Comment on "Revisiting the carrageenan controversy: do we really understand the digestive fate and safety of carrageenan in our foods?" by S. David, C. S. Levi, L. Fahoum, Y. Ungar, E. G. Meyron-Holtz, A. Shpigelman and U. Lesmes, Food Funct., 2018, 9, 1344-1352.

Carrageenan (CGN) is a polysaccharide that is found in various types of sea weed. It is a common food additive used for its gelling and thickening properties and has been used safely throughout the world for decades. CGN is approved as Generally Recognized as Safe (GRAS) by the United States Food and Drug Administration and is also considered safe for the general population by the World Health Organizations Joint Expert Committee on Food Additive (JECFA) and the European Food Safety Authority. CGN has been tested for safety in various animal models for many years and more recently in an array of in vitro or cell-based models. A recent review published by this journal entitled "Revisiting the Carrageenan controversy: Do we really understand the digestive fate and safety of carrageenan in our foods?" has provided the impetus for this commentary (S. David, et al., Food Funct., 2018, 9(3), 1344-1352). It is important that our food is safe, and clearly there are examples of food additives that were found to be unsafe after years of use, but the issue is the need for accurate interpretation of previously published studies and the need for designing and conducting experiments that can be used to make decisions on safety. It is our hope that this commentary brings to light some of the important physical and chemical properties of CGN and how information can be easily misinterpreted.

Clarifying the confusion between poligeenan, degraded carrageenan, and carrageenan: A review of the chemistry, nomenclature, and in vivo toxicology by the oral route.

Carrageenan (CGN) is a common food additive that has been widely used for decades as a gelling, thickening and stabilizing agent. Carrageenan has been proven safe for human consumption; however, there has been significant confusion in the literature between CGN and the products of intentional acid-hydrolysis of CGN, which are degraded CGN (d-CGN) and poligeenan (PGN). In part, this confusion was due to the nomenclature used in early studies on CGN, where poligeenan was referred to as "degraded carrageenan" (d-CGN) and "degraded carrageenan" was simply referred to as carrageenan. Although this nomenclature has been corrected, confusion still exists resulting in misinterpretation of data and the subsequent dissemination of incorrect information regarding the safe dietary use of CGN. The lack of understanding of the molecular weight distribution of CGN has further exacerbated the issue. The significant differences in chemistry, manufacture, and protein reactivity of CGN versus d-CGN and PGN are reviewed, in addition to the in vivo toxicological profiles of CGN, d-CGN, and PGN. As CGN cannot be hydrolyzed to PGN in vivo, concerns over the use of CGN as a food additive are unfounded, particularly since current studies support the lack of oncogenic and tumorigenic activity of CGN in humans.

Addendum to Weiner, M.L. (2016) Parameters and Pitfalls to Consider in the Conduct of Food Additive Research, Carrageenan as a Case Study. Food Chemical Toxicology 87, 31-44.

This paper is an addendum to a 2016 paper outlining pitfalls and parameters to consider in the conduct of food additive research with carrageenan (Fd. Chem. Tox. 87, 31-44 (2016)). The literature on the food additive, "carrageenan," contains many publications which either erroneously misuse the name, carrageenan, for a sample which is not carrageenan, but "degraded carrageenan" or "poligeenan" and also conduct studies without understanding the physical/chemical properties of carrageenan. Degraded carrageenan and poligeenan are not food additives and have a completely different physical/chemical and toxicological properties from carrageenan. Two recent publication examples, one in vivo and one in vitro, demonstrate the serious misunderstanding promulgated by incorrect sample identity/purity and poor study conduct. These new publication examples reiterate the problems in the literature summarized by the Weiner (2016). It is important to have thorough, rigorous peer review of all studies using carrageenan in vivo or in vitro.

Effects of carrageenan on cell permeability, cytotoxicity, and cytokine gene expression in human intestinal and hepatic cell lines.

