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There are now numerous in vitro and in silico ADME alternatives to in vivo assays but how do different industries incorporate them into their decision tree approaches for risk assessment, bearing in mind that the chemicals tested are intended for widely varying purposes? The extent of the use of animal tests is mainly driven by regulations or by the lack of a suitable in vitro model. Therefore, what considerations are needed for alternative models and how can they be improved so that they can be used as part of the risk assessment process? To address these issues, the European Partnership for Alternative Approaches to Animal Testing (EPAA) working group on prioritization, promotion and implementation of the 3Rs research held a workshop in November, 2008 in Duesseldorf, Germany. Participants included different industry sectors such as pharmaceuticals, cosmetics, industrial- and agro-chemicals. This report describes the outcome of the discussions and recommendations (a) to reduce the number of animals used for determining the ADME properties of chemicals and (b) for considerations and actions regarding in vitro and in silico assays. These included: standardisation and promotion of in vitro assays so that they may become accepted by regulators; increased availability of industry in vivo kinetic data for a central database to increase the power of in silico predictions; expansion of the applicability domains of in vitro and in silico tools (which are not necessarily more applicable or even exclusive to one particular sector) and continued collaborations between regulators, academia and industry. A recommended immediate course of action was to establish an expert panel of users, developers and regulators to define the testing scope of models for different chemical classes. It was agreed by all participants that improvement and harmonization of alternative approaches is needed for all sectors and this will most effectively be achieved by stakeholders from different sectors sharing data.

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The herbicide paraquat has been widely used throughout the world for almost 50 years and is important in sustainable agriculture. When used correctly the chemical poses no known risk to human health. However, it is acutely toxic, and can be fatal, if the concentrated product is ingested orally. Despite many years of research there is no successful treatment for paraquat intoxication. In recent years we have turned our attention to understanding how we can make the product safer, if it is accidentally or intentionally consumed. We present in this paper a novel approach aimed at safening the paraquat product, Gramoxone. Following our previous research on the site and mechanism of paraquat absorption from the gastrointestinal tract we have identified a new formulation of paraquat, Gramoxone INTEON that reduces the absorption of paraquat into the blood. This new formulation contains the polysaccharide, alginate, a natural product extracted from sea-weed. We have designed a preparation of paraquat and alginate with surfactants that is herbicidally active but has the unique property that it gels on contact with gastric acid in the stomach. The resulting mixture slows the dispersion and delivery of the toxic chemical to its site of absorption in the small intestine. Alginates also protect the mucosa against the damaging influence of topical gastric irritants, like paraquat. Our studies have shown that increasing the loading of alginate between 7 and 17 g/L causes a dose-related reduction in paraquat absorption in vitro in isolated rat ileum. This is also observed in vivo, as measured by paraquat plasma kinetics in the rabbit where the Area Under Curve (AUC 0-24h) was reduced from 33.8+/-3 for Gramoxone to 12.5+/-6 (microg/mL)h for a formulation containing 17 g/L alginate. Such a reduction in systemic exposure to paraquat is expected to reduce the acute oral toxicity of the formulation. This should be particularly effective in a vomiting species such as man since we have shown in this investigation that alginates not only reduce the peak plasma paraquat values but also delay the time to peak levels. This provides the opportunity for a more effective emetic response since the highly viscous gelled material should remain in the stomach for longer than the liquid Gramoxone. Further research is required to understand and optimise the safening and herbicidal characteristics of these alginate acid-triggered gel formulations of paraquat. However, we anticipate that this alginate technology in Gramoxone INTEON could have significant benefit in reducing human mortalities associated with the herbicide.

