Metabolic Biotransformation Research Articles

Erratum to: Computer-Assisted HPLC Method Development for Determination of Tolmetin and Possible Kinetic Modulators of Its Oxidative Metabolism in Vivo

Following administration of the acidic drug tolmetin (TOL) anaphylactic reactions occurred, which have been hypothesized to be related to the formation of reactive acyl glucuronides. Recently, glutathione adducts have been detected upon incubation of TOL with human liver microsomal preparations, which proved that oxidative activation might also be a pathway of formation of reactive—possibly toxic—glutathione metabolites of TOL. The aim of this work was to develop a new and robust HPLC method to investigate the in vivo effect of 2 coadministered drugs/nutritional supplements on the kinetics of TOL in rats (cimetidine; CIM) known to be a potent inhibitor of CYP3A4, an enzyme that catalyzes the oxidative metabolism and Quercetin; and QUE which induces UGT1A6, an enzyme involved in glucuronidation of acidic drugs. DryLab®, a computer simulation software package, was used to assist in the development and optimization of the HPLC method used for separation of TOL and the two potential kinetic modulators together with three potential internal standards (zomepirac, carvedilol and fexofenadine). The method was validated in biological samples obtained from rats. Non-compartmental pharmacokinetic analysis of data obtained from plasma and rat liver tissue showed significantly higher concentrations of TOL in the presence of CIM; and significantly longer elimination half-life lives in presence of QUE, which implies that drugs or food components interacting with CYP3A4 cause alteration in the metabolic oxidative biotransformation of TOL in vivo leading to accumulation of TOL in the body through a decrease of its clearance. These findings might account for to the side-effects associated with TOL when co-administered with such kinetic modulators.

Investigation of the metabolic biotransformation of substance P in liver microsomes by liquid chromatography quadrupole ion trap mass spectrometry

Substance P (SP) belongs to the tachykinin family and plays an essential role in pain transmission and in neurogenic inflammation. It can be detected in the central and peripheral nervous systems. The objectives of this study were to establish SP metabolic stability in liver microsomes in three species (rat, mouse and human), and identify and characterize SP metabolites by LC-MS/MS. Endogenous peptide metabolism is not well documented and this is particularly true for neuropeptides participating in neurogenic inflammation. In vitro, T(1/2) results in pooled liver microsomes were 9.2, 5.6 and 18.6 min for rat, mouse and human liver microsomes, respectively. Five major SP metabolites were identified and quantified, including C-terminal SP fragments SP(3-11) , SP(5-11) , SP(6-11) , SP(8-11) as well as N-terminal fragment SP(1-7) . The results suggest significant differences between species in SP metabolite concentrations. Consequently, the metabolic profile of each species is distinctive and may have a significant impact on biomolecular mechanisms involved in specific pathophysiological changes.

Modeling Chemical Interaction Profiles: I. Spectral Data-Activity Relationship and Structure-Activity Relationship Models for Inhibitors and Non-inhibitors of Cytochrome P450 CYP3A4 and CYP2D6 Isozymes