Carrageenan (CGN) is a common food additive used for its gelling and thickening properties. The present study was done to evaluate intestinal permeability, cytotoxicity, and CGN-mediated induction of proinflammatory cytokines. A standard Caco-2 absorption model showed no CGN permeability or cytotoxicity at concentrations of 100, 500, and 1000 µg/mL. In two human intestinal cell lines (HT-29 and HCT-8) CGN (0.1, 1.0, and 10.0 µg/mL) did not induce IL-8, IL-6, or MCP-1 (CCL2) or produce cellular toxicity after 24 h. The TLR4 agonist LPS produced weak induction of IL-8 in HT-29 cells and no induction in HCT-8 cells. The effects of κ-CGN (0.1, 1.0, and 10 µg/mL) on cellular oxidative stress was assessed in HT-29 cells using CM-H2DCFDA as the probe. No effect on oxidative stress was observed after 24 h. In the human (HepG2) liver cell line, ʎ-CGN (0.1, 1.0, 10.0 and 100.0 µg/mL) had no effect on the expression of IL-8, IL-6, or MCP-1 (CCL2) after 24 h. In conclusion, CGN was not absorbed, and was not cytotoxic. It did not induce oxidative stress, and did not induce proinflammatory proteins.

Probabilistic hazard assessment for skin sensitization potency by dose-response modeling using feature elimination instead of quantitative structure-activity relationships.

Supervised learning methods promise to improve integrated testing strategies (ITS), but must be adjusted to handle high dimensionality and dose-response data. ITS approaches are currently fueled by the increasing mechanistic understanding of adverse outcome pathways (AOP) and the development of tests reflecting these mechanisms. Simple approaches to combine skin sensitization data sets, such as weight of evidence, fail due to problems in information redundancy and high dimensionality. The problem is further amplified when potency information (dose/response) of hazards would be estimated. Skin sensitization currently serves as the foster child for AOP and ITS development, as legislative pressures combined with a very good mechanistic understanding of contact dermatitis have led to test development and relatively large high-quality data sets. We curated such a data set and combined a recursive variable selection algorithm to evaluate the information available through in silico, in chemico and in vitro assays. Chemical similarity alone could not cluster chemicals' potency, and in vitro models consistently ranked high in recursive feature elimination. This allows reducing the number of tests included in an ITS. Next, we analyzed with a hidden Markov model that takes advantage of an intrinsic inter-relationship among the local lymph node assay classes, i.e. the monotonous connection between local lymph node assay and dose. The dose-informed random forest/hidden Markov model was superior to the dose-naive random forest model on all data sets. Although balanced accuracy improvement may seem small, this obscures the actual improvement in misclassifications as the dose-informed hidden Markov model strongly reduced " false-negatives" (i.e. extreme sensitizers as non-sensitizer) on all data sets.

The common food additive carrageenan is not a ligand for Toll-Like- Receptor 4 (TLR4) in an HEK293-TLR4 reporter cell-line model.

Carrageenan (CGN) is widely used in the food manufacturing industry as an additive that stabilizes and thickens food products. Standard animal safety studies in which CGN was administered in diet showed no adverse effects. However, several in vitro studies have reported that intestinal inflammation is caused by CGN and that this effect is mediated through Toll-Like-Receptor 4 (TLR4). The purpose of this study was to evaluate the ability of different types of CGN to bind and activate TLR4 signaling. To accomplish this a TLR4/MD-2/CD14/NFκB/SEAP reporter construct in a HEK293 cell line was used. The reporter molecule, secretable alkaline phosphatase (SEAP), was measured as an indicator of TLR4 activation. The test compounds were exposed to this system at concentrations of 0.1, 1, 10, 50, 100, 500, 1000, and 5000 ng/mL for 24 h. Cytotoxicity was evaluated following the 24 h exposure period by LDH leakage and ATP. CGN binding to serum proteins was characterized by Toluidine Blue. The results show that CGN does not bind to TLR4 and is not cytotoxic to the HEK293 cells at the concentrations and experimental conditions tested and that CGN binds tightly to serum proteins.

Food additive carrageenan: Part I: A critical review of carrageenan in vitro studies, potential pitfalls, and implications for human health and safety.