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Topical aciclovir cream (ACV, Zovirax Cream) containing 40% propylene glycol (PG), the optimum found for skin penetration, is clinically effective in the treatment of recurrent herpes labialis. One hundred and thirty-nine ACV generic creams were analysed and 80% of these contained less than 20% PG. From this, we hypothesised that these generics might be bioinequivalent to the innovator cream. A pilot in vitro skin permeation study compared the innovator cream with two generics containing about 15% PG. Next, 10 generics containing 0-15% PG were tested in an independent laboratory. Finally, a PG dose-ranging study was conducted in Zovirax cream base. In all studies, human skin was used and ACV analysed by LC-MS-MS. In the pilot study, the innovator cream delivered 7.5-fold more ACV than the two generics. Superiority was confirmed in the second study against all 10 ACV generic creams. By grouping the creams according to PG content, a relationship to ACV skin permeation was suggested. The PG dose effect was confirmed in the third study. These studies suggest that not all marketed ACV creams are bioequivalent to the clinically proven innovator. Given the magnitude of the differences seen, there is concern over therapeutic inequivalence of generic ACV creams to the innovator cream.

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Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulose diphosphate carboxylase, and soybean trypsin inhibitor. A ratio of 10U of pepsin activity/microg test protein was selected for all tests (3:1 pepsin to protein, w:w). Digestions were performed at pH 1.2 and 2.0, with sampling at 0.5, 2, 5, 10, 20, 30, and 60min. Protein digestibility was assessed from stained gels following SDS-PAGE of digestion samples and controls. Results. Results were relatively consistent across laboratories for the full-length proteins. The identification of proteolytic fragments was less consistent, being affected by different fixation and staining methods. Overall, assay pH did not influence the time to disappearance of the full-length protein or protein fragments, however, results across laboratories were more consistent at pH 1.2 (91% agreement) than pH 2.0 (77%). Conclusions. These data demonstrate that this common protocol for evaluating the in vitro digestibility of proteins is reproducible and yields consistent results when performed using the same proteins at different laboratories.

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Assessment of percutaneous absorption in vitro provides key information when predicting dermal absorption in vivo. Confirmation of skin membrane integrity is an essential component of the in vitro method, as described in test guideline OECD 428. Historically, assessment of the membrane's permeability to tritiated water (T2O) and the generation of a permeability coefficient (Kp) were used to confirm that the skin membrane was intact prior to application of the test penetrant. Measuring electrical resistance (ER) across the membrane is a simpler, quicker, safer and more cost effective method. To investigate the robustness of the ER integrity measure, the Kp values for T2O for a range of human and animal skin membranes were compared with corresponding ER data. Overall, for human, rat, pig, mouse, rabbit and guinea pig skin, the ER data gave a good inverse association with the corresponding Kp values; the higher the Kp the lower the ER values. In addition, the distribution across a large dataset for individual skin samples was similar for Kp and ER, allowing a cut-off value for ER to be established for each skin type. Based on CTL's (Syngenta Central Toxicology Laboratory) standard static diffusion cells and databridge, we propose that intact skin should have an ER equal to or above (in kOmega): human (10), mouse (5) guinea pig (5), pig (4) rat (3), and rabbit (0.8). We conclude that measurement of ER across in vitro skin membranes provides a robust measurement of skin barrier integrity and is an appropriate alternative to Kp for T2O in order to identify intact membranes that have acceptable permeability characteristics for in vitro percutaneous absorption studies.

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A prevalidation study sponsored by the European Centre for the Validation of Alternative Methods (ECVAM) on in vitro tests for acute skin irritation is aimed at identifying non-animal tests capable of discriminating irritants (I) from non-irritants (NI), as defined according to European Union and OECD. This paper reports on Phase III for one of the methods, the skin integrity function test (SIFT), assessing the protocol performance of the SIFT, in terms of reproducibility and predictive ability, in three laboratories. The barrier function properties of excised mouse skin were determined using a set of 20 coded chemicals (10 I, 10 NI), using the endpoints of trans-epidermal water loss (TEWL) and electrical resistance (ER). The basis of the SIFT prediction model is if the ratios of the pre- and post-application values for either TEWL or ER are greater than five-fold, then the test chemical is deemed irritant (I). If the ratio of both parameters is less than five-fold then the chemical is deemed non-irritant (NI). Analysis of variance (ANOVA) indicated that the intra-lab reproducibility was acceptable but that the inter-lab reproducibility was not. Overall, the SIFT test under-predicted the irritancy of the test chemicals chosen for Phase III with an overall accuracy of only 55%. The sensitivity value (ability to correctly predict I) was only 30%. The specificity (ability to predict NI) of the test was better at 80%. A retrospective examination of the SIFT results was undertaken using Student's t-test and a significance level of P<0.05 to predict an irritant based on changes in the TEWL ratio values. This improved the predictivity of the SIFT test, giving a specificity of 60%, a sensitivity of 80% and an overall accuracy of 70%. Appropriate modifications to the prediction model have now been made and the SIFT will be re-examined in a new validation exercise to investigate the potential of this non-animal method to predict acute skin irritation potential.