An interagency collaboration was established to model chemical interactions that may cause adverse health effects when an exposure to a mixture of chemicals occurs. Many of these chemicals—drugs, pesticides, and environmental pollutant—interact at the level of metabolic biotransformations mediated by cytochrome P450 (CYP) enzymes. In the present work, spectral data-activity relationship (SDAR) and structure-activity relationship (SAR) approaches were used to develop machine-learning classifiers of inhibitors and non-inhibitors of the CYP3A4 and CYP2D6 isozymes. The models were built upon 602 reference pharmaceutical compounds whose interactions have been deduced from clinical data, and 100 additional chemicals that were used to evaluate model performance in an external validation (EV) test. SDAR is an innovative modeling approach that relies on discriminant analysis applied to binned nuclear magnetic resonance (NMR) spectral descriptors. In the present work, both 1D 13C and 1D 15N-NMR spectra were used together in a novel implementation of the SDAR technique. It was found that increasing the binning size of 1D 13C-NMR and 15N-NMR spectra caused an increase in the tenfold cross-validation (CV) performance in terms of both the rate of correct classification and sensitivity. The results of SDAR modeling were verified using SAR. For SAR modeling, a decision forest approach involving from 6 to 17 Mold2 descriptors in a tree was used. Average rates of correct classification of SDAR and SAR models in a hundred CV tests were 60% and 61% for CYP3A4, and 62% and 70% for CYP2D6, respectively. The rates of correct classification of SDAR and SAR models in the EV test were 73% and 86% for CYP3A4, and 76% and 90% for CYP2D6, respectively. Thus, both SDAR and SAR methods demonstrated a comparable performance in modeling a large set of structurally diverse data. Based on unique NMR structural descriptors, the new SDAR modeling method complements the existing SAR techniques, providing an independent estimator that can increase confidence in a structure-activity assessment. When modeling was applied to hazardous environmental chemicals, it was found that up to 20% of them may be substrates and up to 10% of them may be inhibitors of the CYP3A4 and CYP2D6 isoforms. The developed models provide a rare opportunity for the environmental health branch of the public health service to extrapolate to hazardous chemicals directly from human clinical data. Therefore, the pharmacological and environmental health branches are both expected to benefit from these reported models.

Identification and Characterization of Reactive Metabolites in Natural Products-Driven Drug Discovery

Toxicity of natural products arising from their metabolic biotransformation into reactive chemical intermediates is an important reason for high attrition rates in early drug discovery efforts. Screening promising natural products for their likelihood to form such metabolites is therefore an important step in identifying potential liabilities in the drug development process. However, such screening is complicated by the need to have test methods that are sensitive, reliable, accurate, efficient, and cost-effective enough to allow for routine identification and characterization of the reactive metabolites. These metabolites are typically formed in minute quantities, usually through minor metabolic pathways, and, due to their highly reactive and therefore transient chemical nature, pose considerable analytical challenges in attempts to determine their properties. Understanding the formation of reactive metabolites may be used as the basis for synthetic chemical modification of parent natural products aimed at bypassing such harmful bioactivation. This paper highlights the general principles and protocols commonly used to predict and study the formation of reactive metabolites in vitro and how the data obtained from such studies can be used in the development of safer drugs from natural products.

Metabolic Patterns and Biotransformation Activities of Resveratrol in Human Glioblastoma Cells: Relevance with Therapeutic Efficacies

Background Trans-resveratrol rather than its biotransformed monosulfate metabolite exerts anti-medulloblastoma effects by suppressing STAT3 activation. Nevertheless, its effects on human glioblastoma cells are variable due to certain unknown reason(s).Methodology/Principal FindingsCiting resveratrol-sensitive UW228-3 medulloblastoma cell line and primarily cultured rat brain cells/PBCs as controls, the effect of resveratrol on LN-18 human glioblastoma cells and its relevance with metabolic pattern(s), brain-associated sulfotransferase/SULT expression and the statuses of STAT3 signaling and protein inhibitor of activated STAT3 (PIAS3) were elucidated by multiple experimental approaches. Meanwhile, the expression patterns of three SULTs (SULT1A1, 1C2 and 4A1) in human glioblastoma tumors were profiled immunohistochemically. The results revealed that 100 µM resveratrol-treated LN-18 generated the same metabolites as UW228-3 cells, while additional metabolite in molecular weight of 403.0992 in negative ion mode was found in PBCs. Neither growth arrest nor apoptosis was found in resveratrol-treated LN-18 and PBC cells. Upon resveratrol treatment, the levels of SULT1A1, 1C2 and 4A1 expression in LN-18 cells were more up-regulated than that expressed in UW228-3 cells and close to the levels in PBCs. Immunohistochemical staining showed that 42.0%, 27.1% and 19.6% of 149 glioblastoma cases produced similar SULT1A1, 1C2 and 4A1 levels as that of tumor-surrounding tissues. Unlike the situation in UW228-3 cells, STAT3 signaling remained activated and its protein inhibitor PIAS3 was restricted in the cytosol of resveratrol-treated LN-18 cells. No nuclear translocation of STAT3 and PIAS3 was observed in resveratrol-treated PBCs. Treatment with STAT3 chemical inhibitor, AG490, committed majority of LN-18 and UW228-3 cells but not PBCs to apoptosis within 48 hours.Conclusions/SignificanceLN-18 glioblastoma cells are insensitive to resveratrol due to the more inducible brain-associated SULT expression, insufficiency of resveratrol to suppress activated STAT3 signaling and the lack of PIAS3 nuclear translocation. The findings from PBCs suggest that an effective anticancer dose of resveratrol exerts little side effect on normal brain cells.

