Toxicokinetic (TK) behavior of most chemicals is determined to a large extent by metabolism (xenobiotic-metabolizing enzymes). If metabolic processes were to be measured in in vitro cellular systems, it is of utmost importance to ensure that they are actually functional or at least incorporated in one way or another and at levels similar to those in vivo.In this chapter, current and future experimental possibilities to develop and characterize cells capable of metabolism and having other important dispositional characteristics mimicking the in vivo situation are described. Consideration is also given to incorporating this information into tissue models, and some examples are presented, with special reference to the metabolic and disposition competence of the systems. In conclusion, in vitro cellular systems should be developed and validated in a much more detailed way, taking into account the early characterization of metabolism and other important kinetic processes, because the thorough characterization is the prerequisite for the validation and use of cellular systems for pharmacological and toxicological studies.

Ochratoxin A (OTA) is a common foodborne mycotoxin. Besides its classical toxicities, it is also associated with the impairment of steroidogenesis in rats. It is hypothesized that OTA may act as an endocrine disruptor by intervening 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD). To address this hypothesis, human placental cells JEG-3 were used in vitro to examine the effects of short- and long-term OTA exposures on expression levels of 3β-HSD1 and progesterone secretion at 24-96h. Results showed that both cytotoxic and non-cytotoxic levels of OTA induced 3β-HSD1 mRNA expression by 281-378% at 72 and 96h. A significant induction (43-316%) of 3β-HSD1 protein expression was observed at 48, 72 and 96h, and the progesterone production with the involvement of 3β-HSD1 was significantly increased by 22-89% after 48-96h. This is the first study to demonstrate OTA up-regulates 3β-HSD1 expression in human placental cells, indicating the potential endocrine-disrupting property of OTA.

Liver is the most important organ involved in biotransformation of xenobiotics. Within the main organisational unit, the hepatocyte, is an assembly of enzymes commonly classified as phase I and phase II enzymes. The phase I enzymes principally cytochrome P450 catalyse both oxidative and reductive reactions of a bewildering number of xenobiotics. Many of the products of phase I enzymes become substrates for the phase II enzymes, which catalyse conjugation reactions making use of endogenous cofactors. As xenobiotic metabolising enzymes are responsible for the toxicity of many chemicals and drugs, testing the role of the biotransformation enzymes and the transporters within the hepatocyte is critical. New methodologies may be able to provide information to allow for better in vitro to in vivo extrapolation of data.

1 Self-administration of complementary products concurrently with conventional medication is increasingly common The potential for cytochrome P450 (CYP) inhibition requires investigation The N-in-one assay with ten probe substrates for nine CYPs was used with human liver microsomes to investigate ten products. CYP inhibition was measured in a single liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis Estimated IC50-values were determined for the extracts that produced significant inhibition (less than 100 mu g ml(-1))2. Inhibition of CYP2C19 by dong quai (IC50=13.7-14.3 mu g ml(-1) for the methanolic extract) and CYP2D6 by goldenseal (IC50 = 67 and 6.3 mu g ml(-1) for the aqueous and methanolic extracts, respectively), are of particular concern as the potential for adverse interactions is high The inhibition of CYP2C8 by horsetail (IC50 = 93 pg ml(-1) for the aqueous extract) requires further investigation, as the potential for concurrent use with products that require CYP2C8 for metabolism is significant CYP3A4 inhibition varied depending on the probe reaction being monitored3 The earlier reported findings of inhibition by black cohosh, goldenseal and gotu kola were confirmed. The present work has shown that the N-in-one cocktail is a rapid and reliable method that can be used as an initial screen to help prioritize products that require more detailed investigations and it can also be applied to monitor product variability