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One of the in vitro models involved in an ECVAM-sponsored prevalidation study for acute skin irritation is the skin integrity function test (SIFT), which utilises full-thickness mouse skin. We have evaluated nine different skin types in order to identify the most useful model for assessing skin barrier function using transepidermal water loss (TEWL), electrical resistance (ER) and tritiated water flux (TWF) and sodium lauryl sulphate (SLS) as a standard skin irritant. Tissues were: human skin (epidermis and whole), reconstituted human epidermis (RHE), pig (dermatomed and whole), rabbit (whole), rat (epidermis and whole) and mouse (whole). Barrier function was measured following sodium lauryl sulphate (SLS) exposure and expressed as a damage ratio. Human epidermis gave good responses at high doses of SLS only. RHE had abnormally high permeability to water and therefore had little or no response to SLS. Pig skin gave low TEWL ratios and rabbit skin was a poor responder to SLS. Mouse whole skin performed best in this study, giving consistent high damage ratios to TEWL, ER and TWF following SLS treatment. Rat whole skin also performed well but was generally less responsive. We conclude that mouse skin is the best and most practical in vitro model for the SIFT approach for skin irritation prediction.

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A prevalidation study on in vitro tests for acute skin irritation was conducted during 1999 and 2000. The overall objective of validation in this area, of which this prevalidation study is an initial stage, is to identify tests capable of discriminating irritants (I) from non-irritants (NI), as defined according to European Union (EU) risk phrases ("R38"; no classification) and the harmonised OECD criteria ("Irritant"; no label). This prevalidation study specifically addressed aspects of: protocol refinement (phase I), protocol transfer (phase II), and protocol performance (phase III), in accordance with the prevalidation scheme defined by the European Centre for the Validation of Alternative Methods (ECVAM). The tests evaluated were: EpiDerm (phases I, II and III), EPISKIN (phases I, II and III), PREDISKIN (phases I and II, and additional protocol refinement), the non-perfused pig ear method (phases I and II, and additional protocol refinement), and the mouse skin integrity function test (SIFT; phases I and II). Modified, standardised test protocols and well-defined prediction models were available for each of the tests at the end of phase I. The results of phase I (intralaboratory reproducibility) were sufficiently promising for all of the tests to progress to phase II. Protocol transfer between the Lead Laboratory and Laboratory 2 was undertaken for all five tests during phase II, and additional refinements were made to the test protocols. For EpiDerm, EPISKIN and the SIFT, the intralaboratory and interlaboratory reproducibilities were acceptable; however, better standardisation of certain aspects of the test protocols was needed prior to commencing phase III. Neither PREDISKIN nor the pig ear test performed sufficiently well in phase II to progress to phase III. The PREDISKIN protocol was overly sensitive, resulting in the prediction of all the NI chemicals as I. The variability in the pig ear test results was too great, indicating that the test would show limited predictive ability. In additional studies (a repeat of phase I), further modification of the PREDISKIN protocol and a change in the prediction model considerably improved the ability of the test to distinguish I from NI chemicals. However, attempts to improve the intralaboratory reproducibility of the pig ear test were unsuccessful. In phase III an initial assessment of the reproducibility and predictive ability, in three independent laboratories per test, was undertaken for the EpiDerm and EPISKIN tests (the SIFT was a late inclusion in the prevalidation study, and is being evaluated in a separate phase III study). A set of 20 coded chemicals (10 I, 10 NI) were tested with the final, refined, test protocols. The intralaboratory reproducibility was acceptable for both EpiDerm and EPISKIN. The interlaboratory reproducibility was considered to be acceptable for EPISKIN; however, for EpiDerm, analysis of variance (ANOVA) indicated that there was a statistically significant laboratory effect on the overall variability, suggesting that the interlaboratory transferability of the test needs to be improved. The EpiDerm test had an overall accuracy of 58%, with an over-prediction rate of 37% and an under-prediction rate of 47%. The EPISKIN test had an overall accuracy of 58%, showing an under-prediction rate of 23% and an over-prediction rate of 60%. It is concluded that, as yet, none of the tests evaluated in this prevalidation study are ready for inclusion in a formal validation study on in vitro tests for acute skin irritation. Overall protocol performance of the SIFT is currently being evaluated in a phase III study. Further studies are also in progress to improve the test protocols and prediction models for EpiDerm and EPISKIN.