Assessing the Chronic Aquatic Toxicity of Phthalate Ester Plasticizers

ABSTRACT Phthalic acid esters (PAE) are a class of chemicals varying greatly in terms of uses, properties, and toxicity. C1 to C4 PAE are used in non-vinyl commercial products and pharmaceuticals. C8 to C10 PAE are additives imparting flexibility to vinyl resins. The purpose of the present study is to assess chronic effects of PAE on aquatic organisms. Studies show that populations of fish and invertebrates may be adversely affected by exposure to C1 to C4 PAE, but are not adversely affected by exposure to C8 or higher PAE. Secondary endpoints, including molecular, biochemical, and/or histological responses to chemical exposure, do not appear to predict effects related to primary endpoints of survival, growth and development, or reproductive fitness. A previously published risk assessment for C1 to C4 PAE demonstrated low risks in North American and Western European surface waters. Risk assessments conducted by authorities in Europe with di-2-ethylhexyl-, di-isononyl-, and di-isodecyl phthalates have concluded no risks to aquatic organisms due to aqueous solubility constraints, low expected surface water concentrations, and metabolic biotransformation capabilities of aquatic organisms. The present review of chronic aquatic toxicity data, which includes data from studies performed subsequent to the risk assessments, confirms these earlier conclusions.

Differentiation of human embryonic stem cells along a hepatic lineage

The limited availability of hepatic tissue suitable for the treatment of liver disease and drug discovery research advances the generation of hepatic-like cells from alternative sources as a valuable approach. In this investigation we exploited a unique hepatic differentiation approach to generate hepatocyte-like cells from human embryonic stem cells (hESCs). hESCs were cultured for 10–20 days on collagen substrate in highly defined and serum free hepatocyte media. The resulting cell populations exhibited hepatic cell-like morphology and were characterized with a variety of biological endpoint analyses. Real-time PCR analysis demonstrated that mRNA expression of the ‘stemness’ marker genes NANOG and alkaline phosphatase in the differentiated cells was significantly reduced, findings that were functionally validated using alkaline phosphatase activity detection measures. Immunofluorescence studies revealed attenuated levels of the ‘stemness’ markers OCT4, SOX2, SSEA-3, TRA-1-60, and TRA-1-81 in the hepatic-like cell population. The hepatic character of the cells was evaluated additionally by real-time PCR analyses that demonstrated increased mRNA expression of the hepatic transcription factors FOXA1, C/EBPα, and HNF1α, the nuclear receptors CAR, RXRα, PPARα, and HNF4α, the liver-generated plasma proteins α-fetoprotein, transthyretin, transferrin, and albumin, the protease inhibitor α-1-antitrypsin, metabolic enzymes HMGCS2, PEPCK, and biotransformation enzymes CYP3A7, CYP3A4, CYP3A5, and CYP2E1. Indocyanine green uptake albumin secretion and glycogen storage capacity further confirmed acquisition of hepatic function. These studies define an expeditious methodology that facilitates the differentiation of hESCs along a hepatic lineage and provide a framework for their subsequent use in pharmacological and toxicological research applications requiring a renewable supply of human hepatocytes.

