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The aim of this study was to investigate a range of poly(amidoamine) (PAMAM) dendrimer generations against Gram-positive and Gram-negative skin pathogens and to determine any differences in antimicrobial potency for different generations, characterising how differences in physicochemical properties influence antimicrobial efficacy. A range of tests were carried out, including viable count assays to determine half maximal inhibitory concentration (IC 50 ) values for each dendrimer, membrane integrity studies and an inner membrane permeabilisation assay. This is supported by scanning electron microscopy imaging of the interactions observed between dendrimers and bacteria. The results of this study indicate that the antimicrobial efficacy of native PAMAM dendrimers is dependent on generation, concentration and terminal functionalities, for example, the concentration at 50% growth inhibition (MIC 50 ) (µg/mL), against Staphylococcus aureus was between 26.77 for the G2-PAMAM-NH 2 dendrimer and 2.881 for the G5-PAMAM-NH 2 dendrimer. There was a strong correlation between membrane disruption and the determined biocidal activity, making it a key contributing mechanism of action. This study demonstrates that selection of the type of PAMAM dendrimer is important as their inherent antimicrobial efficacy varies according to their individual physicochemical properties. This understanding may pave the way for the development of enhanced dendrimer-based antimicrobial formulations and drug-delivery systems., (Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

The OECD test guideline 428 for the assessment of dermal absorption in vitro has been in force for more than a decade. Various sectors of industry utilise the method for the registration of chemical products. These include the Agrochemical and Cosmetic sectors where the OECD test guideline and industry-specific guidance forms a key part of the human risk assessment process for new and existing products. This investigation has compared the dermal absorption characteristics of one of the OECD 428 reference chemicals, testosterone, in human and pig dermatomed skin. We used identical dosing and skin decontamination conditions for testosterone in Franz static diffusion cells. This included a full mass balance recovery of the dose applied and distribution of the compound in the different layers of the skin. Our investigation has shown that intact human skin provides a more effective barrier to the dermal absorption of testosterone compared with pig skin, when studied according to modern day in vitro dermal absorption guidance., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Although an internationally-adopted in vitro dermal absorption test guideline is available (OECD Test Guideline 428), the replacement of the in vivo approach in North America for pesticide formulations has not occurred due to concern over the reliability and consistency of the in vitro results. A 2012 workshop convened a panel of experts in the conduct of in vitro studies used for pesticide risk assessment, together with North American regulators, to examine techniques for in vitro dermal absorption testing. Discussions led to the recommended "best practices" for the conduct of in vitro dermal absorption studies provided herein. The workshop participants also developed recommendations for reporting study results in order to improve the quality and consistency of the data submitted to regulatory agencies in North America. Finally, a case study is presented that illustrates the use of the "triple-pack" approach; the studies, conducted for the registration of sulfoxaflor, follow the standardized recommendations provided at the workshop. In addressing the concerns of these regulators and of the regulated community, and providing harmonized recommendations to facilitate comparative data analyses, it is anticipated that wider acceptance of in vitro dermal absorption studies alone can be achieved for pesticide risk assessment., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Skin penetration and localisation of chlorhexidine digluconate (CHG) within the skin have been investigated in order to better understand and optimise the delivery using a nano polymeric delivery system of this topically-applied antimicrobial drug. Franz-type diffusion cell studies using in vitro porcine skin and tape stripping procedures were coupled with Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) to visualise the skin during various treatments with CHG and polyamidoamine dendrimers (PAMAM). Pre-treatment of the skin with PAMAM dendrimers significantly increased the amount and depth of permeation of CHG into the skin in vitro. The effect observed was not concentration dependant in the range 0.5-10mM PAMAM. This could be important in terms of the efficiency of treatment of bacterial infection in the skin. It appears that the mechanism of enhancement is due to the PAMAM dendrimer disrupting skin barrier lipid conformation or by occluding the skin surface. Franz-type diffusion cell experiments are complimented by the detailed visualisation offered by the semi-quantitative ToF-SIMS method which provides excellent benefits in terms of sensitivity and fragment ion specificity. This allows a more accurate depth profile of chlorhexidine permeation within the skin to be obtained and potentially affords the opportunity to map the co-localisation of permeants with skin structures, thus providing a greater ability to characterise skin absorption and to understand the mechanism of permeation, providing opportunities for new and more effective therapies., (Copyright © 2017. Published by Elsevier B.V.)