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Dinitrochlorobenzene (DNCB) absorption through mouse and rat dorsal skin, pig ear skin and human abdominal skin in vitro was determined, and local metabolism to the glutathione conjugate was related to glutathione transferase activities and glutathione status in the skin. Absorption studies were conducted using skin mounted in a flow-through diffusion cell with tissue culture medium as receptor fluid. DNCB applied to the surface of skin in acetone penetrated through 26-day-old rat skin better than through the skin of the other species investigated. The amounts of absorption through pig and human skin and conjugation formation were similar. In general, occlusion resulted in increased penetration of DNCB but no change in conjugation. Human skin showed the highest gluta-thione-S-transferase activity towards DNCB, followed by 26-day-old rat, pig, mouse and neonatal rat skin. Levels of glutathione were highest in mouse skin, followed by neonatal rat, 26-day-old rat, pig and human skin, with pig and human skin showing similar levels. These studies indicated that the glutathione level in skin was the determining factor influencing the degree of DNCB conjugation during percutaneous absorption, and this was greatly depleted during percutaneous penetration of DNCB.

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What limited evidence there is indicates that the formulation in which a chemical allergen is encountered on the skin can have a marked impact upon the induction of cutaneous immune responses and the subsequent development of contact sensitization. The purpose of the present investigations was to examine further this phenomenon by analysis of the influence of dibutyl phthalate (DBP) on dermal sensitization to fluorescein isothiocyanate (FITC), a skin sensitizing fluorochrome. Addition of DBP augmented very substantially, in a dose-dependent fashion, the ability of topically applied FITC to stimulate proliferative responses in mice by draining lymph node cells (LNC), a correlate of skin sensitizing potential. Under these conditions, exposure of mice to DBP alone failed to elicit significant LNC responses. The influence of DBP on the accumulation of dendritic cells (DC) induced by FITC was examined also. Although 10% DBP had little effect on the numbers of DC found within draining nodes 18 hr following exposure of mice to FITC, the phthalate did result in a very substantial increase in the frequency of lymph node DC bearing detectable antigen (FITC+ DC). Furthermore, in the presence of DBP the median amount of FITC associated with antigen-bearing DC was higher. In vitro skin absorption studies indicated that DBP was associated with a small increase in percutaneous absorption of FITC. Collectively these data demonstrate that the vehicle formulation can exert a marked influence on dermal sensitization and that one mechanism which may be relevant is the increased acquisition of antigen by DC, associated possibly with altered penetration of the allergen into or through the skin.