Event Abstract Back to Event Food-drug interactions: elucidating complex mixtures and individual constituents Olavi Pelkonen1*, Pia Vuorela2, Moshe Finel3 and Jorma T. Ahokas4 1 University of Oulu, Department of Pharmacology and Toxicology, Finland 2 Åbo Akademi University, Finland 3 University of Helsinki, Molecular and biochemical pharmacology, Finland 4 RMIT-University, Australia Certain components in our food may affect the way patients respond to prescribed drugs, leading to either inefficacy of the treatment, or stimulating adverse drug effects. These two apparently opposite outcomes could both be due to interactions of certain food components with one or more of the enzymes that our body has for protection against chemicals from the environment (xenobiotics). These enzymes play important roles in regulating the concentration of most drugs and, therefore, their inhibition by food components, or up-regulating their expression level, could lead to unwanted and harmful consequences. Yet, it is currently very difficult to predict and thereby avoid such food-drug interactions due to a lack of sufficient and specific knowledge. The major goal of our ongoing project is to characterize the risks and reduce future cases of harmful food-drug interactions, particularly among the more sensitive sections of the population like the elderly and patients undergoing extensive drug treatments due to severe diseases. In order to reach this goal it is essential to develop tools and approaches to gain deeper knowledge into this topic. In line with this, we are working in four different but interacting topics: 1. We have studied the in vitro interactions of 10 most popular (in Australia) herbal preparations with human hepatic cytochrome P450 enzymes by employing an LC-MS method (Sevior et al, Xenobiotica 2010; 40(4): 245–254). A number of in vitro interactions were observed, with obvious implications for further more refined studies. 2. We have investigated the metabolism and potential interactions of thujone, a major neurotoxic alkaloid in Artemisia absinthum (absinthe drink), in a number of human liver preparations and identified the participating P450 enzymes. 3. We have studied metabolism and interactions of Angelica archangelica extracts as well as constituent furocoumarins, in order to elucidate possibilities to extrapolate from individual components to a complex mixture containing different amounts of those components. Angelica extract and many furocoumarins seem to be potent inhibitors of at least CYP1A2 and to a lesser extent, many other CYPs. After the above mentioned studies aimed at testing necessary tools and approaches and demonstrating the feasibility of such studies, we plan to proceed to targeted in vivo studies in human volunteers, to elucidate the possibilities to perform in vitro – in vivo extrapolations and to improve risk assessment. Support: the above mentioned studies are being supported by the Academy of Finland (Council for Health Sciences) and the Finnish Agency for Research and Innovation (TEKES). Keywords: Food-Drug Interactions, herbal medicines Conference: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010, Thessaloniki, Greece, 1 Oct - 5 Oct, 2010. Presentation Type: Invited speaker Topic: Food, drugs and environmental xenobiotics Citation: Pelkonen O, Vuorela P, Finel M and Ahokas JT (2010). Food-drug interactions: elucidating complex mixtures and individual constituents. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00184 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 05 Mar 2011; Published Online: 04 Nov 2010. * Correspondence: Dr. Olavi Pelkonen, University of Oulu, Department of Pharmacology and Toxicology, Oulu, Finland, olavi.pelkonen@oulu.fi Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Olavi Pelkonen Pia Vuorela Moshe Finel Jorma T Ahokas Google Olavi Pelkonen Pia Vuorela Moshe Finel Jorma T Ahokas Google Scholar Olavi Pelkonen Pia Vuorela Moshe Finel Jorma T Ahokas PubMed Olavi Pelkonen Pia Vuorela Moshe Finel Jorma T Ahokas Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The hypolipidemic fibrates have been identified as agonists of the peroxisome proliferator-activated receptor alpha (PPARalpha), which plays a critical role in the regulation of cardiac fatty acid metabolism. Despite the widespread clinical use of fibrates, their role in myocardial oxidative stress and fatty acid composition is less known. In this study, male Sprague-Dawley rats were treated with either vehicle (olive oil, 1 ml/kg) or clofibrate (300 mg/kgday i.p.) for 1-14 days. Lipid peroxidation in heart homogenate was determined by thiobarbituric acid reactive substance (TBARS) assay. Results show that hearts from clofibrate-treated rats are more susceptible to FeSO(4)-induced TBARS production. The antioxidants including catalase and glutathione-related enzymes were marginally affected. We demonstrated that myocardial fatty acid composition was dramatically altered by clofibrate treatment. In hearts from clofibrate-treated rats, the principal n-6 polyunsaturated fatty acids (PUFAs), linoleic acid (C18:2 n-6) and arachidonic acid (C20:4 n-6), was significantly reduced, while the content of the principal n-3 PUFA, docosahexaenoic acid (C22:6 n-3), was markedly increased. The overall effect was to reduce n-6/n-3 ratio and increase the unsaturation extent of myocardial fatty acids. Functional study showed that hearts from clofibrate-treated rats had an improved recovery of post-ischemic contractile function and reduced ischemia/reperfusion (I/R)-induced infarct size. The data shows that clofibrate has a profound impact on cardiac fatty acid composition, which may contribute to its cardioprotective effect.