Carrageenan (CGN) has been used as a safe food additive for several decades. Confusion over nomenclature, basic CGN chemistry, type of CGN tested, interspecies biology, and misinterpretation of both in vivo and in vitro data has resulted in the dissemination of incorrect information regarding the human safety of CGN. The issue is exacerbated when mechanistic data obtained from in vitro experiments are directly translated to human hazard and used for risk assessment. This can lead to information that is taken out of experimental context and reported as a definitive effect in humans. In recent years, the use of cell-based models has increased and their ability to provide key information regarding chemical or drug safety is well established. In many instances, these new alternative approaches have started to replace the need to use animals altogether. In vitro systems can be extremely useful for understanding subcellular targets and mechanisms of adverse effects. However, care must be exercised when extrapolating the in vitro findings to in vivo effects. Often, issues such as chemical identity and purity, relevant dose, pharmacokinetic properties, solubility, protein binding, adsorption to plastics, and the use of cell models that are biologically and mechanistically relevant are overlooked or ignored. When this occurs, in vitro findings can provide misleading information that is not causally linked to in vivo events in animals or in humans. To date, there has not been a comprehensive review of the CGN in vitro literature, which has reported a wide range of biochemical effects related to this compound. An extensive effort has been made to evaluate as much of this literature as possible. This review focuses on the in vitro observation, the unique chemistry of CGN, and potential pitfalls of in vitro models used for hazard identification. The discussion of the in vitro studies discussed this review are supported by numerous in vivo studies. This provides a unique opportunity to have both the in vitro and in vivo studies reviewed together.

Discovery of Dap-3 polymyxin analogues for the treatment of multidrug-resistant Gram-negative nosocomial infections.

We report novel polymyxin analogues with improved antibacterial in vitro potency against polymyxin resistant recent clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa . In addition, a human renal cell in vitro assay (hRPTEC) was used to inform structure-toxicity relationships and further differentiate analogues. Replacement of the Dab-3 residue with a Dap-3 in combination with a relatively polar 6-oxo-1-phenyl-1,6-dihydropyridine-3-carbonyl side chain as a fatty acyl replacement yielded analogue 5x, which demonstrated an improved in vitro antimicrobial and renal cytotoxicity profiles relative to polymyxin B (PMB). However, in vivo PK/PD comparison of 5x and PMB in a murine neutropenic thigh model against P. aeruginosa strains with matched MICs showed that 5x was inferior to PMB in vivo, suggesting a lack of improved therapeutic index in spite of apparent in vitro advantages.

Evaluation of gene expression changes in human primary uroepithelial cells following 24-hr exposures to inorganic arsenic and its methylated metabolites.

Gene expression changes in primary human uroepithelial cells exposed to arsenite and its methylated metabolites were evaluated to identify cell signaling pathway perturbations potentially associated with bladder carcinogenicity. Cells were treated with mixtures of inorganic arsenic and its pentavalent or trivalent metabolites for 24 hr at total arsenic concentrations ranging from 0.06 µM to 18 µM. One series (five samples) was conducted with arsenite and pentavalent metabolites and a second (10 samples) with arsenite and trivalent metabolites. Similar gene expression responses were obtained for pentavalent or trivalent metabolites. A suite of eight gene changes was consistently identified across individuals that reflect effects on key signaling pathways: oxidative stress, protein folding, growth regulation, metallothionine regulation, DNA damage sensing, thioredoxin regulation, and immune response. No statistical significance of trend (NOSTASOT) analysis of these common genes identified lowest observed effect levels (LOELs) from 0.6 to 6.0 µM total arsenic and no observed effect levels (NOELs) from 0.18 to 1.8 µM total arsenic. For the trivalent arsenical mixture, benchmark doses (BMDs) ranged from 0.13 to 0.92 µM total arsenic; benchmark dose lower 95% confidence limits (BMDLs) ranged from 0.09 to 0.58 µM total arsenic. BMDs ranged from 0.53 to 2.7 µM and BMDLs from 0.35 to 1.7 µM for the pentavalent arsenical mixture. Both endpoints varied by a factor of 3 across individuals. Thisstudy is the first to examine gene expression response in primary uroepithelial cells from multiple individuals and to identify no effect levels for arsenical-induced cell signaling perturbations in normal human cells exposed to a biologically plausible concentration range.

An in vitro method for detecting chemical sensitization using human reconstructed skin models and its applicability to cosmetic, pharmaceutical, and medical device safety testing.

