swimming pools.

There is an increasing number of indications of an oversimplification in the premise that the cumulative properties of the human drug-metabolizing ensemble represent a simple aggregate of the properties of the constituting enzymes. Recent studies of the functional effects of hetero-association of multiple cytochrome P450 species and their interactions with metabolically related enzymes revealed a tight integration in the drug-metabolizing ensemble. In our opinion, the sources of interindividual variability in drug metabolism can be elucidated only when considering this ensemble as a multienzyme system, the functional parameters of which are determined by interactions between its constituents. In this article, we present a conceptual model providing a mechanistic explanation for the functional effects of the interactions between multiple P450 species and propose a clue to understanding the nonadditive behavior of the drug-metabolizing ensemble.

Efficient aquaculture is depending on sustainable protein sources. The shortage in marine raw materials has initiated a shift to "green aquafeeds" based on staple ingredients such as soy and wheat. Plant-based diets entail new challenges regarding fish health, product quality and consumer risks due to the possible presence of chemical contaminants, natural toxins and bioactive compounds like phytoestrogens. Daidzein (DAI), genistein (GEN) and glycitein (GLY) are major soy isoflavones with considerable estrogenic activities, potentially interfering with the piscine endocrine system and affecting consumers after carry-over. In this context, information on isoflavone biotransformation in fish is crucial for risk evaluation. We have therefore isolated hepatic fractions of Atlantic salmon (Salmo salar), the most important species in Norwegian aquaculture, and used them to study isoflavone elimination and metabolite formation. The salmon liver microsomes and primary hepatocytes were characterized with respect to phase I cytochrome P450 (CYP) and phase II uridine-diphosphate-glucuronosyltransferase (UGT) enzyme activities using specific probe substrates, which allowed comparison to results in other species. DAI, GEN and GLY were effectively cleared by UGT. Based on the measurement of exact masses, fragmentation patterns, and retention times in liquid chromatography high-resolution mass spectrometry, we preliminarily identified the 7-O-glucuronides as the main metabolites in salmon, possibly produced by UGT1A1 and UGT1A9-like activities. In contrast, the production of oxidative metabolites by CYP was insignificant. Under optimized assay conditions, only small amounts of mono-hydroxylated DAI were detectable. These findings suggested that bioaccumulation of phytoestrogens in farmed salmon and consumer risks from soy-containing aquafeeds are unlikely.

Extrapolation of pharmacokinetic (PK) parameters from in vitro or in vivo animal to human is one of the main tasks in the drug development process. Translational approaches provide evidence for go or no-go decision-making during drug discovery and the development process, and the prediction of human PKs prior to the first-in-human clinical trials. In vitro-in vivo extrapolation and allometric scaling are the choice of method for projection to human situations. Although these methods are useful tools for the estimation of PK parameters, it is a challenge to apply these methods since underlying biochemical, mathematical, physiological, and background knowledge of PKs are required. In addition, it is difficult to select an appropriate methodology depending on the data available. Therefore, this review covers the principles of PK parameters pertaining to the clearance, volume of distribution, elimination half-life, absorption rate constant, and prediction method from the original idea to recently developed models in order to introduce optimal models for the prediction of PK parameters.

Titania has properties that enable it to be used in a variety of applications, including self-cleaning surfaces, air and water purification systems, hydrogen evolution, and photoelectrochemical conversion. In order to improve the properties of titanium dioxide, modifications are made to obtain oxide/hybrid systems that are intended to have the properties of both components. In particular, zinc oxide, zirconia and molybdenum disulfide have been proposed as the second component of binary systems due to their antibacterial, electrochemical and photocatalytic properties. This paper presents a review of the current state of knowledge on the synthesis and practical utility of TiO₂-ZnO and TiO₂-ZrO₂ oxide systems and TiO₂-MoS₂ hybrid materials. The first part focuses on the hydrothermal method; then a review is made of the literature on the synthesis of the aforementioned materials using the sol-gel method. In the last section, the literature on the electrospinning method of synthesis is reviewed. The most significant physico-chemical, structural and dispersive-morphological properties of binary hybrid systems based on TiO₂ are described. A key aim of this review is to indicate the properties of TiO₂-ZnO, TiO₂-ZrO₂ and TiO₂-MoS₂ hybrid systems that have the greatest importance for practical applications. The variety of utilities of titania-based hybrid materials is emphasized.