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Effective skin sensitization is dependent upon immune activation of lymph nodes draining the site of exposure. The influence of vehicle formulation on the vigour of lymph node cell proliferative responses to 2,4-dinitrochlorobenzene (DNCB) has been examined. Mice (BALB/c strain) were exposed topically to 0.5% DNCB dissolved in either acetone or propylene glycol (PG). A significantly greater lymph node cell proliferative response was induced by DNCB in acetone. The observed differences were not attributable to variations in the numbers of immunostimulatory dendritic cells accumulating in the draining nodes following sensitization. In parallel studies, the absorption and cutaneous disposition of DNCB dissolved in acetone or PG were measured in vitro using static diffusion cells and full thickness mouse skin. Although flux of DNCB through the skin was comparable with both vehicles over 24 h, the absorption of the allergen during the first 4 h of exposure was significantly faster when acetone was used as the vehicle. Localization of DNCB demonstrated that much less of the chemical allergen was present in the skin at 4 h when applied in PG vehicle. However, there were no measurable vehicle effects on skin disposition of DNCB at 24 h. These data indicate that the sensitization potential of DNCB is influenced significantly by the nature of the vehicle used, possibly due to consequential effects on chemical absorption and disposition. The studies described in this paper reveal that the application vehicle may have a significant influence on the ability of DNCB to induce immune activation of draining lymph nodes and hence skin sensitization and that this may in turn be associated with important changes in the absorption and/or disposition of the chemical within the skin.

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It has previously been established that acute diquat (1,1'-ethylene, 2,2'-bipyridilium) toxicity in the rat is associated with stimulation of net fluid secretion into the gastrointestinal tract. We have examined the possibility that the mechanism of diquat toxicity in the small intestine involves redox cycling of the bipyridyl leading to a disturbance of biochemical function and oxidative stress. Experiments performed in vitro showed that diquat (10 microM to 1 mM) produced an increase in activity of the pentose phosphate pathway in rat small intestinal tissue slices, suggesting that there was oxidation of NADPH even at concentrations of diquat which do not cause intestinal fluid secretion in anaesthetized rats. When the effect of diquat on pentose phosphate activity was measured in rats in situ at a dose which causes maximal fluid secretion [50 mM diquat dibromide (DQBr2)], production of 14CO2 from [1-14C]-glucose increased by 278 +/- 28% (N = 4) within 1 hr of exposure to diquat. Under these same conditions, the tissue content of NADPH in the proximal small intestine was significantly depleted, though there was no corresponding increase in NADP+ concentration. Diquat had no effect on tissue concentrations of either the reduced or oxidized forms of NAD. It is likely that NADPH oxidation at low diquat concentrations can be adequately compensated for by mechanisms within the tissue which protect against oxidative stress. However, the data also suggest that diquat-induced fluid secretion in the rat small intestine is associated with redox cycling of bipyridyl leading to depletion of NADPH.

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Tape stripping is a useful technique to assess the distribution and amount of chemical in the stratum corneum (SC). The aim of this work was to develop an in vitro model that could be used to predict the results of in vivo skin stripping. Tape stripping experiments were carried out in vivo with the lipophilic penetrant fluazifop-butyl (FB) as part of a human volunteer study. Tape stripping was carried out at three time points after dosing. In vitro experiments were performed to match conditions in the in vivo experiment, using human epidermal membranes in static diffusion cells. By analysing the amount of penetrant in each pool of strips, the concentration profiles and the total amount of penetrant within the SC were determined from both in vivo and in vitro experiments. The concentration profiles demonstrate that the amount of penetrant decreases with increasing depth into the stratum corneum. The in vitro and in vivo profiles and total recovery of FB were found to be similar. These data suggest in vitro tape stripping provides a good model for the in vivo situation.

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Flow-through diffusion cells for the measurement of skin permeability in vitro are available commercially. We have designed a new system in our laboratory as an improvement on other cells. The CTL system is capable of running 15 cells simultaneously. In this study the flow-through diffusion system was compared with our well-validated and established static cell system, for ability to measure percutaneous absorption. Two hydrophilic chemicals-water and mannitol-were chosen as test penetrants. The permeability of whole skin, epidermis and dermatomed skin from humans, pigs, and rats was examined. The effect of tissue culture medium as receptor fluid was assessed in comparison with saline. Absorption data from the flow-through cells were similar to results from contemporary static cell. Thus, for each species and skin preparation there was good agreement in the permeability coefficients between the cell types. The absorption of the hydrophilic test penetrants into saline and tissue culture medium receptor fluid was also very similar. Further evaluation of this system, particularly for lipophilic molecules, is required.