Unbalanced biotransformation metabolism and oxidative stress status: implications for deficient fatty acid oxidation

The concept of accumulating xenobiotics within the human body as a health risk is well known. However, these compounds can also be endo-genous, as in the case of inborn errors of me-tabolism, and lead to some of the same symp-toms as seen in xenobiotic intoxication. Bio-transformation of both exogenous and endo-genous toxic compounds is an important function of the liver, and the critical balance between these systems is of fundamental importance for cellular health. We propose a novel model, to describe the critical balance between Phase I and Phase II biotransformation and how a disturbance in this balance will increase the oxidative stress status, with resulting pathological consequences. We further used deficient fatty acid oxidation to verify the proposed model, as deficient fatty acid oxidation is associated with the accumulation of characteristic metabolites. These accumulating metabolites undergo both Phase I and Phase II biotransformation reactions, with resulting depletion of biotransformation substrates and co-factors. Depletion of these important biomolecules is capable of disturbing the balance between Phase I and Phase II reactions, and disturbance of this balance will increase oxidative stress status. The value of the proposed model is illustrated by its application to a clinical case investigated in our laboratory. In this case the possibility of deficient fatty acid oxidation only became evident once the critical balance between Phase I and Phase II biotransformation was restored with oral replenishment of biotransformation substrates. In addition to bio-chemical improvement, there was also significant clinical improvement. The significance of this model lies within the treatment possibilities, as the assessment of biotransformation metabolism and oxidative stress status can lead to the development of nutritional treatment strategies to correct imbalances. This in turn may reduce the chances of, or delay the onset of certain disease states.

Persistent organic pollutants and methoxylated PBDEs in harbour porpoises from the North Sea from 1990 until 2008: Young wildlife at risk?

In the European North Sea, harbour porpoises are top predators with relatively long life spans and a limited capacity for metabolic biotransformation of contaminants compared to some other marine mammal species. As such, they are exposed to a mixture of persistent pollutants, such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), DDT and metabolites (DDXs), hexachlorobenzene (HCB) and chlordanes (CHLs) that bioaccumulate in their tissues. We report here on the levels of persistent organic pollutants and of the naturally-produced methoxylated PBDEs (MeO-PBDEs) in blubber, liver and kidney of harbour porpoise neonates (n = 3), calves (n = 15), juveniles (n = 6) and adults (n = 4) of the southern North Sea. Concentrations of almost all contaminant classes decrease slightly in all age groups over the period 1990–2008. For some classes (e.g. PCBs and DDXs) however, levels seem to increase little in harbour porpoise calves. In all animals, blubber had the highest concentrations, followed by liver and kidney, whereas liver and kidney were the preferred tissues for several compounds, such as octa- and deca-PCBs. Our data suggest that harbour porpoises calves are exposed to higher or comparable concentrations of POPs and of MeO-PBDEs and somewhat different patterns of selected POPs than adults, potentially placing them, and the entire population, at a disproportionate risk for exposure-related health effects.

Use of historic metabolic biotransformation data as a means of anticipating metabolic sites using MetaPrint2D and Bioclipse

BackgroundPredicting metabolic sites is important in the drug discovery process to aid in rapid compound optimisation. No interactive tool exists and most of the useful tools are quite expensive.ResultsHere a fast and reliable method to analyse ligands and visualise potential metabolic sites is presented which is based on annotated metabolic data, described by circular fingerprints. The method is available via the graphical workbench Bioclipse, which is equipped with advanced features in cheminformatics.ConclusionsDue to the speed of predictions (less than 50 ms per molecule), scientists can get real time decision support when editing chemical structures. Bioclipse is a rich client, which means that all calculations are performed on the local computer and do not require network connection. Bioclipse and MetaPrint2D are free for all users, released under open source licenses, and available from http://www.bioclipse.net.

Metabolic bioactivation of oestradiol-17β (E 2β) in mouse colon epithelial cells bearing ApcMin mutation