The screening of potential therapeutic compounds using phenotypic drug discovery (PDD) is being embraced once again by researchers and pharmaceutical companies as an approach to enhance the development of new effective therapeutics. Before the genomics and molecular biology era and the consecutive emergence of targeted-drug discovery approaches, PDD was the most common platform used for drug discovery. PDD, also known as phenotypic screening, consists of screening potential compounds in either in vitro cellular or in vivo animal models to identify compounds resulting in a desirable phenotypic change. Using this approach, the biological targets of the compounds are not taken into consideration. Suitable animal models are crucial for the continued validation and discovery of new drugs, as compounds displaying promising results in phenotypic in vitro cell-based and in vivo small animal model screenings often fail in clinical trials. Indeed, this is mainly a result of differential anatomy, physiology, metabolism, immunology, and genetics between humans and currently used pre-clinical small animal models. In contrast, pigs are more predictive of therapeutic treatment outcomes in humans than rodents. In addition, pigs provide an ideal platform to study cancer due to their similarities with humans at the anatomical, physiological, metabolic, and genetic levels. Here we provide a mini-review on the reemergence of PDD in drug development, highlighting the potential of porcine cancer models for improving pre-clinical drug discovery and testing. We also present precision medicine based genetically defined swine cancer models developed to date and their potential as biomedical models.

Trichloroethylene (TCE) and tetrachloroethylene (PCE) are ubiquitous environmental contaminants and occupational health hazards. Recent health assessments of these agents identified several critical data gaps, including lack of comparative analysis of their effects. This study examined liver and kidney effects of TCE and PCE in a dose-response study design. Equimolar doses of TCE (24, 80, 240, and 800 mg/kg) or PCE (30, 100, 300, and 1000 mg/kg) were administered by gavage in aqueous vehicle to male B6C3F1/J mice. Tissues were collected 24 h after exposure. Trichloroacetic acid (TCA), a major oxidative metabolite of both compounds, was measured and RNA sequencing was performed. PCE had a stronger effect on liver and kidney transcriptomes, as well as greater concentrations of TCA. Most dose-responsive pathways were common among chemicals/tissues, with the strongest effect on peroxisomal β-oxidation. Effects on liver and kidney mitochondria-related pathways were notably unique to PCE. We performed dose-response modeling of the transcriptomic data and compared the resulting points of departure (PODs) to those for apical endpoints derived from long-term studies with these chemicals in rats, mice, and humans, converting to human equivalent doses using tissue-specific dosimetry models. Tissue-specific acute transcriptional effects of TCE and PCE occurred at human equivalent doses comparable to those for apical effects. These data are relevant for human health assessments of TCE and PCE as they provide data for dose-response analysis of the toxicity mechanisms. Additionally, they provide further evidence that transcriptomic data can be useful surrogates for in vivo PODs, especially when toxicokinetic differences are taken into account.

Quantitative characterization of UDP-glucuronosyltransferase (UGT) enzymes is valuable in glucuronidation reaction phenotyping, predicting metabolic clearance and drug-drug interactions using extrapolation exercises based on pharmacokinetic modeling. Different quantitative proteomic workflows have been employed to quantify UGT enzymes in various systems, with reports indicating large variability in expression, which cannot be explained by interindividual variability alone. To evaluate the effect of methodological differences on end-point UGT abundance quantification, eight UGT enzymes were quantified in 24 matched liver microsomal samples by two laboratories using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT) standard, and measurements were assessed against catalytic activity in seven enzymes (n = 59). There was little agreement between individual abundance levels reported by the two methods; only UGT1A1 showed strong correlation [Spearman rank order correlation (Rs) = 0.73, P < 0.0001; R2 = 0.30; n = 24]. SIL-based abundance measurements correlated well with enzyme activities, with correlations ranging from moderate for UGTs 1A6, 1A9, and 2B15 (Rs = 0.52-0.59, P < 0.0001; R2 = 0.34-0.58; n = 59) to strong correlations for UGTs 1A1, 1A3, 1A4, and 2B7 (Rs = 0.79-0.90, P < 0.0001; R2 = 0.69-0.79). QconCAT-based data revealed generally poor correlation with activity, whereas moderate correlations were shown for UGTs 1A1, 1A3, and 2B7. Spurious abundance-activity correlations were identified in the cases of UGT1A4/2B4 and UGT2B7/2B15, which could be explained by correlations of protein expression between these enzymes. Consistent correlation of UGT abundance with catalytic activity, demonstrated by the SIL-based dataset, suggests that quantitative proteomic data should be validated against catalytic activity whenever possible. In addition, metabolic reaction phenotyping exercises should consider spurious abundance-activity correlations to avoid misleading conclusions.