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1. Diquat (1,1'-ethylene-2,2'-bipyridilium) is a non-selective desiccant herbicide which, when administered orally to mammalian species, causes significant secretion of fluid into the lumen of the gastrointestinal tract. In order to characterize this secretory response in more detail the effect of sublethal doses of diquat dibromide (DQBr2) on intestinal secretion was investigated in vivo in the jejunum of anaesthetized rats. 2. Ligated segments of jejunum (10 cm) which were prepared in groups of up to five animals were filled with 500 microliters of isosmotic DQBr2 solutions with concentrations ranging from 1-100 mM and maintained in the anaesthetized rat for 1, 2 or 3 h; in control experiments a solution of 100 mM NaBr was used. 3. It was found that while all of the fluid instilled into the segments was absorbed in the control experiments, there was both a dose- and time-dependent secretory response to DQBr2. Maximal fluid secretion occurred after treatment with 50 mM DQBr2 for 3 h. 4. Histological assessment of the jejunum revealed an increase in cell exfoliation and evidence of luminal distension after incubation with DQBr2. However, no structural damage to the mucosa could be seen to account for the fluid secretion. 5. The model described provides a quantitative means of evaluating intestinal secretion and may be used for elucidating the mechanism by which diquat alters fluid transport processes.

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The gastrointestinal absorption of paraquat (1,1'-dimethyl-4,4'-bipyridylium) was studied using the isolated mucosa from different regions of the gastrointestinal tract of rats. Tissues were stripped of their muscle layers and the viability of the mucosa was maintained in flux chambers by bathing both serosal and luminal membranes with separate oxygenated solutions. Paraquat absorption, transmucosal potential difference (PD), and permeability of the mucosa were studied. Exposure of the luminal side of isolated mucosae to paraquat (100 mg/ml) resulted in greater paraquat absorption across the small intestine compared to other regions of the gastrointestinal tract. The descending order of tissue absorption (as %/cm2 mucosa) was jejunum (17.6 +/- 0.8%), ileum (10 +/- 2.7%), colon (5.7 +/- 3.2%), duodenum (5.5 +/- 1.3%), stomach (2 +/- 0.8%), and esophagus (0.5 +/- 0.7%). Mucosal uptake of paraquat in the ileum was nonlinear over a luminal concentration range 2-200 mg/ml. Three phases to paraquat absorption were identified in this region of the small intestine: (i) a rate which was faster than diffusion (2-20 mg/ml paraquat); (ii) a rate which was slower than diffusion and obeyed saturation kinetics, with an apparent Km = 116 mM and Vmax = 11.3 mumol/g/hr, at paraquat concentrations up to 150 mg/ml: and (iii) a rate similar to that of diffusion at 200 mg/ml paraquat. Paraquat absorption at 200 mg/ml was also associated with an increase in mucosal permeability and reduction in PD. Inhibition of tissue metabolism resulted in a linear or diffusional paraquat absorption over a wide luminal concentration range (2-200 mg/ml). It is suggested, therefore, that paraquat absorption in the rat occurs principally in the small intestine and by a mechanism which consists of facilitated, saturable, and diffusional components. Knowledge of the mechanism by which paraquat gains entry to the bloodstream may offer new approaches to the development of safer formulations of the herbicide.