Fluoroacetate-specific defluorinase (FSD) is a critical enzyme in the detoxication of fluoroacetate. This study investigated whether FSD can be classed as a glutathione S-transferase (GST) isoenzyme with a high specificity for fluoroacetate detoxication metabolism. The majority of FSD and GST activity, using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as GST substrates, in rat liver was cytosolic. GSTT1 specific substrate, EPNP caused a slight non-competitive inhibition of FSD activity. CDNB, a general substrate of GST isoenzyme, was a more potent non-competitive inhibitor of FSD activity. The fluoroacetate defluorination activity by GST isoenzymes was determined in this study. The results showed that the GSTZ1C had the highest fluoroacetate defluorination activity of the various GST isoenzymes studied, while GSTA2 had a limited activity toward fluoroacetate. The human GSTZ1C recombinant protein then was purified from a human GSTZ1C cDNA clone. Our experiments showed that GSTZ1C catalysed fluoroacetate defluorination. GSTZ1 shares many of the characteristics of FSD; however, it accounts only for 3% of the total cytosolic FSD activity. GSTZ1C based enzyme kinetic studies has low affinity for fluoroacetate. The evidence suggests that GSTZ1 may not be the major enzyme defluorinating fluoroacetate, but it does detoxify the fluoroacetate. To clarify the identity of enzymes responsible for fluoroacetate detoxication, further studies of the overall FSD activity are needed.

Two forms of fluoroacetate-specific defluorinase (FSD) were purified from rat hepatic cytosol. The first form, FSD 1 (molecular weight 38 kDa), contained 81% of the total cytosolic fluoroacetate defluorination activity and did not bind to the glutathione-affinity, orange A or mono P columns used in the purification procedures. The second form, FSD2 (molecular weight 27 kDa), contained only 13% of the fluoroacetate defluorination activity, had a pI=7.8, and exhibited a high glutathione S-transferase (GST)-like activity towards dichloroacetic acid. The FSD1 proteins were identified from peptide mass data and best matched with rat sorbitol dehydrogenase (SDH) (short form), although pure sheep liver SDH enzyme did not possess defluorination activity when subsequently investigated. The FSD2 protein was identified from peptide mass data and best matched with the amino acid sequence of mouse and human Zeta 1 of glutathione S-transferase (GSTZ1) and showed a high GSTZ1 specific activity. This study suggests that the major FSD component (FSD1) represents a new and unique dehalogenating or dehydrogenating enzyme present in rat liver cytosol. The minor FSD component (FSD2) is due to the GSTZ1 present in rat liver cytosol. However, it is not yet clear that FSD1 is indeed SDH and FSD2 is indeed GSTZ1, due to sequence homology being less than 60 and 45%, respectively.

RMIT University was the first to offer a comprehensive Masters of Toxicology in Australasia 19 years ago. In 2001 the program was transformed into two stages, leading to a Graduate Diploma and Master of Applied Science in Toxicology. Now, these programs are fully online and suitable for graduates living and working anywhere in the world. The modular distance-learning courses are specifically designed to equip students with essential skills for entering fields such as chemical and drug evaluation; risk assessment of chemicals in the workplace; environmental and food toxicology. RMIT's online course delivery system has made it possible to deliver the toxicology programs, both nationally and internationally. The learning material and interactive activities (tests and quizzes, discussion boards, chat sessions) use Blackboard and WebBoard, each with a different educational function. Students log in to a Learning Hub to access their courses. The Learning Hub enables students to extend their learning beyond the classroom to the home, workplace, library and any other location with Internet access. The teaching staff log in to the Learning Hub to maintain and administer the online programs and courses which they have developed and/or which they teach. The Learning Hub is also a communication tool for students and staff, providing access to email, a diary and announcements. The early experience of delivering a full toxicology program online is very positive. However this mode of teaching continues to present many interesting technical, educational and cultural challenges, including: the design and presentation of the material; copyright issues; internationalization of content; interactive participation; and the assessment procedures.