HepaRG cells are widely used as an in vitro model to assess drug-induced hepatotoxicity. However, only few studies exist so far regarding their suitability to detect the effects of drugs requiring a preceding activation via the cytochrome P450 (CYP) system. A prototypic substance is the anti-tuberculosis agent INH, which is metabolized into N-acetylhydrazine, which then triggers hepatotoxicity. Therefore, the aim of the present study was to test if this effect can also be detected in HepaRG cells and if it can be counteracted by the known hepatoprotectant silibinin. For this purpose, differentiated HepaRG cells were treated with increasing concentrations of INH (0.1-100 mM) or 10 mM INH plus escalating concentrations of silibinin (1-100 µM). After 48 h of treatment, cell morphology and parameters indicating cell vitality, oxidative stress, and liver cell function were assessed. High concentrations of INH led to severe histopathological changes, reduced cell vitality and glutathione content, increased LDH and ASAT release into the medium, enhanced lipid peroxidation, and elevated cleaved caspase-3 expression. Additionally, glycogen depletion and reduced biotransformation capacity were seen at high INH concentrations, whereas at low concentrations an induction of biotransformation enzymes was noticed. Silibinin caused clear-cut protective effects, but with few parameters INH toxicity was even aggravated, most probably due to increased metabolization of INH into its toxic metabolite. In conclusion, HepaRG cells are excellently suited to evaluate the effects of substances requiring prior toxification via the CYP system, such as INH. They additionally enable the identification of complex substance interactions.

Inhibition of cytochrome P450 (P450) enzymes (CYP) has been shown to lower the metabolism of drugs that are P450 substrates and to consequently alter their pharmacokinetic profiles. Curcumin (CUR), piperine (PIP), and capsaicin (CAP) are spice components (SC) that inhibit the activities of a range of P450 enzymes, but the selection of which SC to be prioritized for further development as an adjuvant will depend on the ranking order of the inhibitory potential of the SCs on specific P450 isozymes. We used common human recombinant enzyme platforms to provide a comparative evaluation of the inhibitory activities of CUR, PIP, and CAP on the principal drug-metabolizing P450 enzymes. SC-mediated inhibition of CYP3A4 was found to rank in the order of CAP (IC50 1.84 ± 0.71 µM) ∼ PIP (2.12 ± 0.45 µM) > CUR (11.93 ± 3.49 µM), while CYP2C9 inhibition was in the order of CAP (11.95 ± 4.24 µM) ∼ CUR (14.58 ± 4.57 µM) > PIP (89.62 ± 9.17 µM). CAP and PIP were significantly more potent inhibitors of CYP1A2 (IC50 2.14 ± 0.22 µM and 14.19 ± 4.15 µM, respectively) than CUR (IC50 > 100 µM), while all three SCs exhibited weak activity toward CYP2D6 (IC50 95.42 ± 12.09 µM for CUR, 99.99 ± 5.88 µM for CAP, and 110.40 ± 3.23 µM for PIP). Of the three SCs, CAP thus has the strongest potential for further development into an inhibitor of multiple CYPs for use in the clinic. Data from this study are also useful for managing potential drug-SC interactions.

This study demonstrates that low doses (somewhat above the No Observed Adverse Effect Level, NOAEL) of the mycoestrogen zearalenone (ZEN) and its metabolites display multispecificity towards various biological targets in gilts. The observed responses in gilts were surprising. The presence of ZEN and zearalenols (ZELs) did not evoke a response in the porcine gastrointestinal tract, which was attributed to dietary tolerance. Lymphocyte proliferation was intensified in jejunal mesenteric lymph nodes, and lymphocyte counts increased in the jejunal epithelium with time of exposure. In the distal digestive tract, fecal bacterial counts decreased, the activity of fecal bacterial enzymes and lactic acid bacteria increased, and cecal water was characterized by higher genotoxicity. The accompanying hyperestrogenism led to changes in mRNA activity of selected enzymes (cytochrome P450, hydroxysteroid dehydrogenases, nitric oxide synthases) and receptors (estrogen and progesterone receptors), and it stimulated post-translational modifications which play an important role in non-genomic mechanisms of signal transmission. Hyperestrogenism influences the regulation of the host's steroid hormones (estron, estradiol and progesteron), it affects the virulence of bacterial genes encoding bacterial hydroxysteroid dehydrogenases (HSDs), and it participates in detoxification processes by slowing down intestinal activity, provoking energy deficits and promoting antiporter activity at the level of enterocytes. In most cases, hyperestrogenism fulfils all of the above roles. The results of this study indicate that low doses of ZEN alleviate inflammatory processes in the digestive system, in particular in the proximal and distal intestinal tract, and increase body weight gains in gilts.