Chemical sensitization is a serious condition caused by small reactive molecules and is characterized by a delayed type hypersensitivity known as allergic contact dermatitis (ACD). Contact with these molecules via dermal exposure represent a significant concern for chemical manufacturers. Recent legislation in the EU has created the need to develop non-animal alternative methods for many routine safety studies including sensitization. Although most of the alternative research has focused on pure chemicals that possess reasonable solubility properties, it is important for any successful in vitro method to have the ability to test compounds with low aqueous solubility. This is especially true for the medical device industry where device extracts must be prepared in both polar and non-polar vehicles in order to evaluate chemical sensitization. The aim of this research was to demonstrate the functionality and applicability of the human reconstituted skin models (MatTek Epiderm(®) and SkinEthic RHE) as a test system for the evaluation of chemical sensitization and its potential use for medical device testing. In addition, the development of the human 3D skin model should allow the in vitro sensitization assay to be used for finished product testing in the personal care, cosmetics, and pharmaceutical industries. This approach combines solubility, chemical reactivity, cytotoxicity, and activation of the Nrf2/ARE expression pathway to identify and categorize chemical sensitizers. Known chemical sensitizers representing extreme/strong-, moderate-, weak-, and non-sensitizing potency categories were first evaluated in the skin models at six exposure concentrations ranging from 0.1 to 2500 µM for 24 h. The expression of eight Nrf2/ARE, one AhR/XRE and two Nrf1/MRE controlled gene were measured by qRT-PCR. The fold-induction at each exposure concentration was combined with reactivity and cytotoxicity data to determine the sensitization potential. The results demonstrated that both the MatTek and SkinEthic models performed in a manner consistent with data previously reported with the human keratinocyte (HaCaT) cell line. The system was tested further by evaluating chemicals known to be associated with the manufacture of medical devices. In all cases, the human skin models performed as well or better than the HaCaT cell model previously evaluated. In addition, this study identifies a clear unifying trigger that controls both the Nrf2/ARE pathway and essential biochemical events required for the development of ACD. Finally, this study has demonstrated that by utilizing human reconstructed skin models, it is possible to evaluate non-polar extracts from medical devices and low solubility finished products.

An expert consortium review of the EC-commissioned report "alternative (Non-Animal) methods for cosmetics testing: current status and future prospects - 2010".

The European cosmetics legislation foresees a review in 2011 and possible postponement of the 2013 marketing ban to enforce the testing ban for systemic and repeated-dose animal tests. For this purpose, a 119-page report commissioned by the European Commission was published recently. Here, a group of 17 independent experts from the US, Europe, and Japan was brought together to evaluate the report. The expert panel strongly endorsed the report and its conclusions. A number of important options not considered were identified; these do not, however, affect the overall conclusions regarding the current lack of availability of a full replacement, especially for the areas of repeated dose toxicity, carcinogenicity testing, and reproductive toxicity, though a roadmap for change is emerging. However, some of these options may provide adequate data for replacement of some animal studies in the near future pending validation. Various recommendations expand the original report. The reviewers agree with the report that there is greater promise in the short term for the areas of sensitization and toxicokinetics. Additional opportunities lie in more global collaborations and the inclusion of other industry sectors.

CeeTox™ Analysis of CNB-001 a Novel Curcumin-Based Neurotrophic/Neuroprotective Lead Compound to Treat Stroke: Comparison with NXY-059 and Radicut.

In the present study, we used a comprehensive cellular toxicity (CeeTox) analysis panel to determine the toxicity profile for CNB-001 [4-((1E)-2-(5-(4-hydroxy-3-methoxystyryl-)-1-phenyl-1H-pyrazoyl-3-yl)vinyl)-2-methoxy-phenol)], which is a hybrid molecule created by combining cyclohexyl bisphenol A, a molecule with neurotrophic activity and curcumin, a spice with neuro-protective activity. CNB-001 is a lead development compound since we have recently shown that CNB-001 has significant preclinical efficacy both in vitro and in vivo. In this study, we compared the CeeTox profile of CNB-001 with two neuroprotective molecules that have been clinically tested for efficacy: the hydrophilic free radical spin trap agent NXY-059 and the hydrophobic free radical scavenger edaravone (Radicut). CeeTox analyses using a rat hepatoma cell line (H4IIE) resulted in estimated C(Tox) value (i.e., sustained concentration expected to produce toxicity in a rat 14-day repeat dose study) of 42 µM for CNB-001 compared with >300 µM for both NXY-059 and Radicut. The CeeTox panel suggests that CNB-001 produces some adverse effects on cellular adenosine triphosphate content, membrane toxicity, glutathione content, and cell mass (or number), but only with high concentrations of the drug. After a 24-h exposure, the drug concentration that produced a half-maximal response (TC(50)) on the measures noted above ranges from 55 to 193 µM. Moreover, all CNB-001-induced changes in the markers were coincident with loss of cell number, prior to acute cell death as measured by membrane integrity, suggesting a cytostatic effect of CNB-001. NXY-059 and Radicut did not have acute toxic effects on H4IIE cells. We also found that CNB-001 resulted in an inhibition of ethoxyresorufin-o-deethylase activity, indicating that the drug may affect cytochrome P4501A activity and that CNB-001 was metabolically unstable using a rat microsome assay system. For CNB-001, an estimated in vitro efficacy/toxicity ratio is 183-643-fold, suggesting that there is a significant therapeutic safety window for CNB-001 and that it should be further developed as a novel neuroprotective agent to treat stroke.