A new in vitro model based on the electrical resistance properties of the skin barrier has been established in this laboratory. The model utilises a tape stripping procedure in dermatomed pig skin that removes a specific proportion of the stratum corneum, mimicking impaired barrier function observed in humans with damaged skin. The skin penetration and distribution of chemicals with differing physicochemical properties, namely; Benzoic acid, 3-Aminophenol, Caffeine and Sucrose has been assessed in this model. Although, skin penetration over 24h differed for each chemical, compromising the skin did not alter the shape of the time course profile, although absorption into receptor fluid was higher for each chemical. Systemic exposure (receptor fluid, epidermis and dermis), was marginally higher in compromised skin following exposure to the fast penetrant, Benzoic acid, and the slow penetrant Sucrose. The systemically available dose of 3-Aminophenol increased to a greater extent and the absorption of Caffeine was more than double in compromised skin, suggesting that Molecular Weight and Log P ow , are not the only determinants for assessing systemic exposure under these conditions. Although further investigations are required, this in vitro model may be useful for prediction of dermal route exposure under conditions where skin barrier is impaired., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The conventional safety approach that includes dermal absorption of pharmaceutical or consumer products uses models that are based on intact skin. However, when products are intended for application to skin with a less effective barrier, such as in new-born infants, or in cases where the skin is mildly damaged or diseased, there are instances where absorption through compromised skin is also important. A tape stripping procedure was investigated using dermatomed pig skin to assess if an in vitro model could replicate the typical changes in barrier function observed in humans with compromised skin. The relationship between Trans-Epidermal Water Loss (TEWL), Electrical Resistance (ER) and Tritiated Water Flux(TWF), markers of skin barrier function in OECD 428 studies was investigated. There was a step-wise reduction in ER from normal (control) skin following 5, 10, 15 or 20 tape strips. This was mirrored by increases in both TWF and TEWL. An in vitro experimental protocol using 5 tape strips, ER and dermatomed pig skin provided a rapid, robust and reproducible approach equivalent to the 3–4-fold increases in TEWL observed clinically in compromised skin.

Purpose: In order to increase the efficacy of a topically applied antimicrobial compound the permeation profile, localisation and mechanism of action within the skin must first be investigated., Methods: Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to visualise the distribution of a conventional antimicrobial compound, chlorhexidine digluconate, within porcine skin without the need for laborious preparation, radio-labels or fluorescent tags., Results: High mass resolution and high spatial resolution mass spectra and chemical images were achieved when analysing chlorhexidine digluconate treated cryo-sectioned porcine skin sections by ToF-SIMS. The distribution of chlorhexidine digluconate was mapped throughout the skin sections and our studies indicate that the compound appears to be localised within the stratum corneum. In parallel, tape strips taken from chlorhexidine digluconate treated porcine skin were analysed by ToF-SIMS to support the distribution profile obtained from the skin sections., Conclusions: ToF-SIMS can act as a powerful complementary technique to map the distribution of topically applied compounds within the skin.

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.

There is compelling evidence that contact allergens differ substantially (by 4 or 5 orders of magnitude) with respect to their inherent skin-sensitizing potency. Relative potency can now be measured effectively using the mouse local lymph node assay (LLNA) and such data form the basis of risk assessment and risk management strategies. Such determinations also facilitate distinctions being drawn between the prevalence of skin sensitization to a particular contact allergen and inherent potency. The distinction is important because chemicals that are implicated as common causes of contact allergy are not necessarily potent sensitizers. One example is provided by nickel that is undoubtedly a common cause of allergic contact dermatitis, but is a comparatively weak sensitizer in predictive tests. In an attempt to explore other examples of contact allergens where there may exist a discrepancy between prevalence and potency, we describe here analyses conducted with methyl methacrylate (MMA). Results of LLNA studies have been interpreted in the context of historical clinical data on occupational allergic contact dermatitis associated with exposure to MMA.

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.

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.

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.