Cytochrome P450 is a family of enzymes that catalyze reactions involved in the metabolism of drugs and other xenobiotics. These enzymes are therefore important in pharmacologic and toxicologic studies, and information on their abundances is of value in the process of scaling in vitro data to in vivo metabolic parameters. A meta-analysis was applied to data on the abundance of human hepatic cytochrome P450 enzymes in Caucasian adult livers (50 studies). Despite variations in the methods used to measure the abundance of enzymes, agreement between the studies in 26 different laboratories was generally good. Nonetheless, some heterogeneity was detected (Higgins and Thompson heterogeneity test). More importantly, large interindividual variability was observed in the collated data. Positive correlations between the expression levels of some cytochrome P450 enzymes were found in the abundance data, including the following pairs: CYP3A4/CYP3A5*1/*3 (Rs = 0.70, P < 0.0001, n = 52), CYP3A4/CYP2C8 (Rs = 0.68, P < 0.0001, n = 134), CYP3A4/CYP2C9 (Rs = 0.55, P < 0.0001, n = 71), and CYP2C8/CYP2C9 (Rs = 0.55, P < 0.0001, n = 99). These correlations can be used to demonstrate common genetic transcriptional mechanisms.

In vitro-derived information has been increasingly used to support and improve human health risk assessment for exposure to chemicals. Physiologically based pharmacokinetic (PBPK) modeling is a key component in the movement toward in vitro-based risk assessment, providing a tool to integrate diverse experimental data and mechanistic information to relate in vitro effective concentrations to equivalent human exposures. One of the challenges, however, in the use of PBPK models for this purpose has been the need for extensive chemical-specific parameters. With the remarkable advances in in vitro methodologies in recent years, in vitro-derived parameters can now be easily incorporated into PBPK models. In this study we demonstrate an in vitro data based parameterization approach to develop a physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model, using carbaryl as a case study. In vitro experiments were performed to provide the chemical-specific pharmacokinetic (PK) and pharmacodynamic (PD) parameters for carbaryl in the PBPK model for this compound. Metabolic clearance and cholinesterase (ChE) interaction parameters for carbaryl were measured in rat and human tissues. These in vitro PK and PD data were extrapolated to parameters in the whole body PBPK model using biologically appropriate scaling. The PBPK model was then used to predict the kinetics and ChE inhibition dynamics of carbaryl in vivo. This case study with carbaryl provides a reasonably successful example of utilizing the in vitro to in vivo extrapolation (IVIVE) approach for PBPK model development. This approach can be applied to other carbamates with an anticholinesterase mode of action as well as to environmental chemicals in general with further refinement of the current shortcomings in the approach. It will contribute to minimizing the need for in vivo human data for PBPK model parameterization and evaluation in human risk assessments.

CYP2E1 is an important cytochrome P450 isoform in many endogenous processes and in the metabolism of organic solvents, a number of drugs and pre-carcinogens. Information on the abundance of the enzyme may be valuable in various types of research in the field of toxicology and pharmacology. An indirect ELISA for the quantification of CYP2E1 in human liver microsomes was developed and successfully validated. All samples, including validation samples and calibrators, were diluted to a final concentration of microsomal protein of 10μg/ml. Detection of the antigen was obtained through binding of a polyclonal antibody raised against the full length protein, followed by the addition of horseradish peroxidase conjugated secondary antibodies and enzymatic detection. A five-parameter logistics function with 1/x weighting was used for quantification within the concentration range of 4-256pmol CYP2E1/mg microsomal protein. The method showed acceptable intra- and inter-assay precision, with calculated coefficients of variation of 6.3-15.2% and 11.3-21.0%, respectively. The relative error varied between -2.3 and 8.9%, and the total error between 16.0 and 27.2%. No significant cross reactivity with other abundant CYP isoforms was observed. The method was evaluated through the analysis of samples from a pharmacokinetic study, and the comparison with the CYP2E1 activity in those samples.