A new in vitro method for identifying chemical sensitizers combining peptide binding with ARE/EpRE-mediated gene expression in human skin cells.

Allergic contact dermatitis (ACD) is a significant safety concern for developers of cosmetic, personal care, chemical, pharmaceutical, and medical device products. The guinea pig maximization test (GMPT) and the murine local lymph node assay (LLNA) are accepted methods for determining chemical sensitization. Recent legislative initiatives in Europe require the development of new in vitro alternatives to animal tests for chemical sensitization. The aim of this project was to develop an in vitro screening method that uses a human skin cell line (HaCaT), chemical reactivity, and gene expression profiling to identify positive and negative responses, to place chemicals into potency categories of extreme/strong (ES), moderate (M), weak (W), and nonsensitizers (N), and to provide an estimate of corresponding LLNA values. The method and processing algorithm were developed from a training set of 39 chemicals possessing a wide range of sensitization potencies. Three cationic metals, chromium (Cr), nickel (Ni), and silver (Ag), were also evaluated in this model. Chemical reactivity was determined by measuring glutathione (GSH) depletion in a cell free matrix. Three signaling pathways (Keap1/Nrf 2/ARE/EpRE, ARNT/AhR/XRE, and Nrf1/MTF/MRE) that are known to be activated by sensitizing agents were monitored by measuring the relative abundance of 11 genes whose expression is controlled by one of these 3 pathways. Final exposure concentrations were based on toxicity and solubility. A range-finding experiment was conducted with each compound to determine cytotoxicity and solubility. Six exposure concentrations (0.1 to 2,500 microM) and an exposure time of 24 hours were used in the final experiments. Glutathione depletion alone did not provide the accuracy necessary to differentiate potency categories. However, chemical reactivity combined with gene expression profiles significantly improved the in vitro predictions. A predicted toxicity index (PTI) was determined for each test chemical. A comparison of LLNA values with PTI values revealed an inverse relationship. The large variation in LLNA data for compounds in the same potency category makes direct extrapolation from PTI to LLNA difficult. To challenge the system, 58 additional compounds were submitted in a blinded manner. Compounds placed into ES and M categories were considered positive, whereas compounds classified as W or N were considered negative. Accuracy was approximately 84%, with a sensitivity of 81% and a specificity of 92%. The model correctly identified 2 of 3 cationic metals as positive. In conclusion, the method described here demonstrates a valuable in vitro method for identifying chemicals and metals that induce skin sensitization.

Building a tiered approach to in vitro predictive toxicity screening: a focus on assays with in vivo relevance.

One of the greatest challenges facing the pharmaceutical industry today is the failure of promising new drug candidates due to unanticipated adverse effects discovered during preclinical animal safety studies and clinical trials. Late stage attrition increases the time required to bring a new drug to market, inflates development costs, and represents a major source of inefficiency in the drug discovery/development process. It is generally recognized that early evaluation of new drug candidates is necessary to improve the process. Building in vitro data sets that can accurately predict adverse effects in vivo would allow compounds with high risk profiles to be deprioritized, while those that possess the requisite drug attributes and a lower risk profile are brought forward. In vitro cytotoxicity assays have been used for decades as a tool to understand hypotheses driven questions regarding mechanisms of toxicity. However, when used in a prospective manner, they have not been highly predictive of in vivo toxicity. Therefore, the issue may not be how to collect in vitro toxicity data, but rather how to translate in vitro toxicity data into meaningful in vivo effects. This review will focus on the development of an in vitro toxicity screening strategy that is based on a tiered approach to data collection combined with data interpretation.

Uptake and elimination of ionizable organic chemicals at fish gills: I. Model formulation, parameterization, and behavior.