Epidemiological studies have revealed a protective role of oestrogens against the promotion of colorectal cancer (CRC). Therefore, the oestrogen metabolism status of colonic cells is studied to explain it. Loss of function of adenomatous polyposis coli ( Apc) gene product is an early and frequent event in human colorectal carcinogenesis. Normal ( Apc +/+) and premalignant ( Apc multiple intestinal neoplasia ( Min)/+ ) mouse colonic epithelial cells were used to compare their respective metabolic capabilities towards oestradiol-17β (E 2β), with or without an inducer of the CYP1 family, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). In both cell types, the major metabolite was oestradiol-17β-3-glucuronide. The formation of catechol (CE) metabolites by cytochromes P450 of the CYP1 family and their derivatives was shown. Among these metabolites, several O-methyl-ether derivatives were detected, as unconjugated metabolites in Apc +/+ cells and as glucuroconjugates in Apc Min/+ cells, after TCDD treatment. Apc Min/+ cells are metabolically more competent than Apc +/+ cells to produce different hydroxylated metabolites as well as glucuroconjugates. Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) experiments corroborate these results. Indeed, induction by TCDD has prevailing effects in gene expression of CYP1A1, CYP1A2 and CYP1B1 in Apc Min/+ cells, compared with Apc +/+ ones. Apc Min/+ cells displayed higher rates of oestrogen metabolic biotransformation than Apc +/+ ones, but exhibited two opposite tendencies. Apc Min/+ cells were able to detoxify E 2β mainly by the formation of glucuronides and displayed at the same time a striking potential to bioactivate E 2β by producing only the electrophilic 2-CE derivatives, not the 4-CE ones, even though a significant CYP1B1 mRNA induction was noticed. These specific electrophilic metabolites may form DNA adducts but are not prone to generate new mutations. Interestingly, the ultimate 2- O-methyl-ether metabolite of E 2β may be an endogenous protective factor against CRC promotion given its recognised anti-angiogenic and pro-apoptotic properties.

Mycosporine-like amino acid content in the sea anemones Aulactinia marplatensis, Oulactis muscosa and Anthothoe chilensis

The occurrence of mycosporine-like amino acids (MAAs) in the sea anemones Aulactinia marplatensis (Zamponi, 1977), Oulactis muscosa (Drayton in Dana, 1846) and Anthothoe chilensis (Lesson, 1830), from the rocky intertidal habitats on the coast of Mar del Plata, Argentina, was assessed by HPLC. The pattern of MAAs in the mussel Brachidontes rodriguezi, main component of the diet for A. marplatensis and O. muscosa, was as well determined. The results were comparatively analyzed together and with previously reported MAA content in species mainly of the genus Anthopleura. The correlation between the MAA concentration and light availability of their habitats is in line with the photoprotective role assigned to the compounds. The high proportion of mycosporine-taurine in the three species and the results for the evaluation of MAAs in the mussels point to a non-dietary origin or a regulated biotransformation metabolism of dietary MAAs and/or their precursors that is common to sea anemones.

Effects of exposure to Prestige-like heavy fuel oil and to perfluorooctane sulfonate on conventional biomarkers and target gene transcription in the thicklip grey mullet Chelon labrosus

Thicklip grey mullets Chelon labrosus inhabit coastal and estuarine areas where they can be chronically exposed to commonly released pollutants such as polycyclic aromatic hydrocarbons (PAHs) and perfluorinated compounds. These pollutants can also originate from accidental spills, such as the Prestige oil spill in 2002, which resulted in the release of a heavy fuel oil that affected coastal ecosystems in the Bay of Biscay. Peroxisome proliferation (PP), induced biotransformation metabolism, immunosuppression and endocrine disruption are some of the possible biological effects caused by such chemicals. With the aim of studying the effects of organic toxic chemicals on such biological processes at the transcriptional and at the cell/tissue level, juvenile mullets were exposed to the typical mammalian peroxisome proliferator perfluorooctane sulfonate (PFOS), and to fresh (F) and weathered (WF) Prestige-like heavy fuel oil for 2 and 16 days. First, fragments of genes relevant to biotransformation, immune/inflammatory and endocrine disruption processes were cloned using degenerate primers. Fuel oil elicited a significant PP response as proved by the transcriptional upregulation of palmitoyl-CoA oxidase (aox1), peroxisome proliferator activated receptor α (pparα) and retinoic X receptor, by the AOX1 activity induction and by the increased peroxisomal volume density. PFOS only elicited a significant induction of AOX1 activity at day 2 and of PPARα mRNA expression at day 16. All treatments significantly increased catalase mRNA expression at day 16 in liver and at day 2 in gill. Cyp1a transcription (liver and gill) and EROD activity were induced in fuel oil treated organisms. In the case of phase II metabolism only hepatic glutathione S-transferase mRNA was overexpressed in mullets exposed to WF for 16 days. Functionally, this response was reflected in a significant accumulation of bile PAH metabolites. WF treated fish accumulated mainly high molecular weight metabolites while F exposure resulted in accumulation of mainly low molecular ones. Fuel oil significantly regulated immune response related complement component C3 and hepcidin transcription followed by a significant regulation of inflammatory response related apolipoprotein-A1 and fatty acid binding protein mRNAs at day 16. These responses were accompanied by a significant hepatic inflammatory response with lymphocyte accumulations (IRLA) and accumulation of melanomacrophage centers (MMC). PFOS did not elicit any transcriptional response in the studied biotransformation and immune related genes, although histologically significant effects were recorded in IRLA and MMC. A significant reduction of lysosomal membrane stability was observed in all exposed animals. No endocrine disruption effects were observed in liver while brain aromatase mRNA was overexpressed after all treatments at day 2 and estrogen receptor α was downregulated under WF exposure at day 16. These results show new molecular and cellular biomarkers of exposure to organic chemicals and demonstrate that in mullets PP could be regulated through molecular mechanisms similar to those in rodents, although the typical mammalian peroxisome proliferator PFOS and heavy fuel oil follow divergent mechanisms of action.