Physiologically based pharmacokinetic (PBPK) models are tools for interpreting toxicological data and extrapolating observations across species and route of exposure. Chloroform (CHCl(3)) is a chemical for which there are PBPK models available in different species and multiple sites of toxicity. Because chloroform induces toxic effects in the liver and kidneys via production of reactive metabolites, proper characterization of metabolism in these tissues is essential for risk assessment. Although hepatic metabolism of chloroform is adequately described by these models, there is higher uncertainty for renal metabolism due to a lack of species-specific data and direct measurements of renal metabolism. Furthermore, models typically fail to account for regional differences in metabolic capacity within the kidney. Mischaracterization of renal metabolism may have a negligible effect on systemic chloroform levels, but it is anticipated to have a significant impact on the estimated site-specific production of reactive metabolites. In this article, rate parameters for chloroform metabolism in the kidney are revised for rats, mice, and humans. New in vitro data were collected in mice and humans for this purpose and are presented here. The revised PBPK model is used to interpret data of chloroform-induced kidney toxicity in rats and mice exposed via inhalation and drinking water. Benchmark dose (BMD) modeling is used to characterize the dose-response relationship of kidney toxicity markers as a function of PBPK-derived internal kidney dose. Applying the PBPK model, it was also possible to characterize the dose response for a recent data set of rats exposed via multiple routes simultaneously. Consistent BMD modeling results were observed regardless of species or route of exposure.

To evaluate the effects of surgical weight loss on hepatic lipid peroxidation levels and cytochrome P-450 protein expression in patients with nonalcoholic fatty liver disease (NAFLD).

NAFLD and nonalcoholic steatohepatitis (NASH) affect hepatic cytochrome P-450 (CYP) protein expression and activity, and CYP2E1 may play a role in the pathogenesis of NAFLD and NASH through induction of oxidative stress and lipid peroxidation. NAFLD and NASH are associated with increased systemic lipid peroxidation levels and elevated hepatic CYP2E1 activity, but hepatic CYP3A4/5 activity is decreased.

Liver biopsies from 20 patients with NAFLD who underwent bariatric surgery were obtained intraoperatively and at 15 +/- 7 months following surgery. Hepatic malondialdehyde (MDA) levels (a marker of lipid peroxidation), CYP2E1 and CYP3A4/5 protein expression, and steatosis, as a percent of total area, were measured by immunohistochemistry followed by digital image quantitation.

Following weight loss, as reflected by reduced BMI (54 +/- 9 vs. 37 +/- 9 kg/m2; P < 0.001), features of the metabolic syndrome, grade and stage of liver disease, and liver histology were all significantly improved (P < 0.01). Hepatic MDA staining (35 +/- 18% vs. 23 +/- 14%; P = 0.02), CYP2E1 protein content (68 +/- 9% vs. 56 +/- 11%; P < 0.001), and steatosis (17 +/- 7% vs. 2 +/- 3%; P < 0.001) were significantly reduced following weight loss. CYP3A4/5 protein content was unchanged (57 +/- 13% vs. 55 +/- 13%; P = 0.433). The reduction in lipid peroxidation was independently associated with changes in CYP2E1 protein expression after bariatric surgery (r = 0.477; P = 0.033).

Elevations in hepatic lipid peroxidation and CYP2E1 expression that are seen in NAFLD improve significantly with weight loss induced by bariatric surgery.

Zearalenone (ZEN) is a fusarotoxin converted predominantly into alpha-zearalenol (alpha-Zol) and beta-zearalenol (beta-Zol) by hepatic hydroxysteroid dehydrogenases. The feeding of naturally contaminated grains with ZEN was associated with hyperestrogenic and adverse effects on humans and animals. There is a lack of information on the attribution of the toxic effects of these toxins. One wonders if these effects are due to the parent molecule (ZEN) or to its major metabolites (alpha-Zol and beta-Zol). Using human Caco-2 cells, we looked for the molecular mechanisms of toxicity of ZEN, alpha-Zol, and beta-Zol. Toxicity effects were studied by MTT viability assay and oxidative stress induction by measuring malondialdehyde (MDA) generation. To check whether the oxidative stress induction was associated to DNA lesions, we looked for DNA fragmentation by means of the Comet and the diphenylamine assays. To specify cell death pathway, we investigated caspase-3 activation, confirmed by poly(ADP-ribose) polymerase cleavage and by Bcl-2 depletion. Our results clearly demonstrated that ZEN as well as its two metabolites presented variable toxic effects. They induced cell death and an increase in MDA generation. These effects were associated to DNA fragmentation as well as caspase-3 activation. The observed toxic effects seem to be relieved by the metabolism of ZEN into alpha-Zol and beta-Zol.