A mechanistic model for the uptake and elimination of ionizable organic chemicals at fish gills is presented. This model is a modification of a previous model for nonionizable organic chemicals that addressed the transport of chemical to and from gill surfaces in water and blood, diffusion of chemical across epithelial cells, and binding of chemical to components in water and blood. For ionizable chemicals, three additional processes are included. First, excretory products alter the pH at gill surfaces, affecting the relative amounts of neutral and ionized molecules compared with that in the bulk exposure water. Second, ionized molecules support chemical flux to and from epithelial cell membranes and help maintain high diffusion gradients of neutral molecules across these membranes, thereby contributing to uptake and elimination even if the membranes are impermeable to ionized molecules. Third, membrane barriers are not completely impermeable to ionized molecules, and even limited permeability can have appreciable effects on chemical flux. Approaches for model parameterization are discussed. Model-predicted relationships of uptake and elimination rates to exposure water pH, alkalinity, and chemical properties are presented and discussed in terms of model processes. The model is shown to predict important features of reported effects of pH on uptake rates of weak organic acids.

Uptake and elimination of ionizable organic chemicals at fish gills: II. Observed and predicted effects of ph, alkalinity, and chemical properties.

Effects of exposure-water pH on chemical uptake at rainbow trout (Oncorhynchus mykiss) gills were investigated for nine weakly acidic, chlorinated phenols with different ionization constants and hydrophobicities and for a moderately hydrophobic, nonionizable reference chemical (1,2,4-trichlorobenzene). Uptake rates for all chemicals varied little from pH 6.3 to 8.4, despite ionization of the chlorinated phenols ranging from less than 1 to greater than 99.9% among these pH values and chemicals. At pH 9.2, uptake rates were reduced substantially for the chlorinated phenols but not for the reference chemical. These results indicate greater bioavailability of neutral chemical forms but also considerable bioavailability of ionized forms that varies with pH. Three mechanisms were evaluated regarding such ionized chemical bioavailability. First, reduced pH at the gill surface causes net conversion of ionized molecules to more readily absorbed neutral molecules. This mechanism was tested by increasing exposure-water alkalinity, which increased gill surface pH and reduced uptake of the chlorinated phenols but not of the reference chemical. Magnitudes of these effects were close to predictions from a mathematical model for chemical exchange at fish gills that incorporated this mechanism. Second, ionized molecules contribute to uptake by maintaining high gradients of neutral molecules across epithelial membrane barriers, even if the barriers are impermeable to these ions. This mechanism was demonstrated to explain the similarity of uptake among pH values and chemicals at pH less than 8.4 and the degree to which uptake declined at pH 9.2. Third, membrane barriers can have some permeability to the ionized forms, but this was not important for the chemicals and conditions of the present study. Increased exposure-water pH also was demonstrated to increase elimination rates of these chemicals, which also was in accord with model expectations.

The value of DNA methylation analysis in basic, initial toxicity assessments.

DNA methylation is an epigenetic mechanism regulating patterns of gene expression. Our goal was to see if the assessment of DNA methylation might be a useful tool, when used in conjunction with initial, basic in vitro tests, to provide a more informative preliminary appraisal of the toxic potential of chemicals to prioritize them for further evaluation. We sought to give better indications of a compound's toxic potential and its possible mechanism of action at an earlier time and, thereby, contribute to a rational approach of an overall reduction in testing by making improved early decisions. Global and GC-rich patterns of DNA methylation were evaluated along with more traditional cytolethality measurements, e.g., cytolethality and genotoxicity assessments, on rat hepatoma (H4IIE) cells. The relative toxic potential of model compounds camptothecin, 5-fluorouracil, rotenone, and staurosporine was estimated by employing DNA methylation assessments combined with our cytolethality data plus genotoxicity information gleaned from the literature. The overall contribution of the methylation assessment was threefold; it (1) strengthened a ranking based on genotoxicity; (2) provided an indication that a compound might be more potentially problematic than what cytolethality and genotoxicity assessments alone would indicate; and (3) suggested that compounds, particularly nongenotoxins, that are more potent regarding their ability to alter methylation, especially at noncytolethal concentrations, may be more potentially toxic. Altered methylation per se is not proof of toxicity; this needs to be viewed as a component of an evaluation.

Effects of anesthesia (tricaine methanesulfonate, MS222) on liver biotransformation in rainbow trout (Oncorhynchus mykiss).

