Adaption of a dermal in vitro method to investigate the uptake of chemicals across amphibian skin

Abstract

BackgroundLiterature data indicate that terrestrial life stages of amphibians may be more sensitive to xenobiotics than birds or mammals. It is hypothesized that dermal exposure could potentially be a significant route of exposure for amphibians, as there is evidence that their skin is more permeable than the skin of other vertebrate species. Thus, higher amounts of xenobiotics might enter systemic circulation by dermal uptake resulting in adverse effects. Heretofore, no guidelines exist to investigate dermal toxicity of chemicals to amphibians. In order to minimize vertebrate testing, this work was targeted to develop an in vitro test system as a possible model to assess the dermal uptake of chemicals across amphibian skin.ResultsThe dermal absorption in vitro method (OECD guideline 428), an established toxicological (mammal) test procedure, was adapted to amphibian skin, in a first approach using the laboratory model organism Xenopus laevis and reference compounds (caffeine and testosterone). Skin permeability to both reference substances was significantly higher compared to published mammalian data. Caffeine permeated faster across the skin than testosterone, with ventral skin tending to be more permeable than dorsal skin. As usage of frozen mammalian skin is accepted, frozen skin of X. laevis was tested in parallel. To the freshly excised skin, however, freezing led to increased skin permeability, in particular to caffeine, indicating a loss of skin integrity due to freezing (without additional preservation measures).ConclusionsThis work has demonstrated that the chosen method can be applied successfully to amphibian skin, providing the basis for further investigations. In future, well-established in vitro test systems and a broad dataset for many chemicals may help assess potential amphibian risk from xenobiotics without the need for extensive vertebrate testing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12302-016-0080-y) contains supplementary material, which is available to authorized users.

Effects of skin region and relative lipophilicity on percutaneous absorption in the toad Rhinella marina

“…In contrast to the findings in L. caerulea , which showed a parabolic relationship between logFlux and log P for dorsal absorption and a linear relationship for ventral absorption (Llewelyn et al ), the impact of lipophilicity on flux was consistent between skin regions in R. marina , demonstrating a decline with increasing lipophilicity. This finding corresponds with the findings of Kaufmann and Dohmen (), who reported a reduction in flux for testosterone (log P = 3.3) compared with caffeine in X. laevis . A reduction in flux with increasing log P has also been reported in mammalian species (Dal Pozzo and Pastori ; Cross et al ).…”

“…It is therefore likely that the increased water absorption from this region would facilitate concurrent absorption of hydrophilic chemicals such as caffeine. This finding is not unique; an increase in caffeine flux through the ventral pelvic skin is consistent with findings in both L. caerulea and X. laevis , with both studies reporting the highest and most variable flux through the ventral pelvic skin for caffeine (Kaufmann and Dohmen ; Llewelyn et al ). However, the magnitude of the difference between skin regions differed between species, with the present study finding a 1.2‐fold increase in flux, whereas a 1.5‐fold increase was reported in X. laevis and a 33‐fold increase in ventral flux compared to dorsal flux was reported in L. caerulea .…”

“…Absorption rates reported in these studies reflected these physiological differences in skin structure, with significantly higher flux of all chemicals through the ventral skin compared with the dorsum in L. caerulea (Llewelyn et al ). A slight increase in ventral flux was also reported in X. laevis (Kaufmann and Dohmen ); however, the magnitude of the difference was far less than that reported in L. caerulea , being no more than a 2.3‐fold increase for testosterone, compared to a 33‐fold increase between regions seen for ventral versus dorsal flux of caffeine in L. caerulea (Llewelyn et al ).…”

“…Two recent studies (Kaufmann and Dohmen ; Llewelyn et al ) investigated the in vitro transdermal kinetics of various standard chemicals in isolated frog skin using static diffusion cells. Each study investigated a species from a different habitat: Kaufmann and Dohmen () conducted investigations in the aquatic frog Xenopus laevis , whereas Llewelyn et al () used the arboreal frog Litoria caerulea .…”

“…Two recent studies (Kaufmann and Dohmen ; Llewelyn et al ) investigated the in vitro transdermal kinetics of various standard chemicals in isolated frog skin using static diffusion cells. Each study investigated a species from a different habitat: Kaufmann and Dohmen () conducted investigations in the aquatic frog Xenopus laevis , whereas Llewelyn et al () used the arboreal frog Litoria caerulea . Skin morphology in these species differs substantially, with X. laevis demonstrating a thicker skin overall with reduced vascularization across the entire body (Roth ) and L. caerulea having a relatively thick dorsum compared to its thin and highly vascularized ventral skin (personal observation).…”