Chemical carcinogenesis is a complex, multi-stage process and the relationship between dose and tumour formation is an important consideration in the risk assessment of chemicals. Extrapolation from empirical dose-response relationships obtained in experimental studies has been criticized, as it fails to take into account information on mode of action. Strategies for incorporating mode of action information into the risk assessment of chemical carcinogens are described, with a focus on hepatic cancer. Either toxicokinetic or toxicodynamic processes can be addressed. Whilst the former have been the focus of more attention to date, for example by using physiologically based modelling, there is increasing interest in the development of mode of action-based toxicodynamic models. These have the advantage that they do not require extreme assumptions, and may be amenable to paramaterization using human data. This is rarely if ever possible when using conventional dose-tumour response relationships. The approaches discussed are illustrated using chloroform as a case study. This compound is converted to a cytotoxic metabolite, phosgene, by CYP2E1 in liver and/or kidney. Cytotoxicity results in proliferative regeneration, with increased probability of tumour formation. Both physiologically based toxicokinetic and toxicodynamic models have been developed, and it is possible to use probabilistic approaches incorporating, for example, data on the distribution of hepatic CYP2E1 levels. Mode of action can provide an invaluable link between observable, experimental data, on both toxicokinetics and toxicodynamics, and chemical-specific risk assessment, based on physiological approaches.

A search of the scientific literature was carried out for physiochemical and biological data [i.e., IC50, LD50, Kp (cm/h) for percutaneous absorption, skin/water and tissue/blood partition coefficients, inhibition ki values, and metabolic parameters such as Vmax and Km] on 31 organophosphorus pesticides (OPs) to support the development of predictive quantitative structure-activity relationship (QSAR) and physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models for human risk assessment. Except for work on parathion, chlorpyrifos, and isofenphos, very few modeling data were found on the 31 OPs of interest. The available percutaneous absorption, partition coefficients and metabolic parameters were insufficient in number to develop predictive QSAR models. Metabolic kinetic parameters (Vmax, Km) varied according to enzyme source and the manner in which the enzymes were characterized. The metabolic activity of microsomes should be based on the kinetic activity of purified or cDNA-expressed cytochrome P450s (CYPs) and the specific content of each active CYP in tissue microsomes. Similar requirements are needed to assess the activity of tissue A- and B-esterases metabolizing OPs. A limited amount of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CaE) inhibition and recovery data were found in the literature on the 31 OPs. A program is needed to require the development of physicochemical and biological data to support risk assessment methodologies involving QSAR and PBPK/PD models.

Intestinal cytochrome P450 (P450) proteins play an important role in the biotransformation of drugs and may significantly limit their oral absorption and bioavailability. Therefore, we have investigated the amount of P450 proteins via Western blot analysis along the entire intestine of male and female rats. Despite of the use of an inbred rat strain, controlled housing conditions for the animals, and a timed sample preparation, high interindividual differences in the expression of all P450 proteins was observed. CYP3A (135-243 fmol/mg of protein) and CYP2B1 (107-645 fmol/mg of protein) were the most abundant P450 isoforms in the duodenum and jejunum of rat intestine but were present in neither the ileum nor the colon. Compared with CYP2B1 and CYP3A, CYP2D1 (25-71 fmol/mg of protein) and CYP2C6 (3-10 fmol/mg of protein) were only expressed in minor amounts. CYP2C11 could not be identified in the entire rat intestine. In conclusion, this is the first systematic evaluation and quantification of the expression of P450 proteins along the entire length of the intestine in both male and female rats. These data will provide a basis for a better understanding of the extent of intestinal metabolism along the gastrointestinal tract.