The European Centre for the Validation of Alternative Methods (ECVAM) Skin Irritation Task Force was established in 1996, to review the status of the development and validation of alternative tests for skin irritation and corrosion, and to identify appropriate non-animal tests for predicting human skin irritation that were sufficiently well-developed to be prevalidated and validated by ECVAM. The EpiDerm method, based on a reconstituted human skin model, was proposed as being sufficiently well advanced to enter a prevalidation (PV) study. Based on a review of test protocols, prediction models (PMs), and data submitted by test developers on ten specified chemicals, with 20% sodium lauryl sulphate as a reference standard, the task force recommended the inclusion of four other tests: EPISKIN and PREDISKIN, based on reconstituted human epidermis or on human skin; the non-perfused pig-ear test, based on pig skin; and the skin integrity function test (SIFT), with ex vivo mouse skin. The prevalidation study on these methods was funded by ECVAM, and took place during 1999-2000. The outcome of the PV study was that none of the methods was ready to enter a formal validation study, and that the protocols and PMs of the methods had to be improved in order to increase their predictive abilities. Improved protocols and PMs for the EpiDerm and EPISKIN methods, the pig ear test, and the SIFT were presented at an extended Task Force meeting held in May 2001. It was agreed that, in the short term, the performance of the revised and harmonised EpiDerm and EPISKIN methods, as well as the modified SIFT, should be evaluated in a further study with a new set of 20 test chemicals. In addition, it was decided that the SIFT and the pig ear test would be compared to see if common endpoints (transepidermal water loss, methyl green-pyronine stain) could be identified.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Effects of antiinflammatory agents and prostaglandins on H+ and HCO-3 secretions and electrical properties were investigated in the amphibian-isolated gastric mucosa. Gastric HCO-3 transport was studied in Rana temporaria fundus, in which H+ secretion had been inhibited with the histamine H2-receptor antagonists metiamide or cimetidine (10(-3) M), and in Necturus antrum, which secreted HCO-3 spontaneously. Hydrocortisone (100-500 microgram/ml) had no effect on H+ or HCO-3 secretion in the fundus. Indomethacin (10(-4) M) was a considerably more potent inhibitor of HCO-3 secretion than of H+ secretion in the fundus and also inhibited HCO-3 transport in the antrum. Fenclofenac (3 x 10(-3) M) almost abolished fundic HCO-3 transport and also depressed H+ secretion. There was a marked fall in transmucosal potential difference and a decrease in electrical resistance in fenclofenac-treated mucosae whereas indomethacin had less effect on electrical properties at the concentrations used here. The prostaglandins, E2, 16,16-dimethyl E2 and I2 all inhibited H+ secretion but only 16,16-dimethyl E2 stimulated HCO-3 secretion. The inhibitory action of indomethacin on HCO-3 secretion was prevented by co-administration of 16,16-dimethyl PGE2 (10(-6) M). It is proposed that the inhibitory action of nonsteroidal antiinflammatory drugs and the stimulatory action of some prostaglandins on HCO-3 secretion contributes to their ulcerogenic and anti-ulcer actions on the gastric mucosa.

Segments of proximal duodenum from Rana catesbeiana, stripped of external muscle and mounted as a tube in a glass chamber, alkalinized the luminal-side bathing solution at a rate of 1.70 +/- 0.16 microEq/cm . h (n = 18, gross surface area approximately 1.5 cm2/cm). A single change of the serosal-side bathing solution for fresh solution reduced the rate of titratable alkaline secretion, which achieved a new steady state after 45 min amounting to 66% +/- 2% of the initial rate; transmucosal potential difference (lumen negative) fell from 10.6 +/- 1.2 to 8.8 +/- 1.1 mV. Concentrations of E-type prostaglandins in the serosal-side solution measured by radioimmunoassay were 8.5 nM (3 ng/ml) before, 0.17 nM 5 min after, and 1.7 nM 90 min after the solution change (n = 8). Reapplication of the original bathing solution 90 min after the initial change reestablished original secretory rate and potential difference. The increases in alkaline secretion and potential difference were comparable in magnitude and profile to those induced by serosal administration of 10 nM prostaglandin E2. Addition of the metabolic inhibitor 2,4-dinitrophenol (100 microM, serosal side) reduced basal alkaline secretion to 30% +/- 7% of the initial rate and abolished the potential difference (n = 8). These data demonstrate that endogenous prostaglandin E production by an isolated preparation of amphibian duodenum accounts for a proportion of alkaline secretion that is equivalent to 50% of metabolism-dependent basal secretion.