Differential transcription of genes involved in peroxisome proliferation in thicklip grey mullets Chelon labrosus injected with benzo( a)pyrene

Benzo(a)pyrene (B( a)P) is a mutagenic polycyclic aromatic hydrocarbon (PAH) commonly released into the environment. B( a)P induces phase I biotransformation metabolism and peroxisome proliferation which is characterised in rodents by increased peroxisomal volume density, accompanied by the transcriptional induction of peroxisomal proteins. The aim of the present work was to study peroxisome proliferation at the transcriptional level, in comparison to the transcription of the well-characterised cytochrome P450 1A gene ( cyp1a) in the thicklip grey mullet Chelon labrosus. For this purpose, genes coding for the major peroxisomal membrane protein PMP70 and CYP1A were cloned using degenerate primers. Then juvenile mullets were single injected with B( a)P (5 mg/kg) and transcription of palmitoyl-CoA oxidase (aox1), multifunctional protein (mfp1), 3-ketoacyl-CoA thiolase (thio), Δ 2 ,Δ 4 dienoyl-CoA reductase 2, pmp70, catalase and cyp1a was semi-quantified in liver and gills 1 and 7 days after the injection. Transcription of aox1 and cyp1a was induced in gills 1 day after B( a)P injection. Cyp1a transcription was also induced in mullet liver one day after injection, indicating that B( a)P was available for the liver. This was further proved by the significant accumulation of B( a)P-like metabolites in bile 7 days after the injection. In liver, aox1, mfp1 and thio transcription was induced at day 1 followed by the induction of catalase transcription at day 7 that may reflect a response to an oxidative stress caused by B( a)P itself or by oxyradicals produced through the biotransformation metabolism and the peroxisomal β-oxidation. These hepatic responses were not reflected at AOX1 activity level. In conclusion, it has been shown for the first time that the three enzymes in the fish peroxisomal β-oxidation pathway are transcriptionally induced by B( a)P. It remains to be tested whether this enhanced transcription is reflected in an increase in the volume of peroxisomes.

First evidence of Okadaic acid acyl-derivative and Dinophysistoxin-3 in mussel samples collected in Chiloe Island, Southern Chile