It is now widely accepted that the fraction of the dose metabolized by a given drug-metabolizing enzyme is one of the major factors governing the magnitude of a drug interaction and the impact of a polymorphism on (total) drug clearance. Therefore, most pharmaceutical companies determine the enzymes involved in the metabolism of a new chemical entity (NCE) in vitro, in conjunction with human data on absorption, distribution, metabolism and excretion. This so called reaction-phenotyping, or isozyme-mapping, usually involves the use of multiple reagents (e.g., recombinant proteins, liver subcellular fractions, enzyme-selective chemical inhibitors and antibodies). For the human CYPs, reagents are readily available and in vitro reaction-phenotyping data are now routinely included in most regulatory documents. Ideally, the various metabolites have been definitively identified, incubation conditions have afforded robust kinetic analyses, and well characterized (high quality) reagents and human tissues have been employed. It is also important that the various in vitro data are consistent (e.g., scaled turnover with recombinant CYP proteins, CYP inhibition and correlation data with human liver microsomes) and enable an integrated in vitro CYP reaction-phenotype. Results of the in vitro CYP reaction-phenotyping are integrated with clinical data (e.g., human radiolabel and drug interaction studies) and a complete package is then submitted for regulatory review. If the NCE receives market approval, information on key routes of clearance and their associated potential for drug-drug interactions are included in the product label. The present review focuses on in vitro CYP reaction-phenotyping and the integration of data. Relatively simple strategies enabling the design and prioritization of follow up clinical studies are also discussed.

trans-Bromuconazole is a chiral chemical representative of a class of triazole derivatives known to inhibit specific fungal cytochrome P-450 (CYP) reactions. Kinetic measurements and delineation of metabolic pathways for triazole chemicals within in vitro hepatic microsomes are needed for accurate risk assessment and predictive in vivo physiological modeling. The studies described here were conducted with rat liver microsomes to determine Michaelis-Menten saturation kinetic parameters (Vmax and KM) for trans-bromuconazole using both substrate depletion and product formation reaction velocities. Kinetic parameters determined for trans-bromuconazole depletion at varying protein levels incubated at physiological temperature 37 degrees C resulted in a KM value of 1.69 microM and a Vmax value of 1398 pmol/min/mg protein. The concomitant linear formation of two metabolites identified using liquid chromatography/time-of-flight mass spectrometry (LC/MS-TOF) and LC-MS/MS indicated hydroxylation of the trans-bromuconazole dichlorophenyl ring moiety. KM values determined for the hydroxylated metabolites were 0.87 and 1.03 microM, with Vmax values of 449 and 694 pmol/min/mg protein, respectively. Chemical inhibition assays and studies conducted with individual purified human recombinant enzymes indicated the CYP3A subfamily was primarily responsible for biotransformation of the parent substrate. Additionally, trans-bromuconazole was found to undergo stereoselective metabolism as evidenced by a change in the enantiomeric ratio (trans-/trans+) with respect to time.

The therapeutic effects of immunosuppressive drugs are known to deviate largely between patients, but efficient strategies for the differentiation of patients who show clinical resistance to immunosuppressive therapies have not been established. Accordingly, a considerable number of patients receive treatment with immunosuppressive drugs despite the onset of serious side effects and poor responses. Cellular pharmacodynamics of immunosuppressive drugs in vitro using peripheral lymphocytes derived from each patient, an attractive way to distinguish resistant patients, is respected and has been applied to the carrying out of individualized immunosuppressive therapy. In this article, I summarize experimental procedures for assaying immune cell responses to immunosuppressive drugs in vitro, and highlight the relationship between cellular sensitivity to immunosuppressive drugs and the therapeutic efficacy of drugs in organ transplantation and several immunological disorders. I will also overview the molecular mechanisms and genetic bases for cellular and clinical resistance to immunosuppressive drugs. Lastly, the future clinical prospects for the application of in vitro drug sensitivity tests for "patient-tailored" immunosuppressive therapies are discussed.

Much progress has been made in understanding the complex pharmacokinetics of trichloroethylene (TCE) . Qualitatively, it is clear that TCE is metabolized to multiple metabolites either locally or into systemic circulation. Many of these metabolites are thought to have toxicologic importance. In addition, efforts to develop physiologically based pharmacokinetic (PBPK) models have led to a better quantitative assessment of the dosimetry of TCE and several of its metabolites. As part of a mini-monograph on key issues in the health risk assessment of TCE, this article is a review of a number of the current scientific issues in TCE pharmacokinetics and recent PBPK modeling efforts with a focus on literature published since 2000. Particular attention is paid to factors affecting PBPK modeling for application to risk assessment. Recent TCE PBPK modeling efforts, coupled with methodologic advances in characterizing uncertainty and variability, suggest that rigorous application of PBPK modeling to TCE risk assessment appears feasible at least for TCE and its major oxidative metabolites trichloroacetic acid and trichloroethanol. However, a number of basic structural hypotheses such as enterohepatic recirculation, plasma binding, and flow- or diffusion-limited treatment of tissue distribution require additional evaluation and analysis. Moreover, there are a number of metabolites of potential toxicologic interest, such as chloral, dichloroacetic acid, and those derived from glutathione conjugation, for which reliable pharmacokinetic data is sparse because of analytical difficulties or low concentrations in systemic circulation. It will be a challenge to develop reliable dosimetry for such cases.