Duodenal surface epithelial transport of HCO3(-) was measured by direct titration in anesthetized animals. Alkalinization of the lumen occurred in all species, although basal rates varied considerably: rats (approximately 10), cats (approximately 15), pigs (approximately 25), dogs (approximately 25), guinea pigs (approximately 40), and rabbits (approximately 170 mueq.cm-1.h-1). In cats duodenum transported HCO3(-) at a greater basal rate than jejunum (approximately 5 mueq.cm-2.h-1) and developed a higher transmucosal electrical potential difference (PD, lumen negative). Luminal application of 10 mM HCl for 5 min produced a sustained increase in the rate of duodenal HCO3(-) transport that was accompanied by a rise in appearance of E-like prostaglandin immunoreactivity in the lumen and a decrease in DNA release. In cats pretreated with indomethacin (10 mg/kg iv), acid caused only a transient increase in HCO3(-) transport. Exogenous prostaglandin E2 (1-12 microM, luminal) increased basal HCO3(-) transport in cats, rats, and dogs but had no effect on this transport in guinea pigs and rabbits. However, prostaglandin E2 increased HCO3(-) transport and PD in guinea pigs pretreated with inhibitors of tissue cyclooxygenase activity (indomethacin or aspirin) or gastric H+ secretion (cimetidine). Thus the continuous exposure of the duodenum of herbivores to HCl discharged from the stomach may itself stimulate HCO3(-) transport via an increase in endogenous prostaglandin levels and render exogenous prostaglandins ineffective. Secretin (1-15 CU/kg iv) was without effect in both cats and guinea pigs. In guinea pigs, intravenous glucagon (120-360 micrograms.kg-1.h-1) or gastric inhibitory peptide (5 micrograms/kg) both increased HCO3(-) transport but not PD. Hence, prostaglandin-stimulated and hormone-stimulated mechanisms of HCO3(-) transport probably occur in mammalian duodenum as found previously in the isolated amphibian duodenum. The results suggest that epithelial HCO3(-) transport is a major mechanism of acid disposal, and thus mucosal protection, in mammalian duodenum under the control of hormones and endogenous prostaglandins.

Luminal application of acid was recently shown to stimulate surface epithelial HCO3(-) transport in stomach and duodenum. Effects of some potential transmitters of this response were therefore studied in amphibian gastric fundic and proximal duodenal mucosa in vitro. Duodenal HCO3- transport, which could be titrated directly, was stimulated by dibutyryl cAMP (DBcAMP, 10(-6) M), the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (10(-6) M), noradrenaline (10(-6) M), pancreatic glucagon (10(-8) M), and gastric inhibitory peptide (GIP, 10(-10) M). Stimulation by glucagon, but not by prostaglandin E2 (PGE2, 10(-6) M), required Cl- in the luminal solution and was prevented by furosemide (10(-3) M). This suggests that glucagon may affect HCO3(-)-Cl- exchange at the luminal membrane while transport stimulated by prostaglandins may be electrogenic. Stimulatory effects of glucagon and PGE2 were also additive. Gastric HCO3- transport, studied in tissues after inhibition of H+ secretion by histamine H2-antagonists, clearly differed from duodenum in that noradrenaline and GIP were inhibitory and DBcAMP was without effect. Stimulation of gastric HCO3- transport was observed with glucagon (10(-8) M), natural cholecystokinin (CCK, 10(-8) M), and CCK octapeptide (10(-7) M), CCK preparations had no effect in the duodenum. Although tested over a wide range of concentrations, no effect on either duodenal or gastric HCO3- transport was observed with histamine, pentagastrin, tetragastrin, urogastrone, ACTH, bombesin, motilin, secretin, serotonin, somatostatin, substance P, or vasoactive intestinal peptide.