The effect of tricaine methanesulfonate (MS222) on rainbow trout liver biotransformation rates was investigated with a microsomal model; an in vitro preparation that can be employed with or without the use of an anaesthetic. Two experimental sets of rainbow trout microsomes were tested; one representing in vivo or surgical tricaine exposures and the other representing in vitro tissue/organ collection tricaine exposures. Microsomal incubations were performed on these two experimental groups with phenol as substrate to assess the effects of tricaine on Phase I (ring-hydroxylation) and II (glucuronidation) liver biotransformation by monitoring production of hydroquinone (HQ), catechol (CAT), and phenylglucuronide (PG). The use of a 2-h 100 mg/l exposure of tricaine for surgical anesthesia with or without 24-h recovery did not significantly (P< or =0.05) affect rates of phenol (Phase I and II) biotransformation rates; nor, did the 5-min 300 mg/l tricaine exposure for isolated organ/tissue collection significantly (P< or =0.05) affect phenol (Phase I and II) biotransformation rates. There were also no significant statistical differences (P< or =0.05) in P450 protein levels, or 7-ethoxyresorufin-O-deethylase (EROD) activity in these microsomal assays between any of the tricaine treated rainbow trout and controls.

An in vivo microdialysis method for the qualitative analysis of hepatic phase I metabolites of phenol in rainbow trout (Oncorhynchus mykiss).

Development of reliable and accurate methodologies for determination of xenobiotic hepatic biotransformation rate and capacity parameters is important to the derivation of precise physiologically-based toxicokinetic (PB-TK) models. Biotransformation data incorporated into PB-TK models has, for the most part, depended on in vitro techniques designed to mimic the in vivo environment; however, data from direct in vitro/in vivo comparisons is limited. In this investigation we describe for the first time a method using in vivo microdialysis (MD) to qualitatively assess hepatic xenobiotic biotransformation of phenol in an unanesthetized fish. MD probes were surgically implanted into the livers of adult rainbow trout which were subsequently confined to respirometer-metabolism chambers. Phenol (1-300 mM) was delivered directly to the liver via the MD probe at a perfusion rate of 1 microl min(-1) which consistently resulted in a relative delivery of 77-85% of the phenol in the perfusate to the tissue over a 3 day experimental time frame. Location of the probe within the liver was also shown to have no effect on the delivery of phenol or on the type or quantity of phase I metabolites formed. Production of hydroquinone (HQ) and catechol (CAT), the primary phase I metabolites of phenol, was monitored through direct sampling of the hepatic extracellular fluid space via the MD probe. HQ and CAT production increased with increasing time of perfusion and with increasing concentration of phenol delivered to the liver. In the future, data obtained through in vivo MD will be useful in resolving uncertainties in biotransformation rate and capacity parameters, which are central to fish PB-TK modeling of chemical disposition.

Dose-response modeling of cytochrome p450 induction in rats by octamethylcyclotetrasiloxane.

Inhalation of octamethylcyclotetrasiloxane (D4) induces CYP2B1/2 protein and causes liver enlargement. We have developed a pharmacodynamic (PD) extension to a physiologically based pharmacokinetic (PBPK) model to characterize these dose-response behaviors. The PD model simulates interactions of D4 with a putative receptor, leading to increased production of cytochrome P450 2B1/2. Induction was modeled with a Hill equation with dissociation constant, Kd, and Hill coefficient, N. Both a 1- and a 5-compartment liver model were evaluated. The PBPK model provided excellent simulations of tissue D4 and hepatic CYP2B1/2 protein concentrations following 6 h/day, 5-day inhalation exposures to 0, 1, 7, 30, 70, 150, 300, 500, 700, or 900 ppm D4. Either the 1- or 5-compartment liver model could accurately simulate increases in CYP2B1/2 protein in the liver. With a 1-compartment liver, Kd and N were 0.67 microM (free liver concentration) and 1.9, respectively. The 5-compartment model used higher N-values (approximately 4.0) and varied Kd between compartments. The fitted 5-compartment model parameters were Kd = 0.67 microM in the midzonal compartment with geometric differences in Kd between compartments of 2.9. On the basis of unbound (free) plasma concentrations, D4 appeared to be a higher potency inducer than phenobarbital (PB). Dose-response curves for increased liver weights had N/mS 1.0 and Kd/mS 3.4 microM, very different values from those for enzyme induction. Exposure concentration leading to a 0.1% increase in CYP2B1/2 protein predicted by the 1- and 5-compartment models were 2.1 ppm and 5.1 ppm, respectively. The 1- and 5-compartment liver models provided very similar fits to the whole liver induction data, excluding the lowest dose, but the 5-compartment liver model had the additional advantage of simultaneously describing the regional induction of CYP2B1/2.