Zearalenone (ZEA), a Fusarium toxin, is frequently found in animal feed materials. It is known to exert oestrogenic effects in all animals tested but susceptibility varies between species, possibly reflecting differences in the metabolic processing of ZEA, which predominantly involves hydroxylations, assumed to be catalysed by 3alpha- and 3beta- hydroxysteroid dehydrogenases, as well as conjugation with glucuronic acid. In this study, the biotransformation of ZEA by hepatic subcellular fractions of various domestic animals was investigated and compared to the rat. Notable inter-species differences in terms of the rate of absolute and relative metabolite production in the different subcellular fractions were identified. The highest amount of alpha-zearalenol (alpha-ZOL) was produced by pig hepatic microsomes (V(max)=795.8+/-122.7pmol/mg/min), whereas in chicken microsomes the highest amounts of beta-zearalenol (beta-ZOL) (V(max)=1524+/-29.7pmol/mg/min) could be measured. Except for sheep and cattle, the efficiency of alpha-ZOL production (expressed as the ratio of apparent V(max)/k(m)) was higher in the microsomal fraction compared to the post-mitochondrial fraction. In contrast, the apparent efficiency of beta-ZOL production was high in pigs, cattle, chickens and rats, but very low in sheep. Conjugation of ZEA with glucuronic acid was investigated, and the results indicated significant inter-species differences in the rate of glucuronidation, which was saturable at low concentrations in all species tested, except pigs. The significant differences between the percentages of glucuronidation of ZEA, alpha-ZOL, and beta-ZOL suggest not only differences in the affinity of the individual substrate, but might also indicate the presence of different isoforms of uridine diphosphate glucuronyl transferases (UDPGTs). The results are of clinical relevance, as they contribute to the understanding of the species-specific susceptibility towards exposure to ZEA.

Acute exposure guideline levels (AEGLs) are derived to protect the human population from adverse health effects in case of single exposure due to an accidental release of chemicals into the atmosphere. AEGLs are set at three different levels of increasing toxicity for exposure durations ranging from 10 min to 8 h. In the AEGL setting for methylene chloride, specific additional topics had to be addressed. This included a change of relevant toxicity endpoint within the 10-min to 8-h exposure time range from central nervous system depression caused by the parent compound to formation of carboxyhemoglobin (COHb) via biotransformation to carbon monoxide. Additionally, the biotransformation of methylene chloride includes both a saturable step as well as genetic polymorphism of the glutathione transferase involved. Physiologically based pharmacokinetic modeling was considered to be the appropriate tool to address all these topics in an adequate way. Two available PBPK models were combined and extended with additional algorithms for the estimation of the maximum COHb levels. The model was validated and verified with data obtained from volunteer studies. It was concluded that all the mentioned topics could be adequately accounted for by the PBPK model. The AEGL values as calculated with the model were substantiated by experimental data with volunteers and are concluded to be practically applicable.

Human cytochrome P450 2E1 (CYP2E1) is a phase I metabolizing enzyme. It is involved in the biotransformation of xenobiotics and endogenous substrates. Inter-individual genetic polymorphisms of the CYP2E1 gene are associated with different cancer diseases as well as alcohol and nicotine dependence. We report here for the first time three novel alternative spliced mRNA transcripts which are more frequently present in lung carcinoma cell lines as in hepatocyte cell lines. They are unexpected detectable in blood leukocytes from healthy volunteers but not in normal and cancerous lung tissue. The full-length wildtype transcript of CYP2E1 is described to be concomitant to an alternatively spliced mRNA transcript. Stimulation with CYP2E1-inducing agents did not change the splicing transcript pattern. The three splicing variants should lead to truncated non-functional proteins. Thus the genetic diversity of CYP2E1 is additionally extended at the transcriptional level of gene expression. The physiological role of the splicing variants is not known, yet, but they seem to be related to the carcinogenic property of the cell lines.