Transport of HCO3- was measured by pH-stat titration in pairs of amphibian fundic or proximal duodenal mucosa using a modified Ussing chamber. Separate unbuffered solutions bathed the luminal sides of two tissues while their serosal surfaces were in contact with a common buffered solution. Lowering luminal pH bathing one fundic mucosa from 7.40 to 1.85 significantly increased HCO3- transport by another fundus. However, acidification of fundic mucosa did not affect duodenal HCO3- transport. In duodenal mucosal pairs, lowering pH from 7.40 to 5.46 caused an increase in HCO3- transport by the other tissue. Luminal H+ ion concentration may therefore regulate HCO3- transport via a humoral mechanism.

Epithelial HCO3 transport by stomach and duodenum is influenced by luminal pH. Acid itself, but not stimulants of gastric HCl secretion, increase mucosal alkaline secretion in a variety of in vitro and in vivo systems. Using pairs of amphibian mucosa, both gastric and proximal duodenal HCO3 transport was stimulated by low luminal pH. This response displayed some tissue specificity and operates by a humoral mechanism. In the anaesthetized cat, the increase in duodenal epithelial HCO3 secretion following exposure of the lumen to acid (pH2) is probably independent of luminal Cl-. Low luminal pH in the stomach did not influence HCO3 secretion by the duodenum either in vitro or in vivo. The process of acid-stimulated epithelial HCO3 transport may be important in maintaining H+ ion disposal in the upper gastrointestinal tract in response to low luminal pH.

Luminal acid (10 mM HCl) is a stimulant of surface epithelial HCO-3 transport in mammalian stomach and duodenum in vivo. To determine whether a humoral mechanism is involved in mediation of this response, amphibian fundic, antral, or proximal duodenal mucosae were mounted in parallel in an in vitro chamber with their nutrient (serosal) surfaces facing a common solution. The mucosal surfaces were bathed by separate solutions and the rate of HCO-3 transport by one mucosa titrated (at pH 7.40) during exposure of the parallel tissue to luminal acid. In studies of fundic HCO-3 transport, H+ secretion was inhibited with the histamine H2-antagonist tiotidine (10(-4) M). Fundic luminal acid stimulated HCO-3 transport by a parallel fundus (27 +/- 6%) or antrum (53 +/- 27%) but had no effect on a parallel duodenum. Antral luminal acid had no effect on a parallel antrum, indicating that the gastric stimulant is of fundic origin. Duodenal luminal acid increased HCO-3 transport by both parallel duodenum (21 +/- 5%) and fundus (109 +/- 32%). Stimulation of HCO-3 transport occurred at higher luminal pH in duodenum (approximately 4.0) than in fundus (approximately 2.0). Thus, exposure to luminal acid releases humoral factor(s) capable of stimulating surface epithelial HCO-3 transport by both stomach and duodenum. The actions of these putative stimulants are in part tissue specific, and they may be important in mediation of mucosal protection against luminal acid.

The mechanism of the gastric cytoprotective action of prostaglandins is unknown but seems to be unrelated to inhibition of acid secretion. In the present study, effects of the prostaglandins, 16,16-dimethyl E2 and F2 alpha on H+ and HCO-3 secretion and electrical properties in amphibian-isolated gastric mucosa were studied. Spontaneous net secretion in fundic mucosa from Rana temporaria and Necturus was acid, whereas Necturus antrum secreted only HCO-3. The histamine H2-receptor antagonist, metiamide (10(-3) M), was used to inhibit acid secretion for studies on fundic alkalinization. Nutrient side administration of 16,16-dimethyl E2 (10(-6) M) for 60 min inhibited H+ secretion and stimulated HCO-3 secretion in Rana temporaria fundus. The drug (10(-5) M) also stimulated antral alkalinization. There was a dose-related increase in HCO-3 secretion in Necturus fundus after administration of F2 alpha (10(-5)-10(-4) M), but this drug had no significant effect on H+ secretion. Inhibition of acid secretion by 16,16-dimethyl E2 was associated with an increase in potential difference (PD), but there was no change in electrical resistance. Neither of the prostaglandins affected PD or resistance in alkaline-secreting tissues. Previous work has suggested that gastric HCO-3 secretion has a physiologic role in protecting the mucosal surface. The ability of prostaglandins to stimulate alkaline secretion may contribute to the cytoprotective action of these drugs in the stomach.