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Stimulation of mucosal alkaline secretion represents an opportunity for discovering novel drugs of potential benefit in maintenance therapy of duodenal ulcer disease. We screened over 200 agents representing the full spectrum of pharmacological categories in order to characterize stimulatory pathways and identify mechanistic leads. A variety of eicosanoids, phospho-diesterase inhibitors and adrenoreceptor agonists together with forskolin, 6-hydroxy-dopamine, 2-chloroadenosine, diazepam, testosterone, dipyridamole and dihydropyridazinone caused a reproducible increase in the metabolism-dependent component of alkaline secretion in bullfrog proximal duodenum. PGE2 (ED50 0.02 microgram/ml) was the most potent agent in vitro and was also the most effective stimulant of duodenal alkalinization in vivo in an anaesthetized cat preparation. Agents without effect on spontaneous alkaline secretion by amphibian duodenum included agonists and antagonists of histamine, 5-hydroxy-tryptamine, gamma-aminobutyric acid, dopamine, muscarinic and nicotinic receptors, inhibitors of amine uptake, monoamine oxidase and cholinesterase, plus various corticoids, diuretics, oestrogens, chemotherapeutic (anticancer) and antimicrobial agents. The major mechanism of stimulating alkaline secretion in the isolated duodenum is by increasing intracellular cyclic AMP levels. This may occur by either inhibiting metabolism of the nucleotide or by stimulating its formation. Additionally, many stimulants appear to act indirectly via liberation of endogenous prostaglandins as judged from the marked attenuation of responses in the presence of indomethacin to all agonists apart from exogenous PGE2, forskolin, ICI 63197 (PDE inhibitor), 2-chloroadenosine and diazepam. Whether purinergic agonists and benzodiazepines act directly on the enterocyte or by releasing other paracrine mediators is unknown.

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This report describes approaches for accurate determination of the comparative activities of stimulants of duodenal alkaline secretion. We screened about 200 standard pharmacological agents using a bullfrog-isolated mucosal preparation in order to characterize fully the mechanisms of duodenal alkaline secretion. A variety of eicosanoids, phosphodiesterase (PDE) inhibitors, adrenoreceptor agonists and benzodiazepines, together with forskolin, 6-hydroxydopamine, 2-chloroadenosine, dipyridamole, dihydropyridazinone and testosterone, caused a reproducible increase in the metabolism-dependent component of duodenal alkaline secretion. Prostaglandin E2 (ED50 of 0.02 micrograms ml-1 in vitro) was the most potent and efficacious stimulant in the isolated mucosa and in the perfused cat duodenum in vivo. In the isolated duodenum, the predominant mechanism for stimulating alkaline secretion appears to be via elevation of intracellular cAMP levels, but in vivo indirect effects, for example on mucosal blood flow, may determine the overall influence of agents on duodenal alkalinization.

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We studied basal and prostaglandin E2 (PGE2)-stimulated duodenal HCO3- transport in the rat in vivo both in the presence and absence of a concentration gradient for HCO3- from blood to lumen. Basal HCO3- transport was not reduced when the luminal solution was changed from one containing 0 mM HCO3- to one containing 22 mM HCO3- either at pH 9.0 or 7.5. Thus basal duodenal HCO3- transport in rats is independent of a blood-to-lumen HCO3- concentration gradient, which indicates an energy-dependent process with little passive flux of HCO3-. Luminal or intravenous administration of PGE2 significantly (P less than 0.01) increased HCO3- secretion into a HCO3(-)-free luminal solution but had no effect on HCO3- secretion into luminal solutions containing 22 mM HCO3-, either at pH 9.0 or 7.5. Therefore prostaglandins may act by increasing passive flux of HCO3- rather than by stimulating energy-dependent duodenal HCO3- transport.

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In dogs beta-adrenoreceptor agonists inhibit gastric acid secretion stimulated by exogenous gastrin to a much greater extent than acid secretion stimulated by exogenous histamine. One possible explanation for this observation is that endogenous histamine is important in gastrin-mediated acid secretion and that isoprenaline and related beta-adrenoreceptor agonists block gastric mucosal histamine release. This possibility was tested in the present study in gastric lumen-perfused anaesthetized rats. Intravenous infusion of isoprenaline (12 microgram kg-1 h-1) inhibited maximal, pentagastrin-stimulated acid output by 50-70% (P less than 0.01), but had no significant inhibitory effect on the maximal acid secretory response to histamine. In contrast to its inhibitory effect on gastrin-stimulated acid output, isoproterenol had no effect on gastric histamine output during pentagastrin infusion. We conclude that isoprenaline selectively inhibits gastrin-stimulated acid secretion in the rat, as in the dog, and by a mechanism other than inhibiting gastric histamine release.

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