The Australian Institute of Marine Science (AIMS) conducted a pilot study around the Harriet A oil production platform on the Northwest Shelf of Australia. We evaluated hepatic ethoxyresorufin-O-deethylase (EROD) activity, fluorescent aromatic compounds (FACs) in bile and immunodetection of CYP1A-like proteins in two Australian tropical fish species, Gold-Spotted Trevally (Carangoides fulvoguttatus) and Bar-Cheeked Coral Trout (Plectropomus maculatus) to assess exposure to petroleum hydrocarbons associated with produced formation water (PFW). Additionally, the incidence of hydrocarbon-degrading bacteria isolated from the liver and bile of all fish captured was examined. Low EROD activity was found in both species, with EROD activity in C. fulvoguttatus showing significant site differences. FACs and CYP1A protein levels in C. fulvoguttatus showed a clear trend in hydrocarbon exposure consistent with hydrocarbon chemistry data: Harriet A>Harriet C>reference site. P. maculatus showed elevated levels of FACs at Harriet A as compared to the reference site and demonstrated detectable levels of CYP1A-like proteins at these two sites. Hydrocarbon-degrading bacteria were found in the liver and bile of both species, yet there was no correlation by sites. Our results demonstrate that C. fulvoguttatus and P. maculatus have potential as indicator species for assessing the effects from exposure to petroleum hydrocarbons. Both FACs and CYP1A are providing warning signs that there is potential for biological effects on fish populations exposed to PFW around the Harriet A production platform.

The surface of Lactobacillus rhamnosus strain GG (LGG) has previously been shown to bind aflatoxin B1 (AFB1) effectively, it being a food-borne carcinogen produced by certain species of Aspergillus fungi. To establish which components of the cell envelope are involved in the AFB1 binding process, exopolysaccharides and a cell wall isolate containing peptidoglycan were extracted from LGG and its AFB1 binding properties were tested. LGG was also subjected to various enzymatic and chemical treatments and their effects on the binding of AFB1 by LGG were examined. No evidence was found for exopolysaccharides, cell wall proteins, Ca2+ or Mg2+ being involved in AFB1 binding. The AFB1 binding activity of the cell wall isolate indicates that AFB1 binds to the cell wall peptidoglycan of LGG or compounds tightly associated with the peptidoglycan.

Defluorination activity in liver subcellular fractions was investigated in species of rodent, marsupials and monotremes. The defluorination activity in the mouse and rat was compared with that of two Australian marsupials, the brushtail possum and tamar wallaby to dtermine the species related differences in fluoroacetate sensitivity. The results indicated that there are clear species differences in the subcellular distribution of defluorination activity in all species, the marsupials possessed a certain amount of defluorination ability in their mitochondrial fractions. The nuclear fractions generally contained a consistent amount of defluorination activity across the four species, indicating the need for further investigation. The single playpus and echidna samples also exhibited a similar distribution of defluorination activity in the liver subcellular fractions. These results clearly localise the fluoroacetate defluorination activity in liver cytosol and provide further insight into the fluoroacetate detoxication ability of Austrlian marsupials and montoremes. The enzyme kinetic study revealed that the Australian marsupials living outside the range of fluoroacetate-containing plants appear to have a lower ability to detoxify fluoroacetate than rodents have. This suggests that marsupials may be more disadvantaged in there metabolic detoxication of some xenobiotics than previously thought.

The papers in these proceedings truly reflect the wide nature of research work that is being undertaken and the advances that have been achieved particularly in the therapeutic application of probiotics. All abstracts are available as pdf and html format.

Specific strains of lactic acid bacteria possessing antimutagenic properties are suggested to remove mutagenic contaminants of foods through binding and an investigation of their substrate specificity is required. The ability of Lactobacillus rhamnosus strains GG and LC-705 in viable and non-viable (heat- and acid-treated) forms to remove both dietary mutagens and other aromatic dietary substrates from solution was studied using HPLC. Overall, removal increased in the order: caffeine = vitamin B-12 = folic acid < ochratoxin A< aflatoxin B-1 = PhIP (2-amino-1-methyl-6-phenyl-imidazo[ 4,5-b] pyridine) <Trp-P-1 (3-amino-1, 4-dimethyl-5H-pyrido[ 4,3-b] indole) (p <0.05). Aflatoxin B-1, Trp-P-1 and PhIP were removed in high amounts (77-95%) and ochratoxin A was removed in moderate amounts (36-76%). By contrast, only minimal amounts of caffeine, vitamin B-12 and folic acid were removed (9-28%). The significant removal of selected mutagens, but not other substrates, suggests these strains may be useful for dietary detoxification. Since exposure to multiple mutagens is likely, the removal of aflatoxin B-1 and Trp-P-1 from a mixture of these substrates was also investigated. Removal of AFB(1) significantly increased (p <0.05) in the presence of Trp-P-1, while removal of Trp-P-1 significantly decreased (p <0.05) in the presence of AFB(1). Overall, no significant differences in removal were found between bacterial strains or between viable, heat- and acid-treated bacteria.