Secretion of HCO3 (amounting to 2-10% of maximum H+ secretion) in conjunction with the adherent mucus gel layer (functioning as a mixing barrier) protects gastric mucosa from luminal acid by a process of surface neutralization. Gastric HCO3 secretion is augmented by cholinergic agonists, prostaglandins and low luminal pH. Ulcerogens attenuate HCO3 secretion although passive diffusion of alkali consequent upon an increase in mucosal permeability may mask these inhibitory actions. Studies in vitro indicate that HCO3 transport in the stomach is dependent on oxidative metabolism, carbonic anhydrase activity and involves a CL exchange mechanism. Mucus, synthesized and released from epithelial cells, adheres to the mucosal surface as a thin (less than 80 microns in rat) but continuous gel layer. Prostaglandins and carbachol induced release of preformed mucus and thereby increase thickness, whereas acute exposure to ulcerogens has little effect on overall dimensions of the surface mucus layer. Measurements of pH gradients adjacent to gastric mucosa indicate that the disposal of luminal H+ occurs by extracellular neutralization. However, the fall in pH at the apical cell membrane when luminal pH is low (pH 1.5) suggests that while a mucus-bicarbonate barrier comprises the first line of mucosal defence, other factors are involved in the overall process of mucosal protection in the stomach.

The influence of carbenoxolone sodium on HCO-3 transport has been examined in spontaneously alkalinizing amphibian antral (Necturus and Rana catesbeiana) and proximal duodenal (Rana catesbeiana) mucosa and in cimetidine-treated fundic mucosa (Rana temporaria) in vivo. Low concentrations of carbenoxolone (10(-6)-10(-4) mol/l, serosal side and 10(-5) mol/l, luminal side) did not affect the secretory rate or electrical properties of these tissues. In the stomach a higher concentration of carbenoxolone (10(-3) mol/l, serosal side) caused an immediate fall in transmucosal potential difference (PD) and electrical resistance. There was an initial decrease in the rate of HCO-3 transport followed by an increase in titratable alkalinization due to passive permeation of base from the serosal bathing solution. The non-steroidal anti-inflammatory agent ibuprofen (3 x 10(-3) mol/l, serosal side) inhibited alkaline secretion while the bile salt sodium taurocholate (10(-4) mol/l, luminal side) converted net alkaline secretion to a titratable acidity in cimetidine-treated fundus. Pretreatment of the mucosa with carbenoxolone (10(-4) mol/l) did not influence the response to taurocholate but when added with ibuprofen it potentiated the inhibitory effect of this drug on fundic alkaline secretion. In contrast, prostaglandin E2 (10(-6) mol/l) markedly reduced the inhibition of fundic alkaline secretion caused by ibuprofen. The anti-ulcer properties of carbenoxolone do not appear to be related to effects on gastroduodenal HCO-3 transport.

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.

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.

This study investigated the action of enprostil, a synthetic analog of PGE2, on gastric HCO3- secretion in humans and on duodenal HCO3- secretion in the anesthetized rat. A previously validated 2-component model was used to calculate gastric HCO3- and H+ secretion in 10 human subjects. Compared to placebo, a single 70 micrograms oral dose of enprostil increased basal gastric HCO3- secretion from 1810 +/- 340 to 3190 +/- 890 mumol/hr (P less than 0.05). In addition, enprostil reduced basal gastric H+ secretion from 5240 +/- 1140 to 1680 +/- 530 mumol/hr (P less than 0.02). Enprostil also increased HCO3- secretion and reduced H+ secretion during intravenous pentagastrin infusion. In the rat, duodenal HCO3- secretion was measured by direct titration in situ using perfused segments of duodenum just distal to the Brunner gland area and devoid of pancreatic and biliary secretions. Addition of enprostil (10 micrograms/ml) to the duodenal bathing solution increased duodenal HCO3- secretion from 6.3 +/- 1.3 to 15.1 +/- 2.0 mumol/cm X hr (P less than 0.01, n = 6). The stimulatory action of enprostil on duodenal HCO3- secretion at 10 micrograms/ml was comparable in magnitude and duration to that of 10 micrograms/ml natural PGE2. In summary, the PGE2 analog enprostil stimulated gastroduodenal HCO3- secretion, effects which may be beneficial in protection of the gastroduodenal mucosa against luminal acid.