This paper shows the detection of Diarrhetic Shellfish Poison (DSP) phycotoxins, using HPLC-FLD with pre-column derivatization procedure and HPLC-MS methods, in the analysis of shellfish extracts tested positive with the official DSP mouse bioassay. The shellfish samples were collected in Chiloe Island, Southern of Chile. The amount of Dinophysistoxin-3 (DTX-3) measured in the shellfish extracts were in average above the international safe limits for DSP content in the shellfish extracts analyzed. As internal control of detection and recovery, DTX-1 analytical standard was spiked into dichloromethane-clean shellfish extracts in order to calculate de extraction recovery of DTX-1. The average recovery was 97%. From all DSP toxins analyzed, the hydrolyzed extract samples appeared mainly DTX-3 in concentrations ranging from 99.40 +/- 1.22 to 257.73 +/- 12.46 ng/g digestive-glands. The acyl-Okadaic Acid (acyl-OA) was also detected in some samples, ranging from 1.02 +/- 1.4 to 3.07. +/- 1.6 ng of DSP toxin/g digestive-glands. This is the first report of acyl-OA ever found in Chilean shellfish samples. This data shows that shellfish samples were contaminated with a complex DSP toxins profile, in which DTX-3 is the major DSP toxin component, followed by DTX-1 and the acyl-OA as the minor one. The important findings showed in this study are the presence of both acyl-derivates (DTX-3 and Acyl-OA) which are the product of a main metabolic biotransformation that occurred inside the shellfish, in order to chelate DTX-1 and OA, transforming them into DTX-3 and the acyl-OA respectively. This metabolic biotransformation must be performed to avoid self-inhibition of their Protein Phosphatase 2A done by DTX-1 and OA, since both acyl-derivates (DTX-3 and acyl-OA) do not inhibit Protein Phosphatase 2A. This complex DSP toxins profile and the permanent presence of both acyl-derivates (DTX-3 and Acyl-OA) could explain the permanent diarrhea symptoms that experience patients who have ingested cooked shellfish in the southern of Chile. This diarrhea is not associated to Vibrio parahaemolyticus or other enteropathogens as had been suggested before. The massive shellfish consumption is an important Chilean cultural habit and now has become a major health issue in the southern of Chile.

Multimedia modeling of human exposure to chemical substances: The roles of food web biomagnification and biotransformation

The Risk Assessment IDentification And Ranking (RAIDAR) model is refined to calculate relative human exposures as expressed by total intake, intake fraction (iF), and total body burden (TBB) metrics. The RAIDAR model is applied to three persistent organic pollutants (POPs) and six petrochemicals using four mode-of-entry emission scenarios to evaluate the effect of metabolic biotransformation estimates on human exposure calculations. When biotransformation rates are assumed to be negligible, daily intake and iFs for the nine substances ranged over six orders of magnitude and TBBs ranged over 10 orders of magnitude. Including biotransformation estimates for fish, birds, and mammals reduced substance-specific daily intake and iF by up to 4.5 orders of magnitude and TBB by more than eight orders of magnitude. The RAIDAR iF calculations are compared to the European Union System for the Evaluation of Substances (EUSES) model iF calculations and differences are discussed, especially the treatment of food web bioaccumulation. Model selection and application assumptions result in different rankings of human exposure potential. These results suggest a need to critically consider model selection and to include reliable biotransformation rate estimates when assessing relative human exposure and ranking substances for priority setting. Recommendations for further model evaluations and revisions are discussed.

Data mining on carcinogenicity of chemical compounds by the JSM method

Prediction of the carcinogenicity of chemical compounds to rats was carried out by data mining analysis based on the logic of John Stuart Mill (JSM) and the fragmentary code of the substructure superposition (FCSS) data presentation language. The learning (608 compounds) and test (156 compounds) samples were taken from the database on the carcinogenicity of substances for laboratory animals developed by the Environmental Protection Agency of the USA (EPA USA). Predictions were made for 44% of the test samples. The prediction accuracy was 71%, sensitivity 73%, and specificity 67%. The causes for erroneous positive and negative predictions were studied, accounting for bioactivation of compounds in the course of metabolic biotransformations.

Cloning and transcription of nuclear receptors and other toxicologically relevant genes, and exposure biomarkers in European hake ( Merluccius merluccius) after the Prestige oil spill

In November 2002 the tanker Prestige released more than 60,000 t of a heavy fuel oil which spread over Galician waters and the Biscay Bay, affecting coastal ecosystems. Polycyclic aromatic hydrocarbons are the main components of the Prestige fuel oil and induce biotransformation metabolism and peroxisome proliferation in marine organisms. In vertebrates, this later response involves peroxisome proliferator-activated receptors (PPARs), transcription factors belonging to the nuclear receptor superfamily, that act upon heterodimerization with the retinoid X receptor (RXR). In order to assess the possible biological effects of the Prestige oil spill in the Biscay Bay, male and female juvenile and adult European hakes Merluccius merluccius were sampled in June and December 2004 and 2005. PCR screening of hake liver cDNA with degenerate primers resulted in cloning and sequencing of cDNA fragments of PPARα (1011 bp), PPARγ (812 bp), RXR (270 bp) and of the PPARα target gene palmitoyl-CoA oxidase (AOX1, 792 bp). Fragments of another 9 toxicologically relevant genes were also cloned and sequenced. PPARα mRNA expression was not significantly different among groups. In juvenile females transcription of PPARγ, RXR and AOX1 significantly increased in June 2005 when compared to June 2004. In adult males levels of AOX1 decreased in the same period. AOX1 and 7-ethoxyresorufin O-deethylase (EROD) activities, measured as exposure biomarkers, differed between years only in males sampled in June. EROD activity was higher in 2004 than in 2005 in adults, whereas both juvenile and adults showed higher AOX1 activity in 2005. The lack of historical data previous to the accident or in areas not affected by the accident did not allow to relate observed variations in gene transcription levels and enzyme activities to the Prestige oil spill. Reported data could be useful for comparison purposes for future studies in European hake and contributes gene sequence information relevant for future toxicological studies.

A quantitative structure‐activity relationship for predicting metabolic biotransformation rates for organic chemicals in fish

An evaluated database of whole body in vivo biotransformation rate estimates in fish was used to develop a model for predicting the primary biotransformation half-lives of organic chemicals. The estimated biotransformation rates were converted to half-lives and divided into a model development set (n=421) and an external validation set (n=211) to test the model. The model uses molecular substructures similar to those of other biodegradation models. The biotransformation half-life predictions were calculated based on multiple linear regressions of development set data against counts of 57 molecular substructures, the octanol-water partition coefficient, and molar mass. The coefficient of determination (r2) for the development set was 0.82, the cross-validation (leave-one-out coefficient of determination, q2) was 0.75, and the mean absolute error (MAE) was 0.38 log units (factor of 2.4). Results for the external validation of the model using an independent test set were r2 = 0.73 and MAE = 0.45 log units (factor of 2.8). For the development set, 68 and 95% of the predicted values were within a factor of 3 and a factor of 10 of the expected values, respectively. For the test (or validation) set, 63 and 90% of the predicted values were within a factor of 3 and a factor of 10 of the expected values, respectively. Reasons for discrepancies between model predictions and expected values are discussed and recommendations are made for improving the model. This model can predict biotransformation rate constants from chemical structure for screening level bioaccumulation hazard assessments, exposure and risk assessments, comparisons with other in vivo and in vitro estimates, and as a contribution to testing strategies that reduce animal usage.

Enantioselective whole-cell biotransformation of acetophenone to S-phenylethanol by Rhodotorula glutinis: Part I. Product formation kinetics and feeding strategies in aqueous media

This work explores product-formation kinetics of the reduction of acetophenone to ( S)-phenylethanol by Rhodotorula glutinis in aqueous media. Different feeding strategies were investigated to improve biotransformation productivity. An empirical kinetic model was formulated considering two different reaction periods: Phase-1, a very fast chemical-shock reaction in the first 5 min; Phase-2, a subsequent slower reaction which, depending on the product level, could be subinhibitory or inhibitory. The kinetics of the biotransformation metabolism were modulated by the levels of glucose and acetic acid in the bioreactor. In addition, two substrate feeding regimes were compared: intermittent and continuous. Although similar overall productivities were found, the intermittent method clearly holds greater potential for enhanced space-time yields as it takes advantage of the Phase-1 high reaction rates – additions of substrate pulses produce reaction rates of the same order of magnitude as those achieved in Phase-1. However, product accumulation to a “toxic limit” leads to the total cessation of biocatalytic activity, thus highlighting the importance of in-situ product removal during the biotransformation. Strategies for in-situ product removal are addressed in Part II of this study.