Isolated Perfused Porcine Skin Flap Research Articles

Modeling gold nanoparticle biodistribution after arterial infusion into perfused tissue: effects of surface coating, size and protein corona

A detailed understanding of the factors governing nanomaterial biodistribution is needed to rationally design safe nanomedicines. This research details the pharmacokinetics of gold nanoparticle (AuNP) biodistribution after arterial infusion of 40 or 80 nm AuNP (1 μg/ml) into the isolated perfused porcine skin flap (IPPSF). AuNP had surface coatings consisting of neutral polyethylene glycol (PEG), anionic lipoic acid (LA), or cationic branched polyethylenimine (BPEI). Effect of a porcine plasma corona (PPC) on 40 nm BPEI and PEG-AuNP were assessed in the IPPSF. Au concentrations were determined by ICP/MS and arterial to venous concentration-time profiles were analyzed over 8 hr (4 hr infusion, 4 hr washout) using a two-compartment pharmacokinetic model. IPPSF viability and vascular function were assessed by change in glucose utilization, vascular resistance, or weight gain after perfusion. All AuNP demonstrated some degree of AuNP arterial extraction and skin flap retention, as well as enhanced kinetic parameters of tissue uptake; with BPEI-AuNP consistently having the greatest biodistribution even with a PPC. Toxicological effects were not detected. Transmission electron microscopy confirmed intracellular uptake of AuNP. These studies paralleled previous in vitro cell culture studies using the same AuNP in human endothelial and renal proximal tubule cells, hepatocytes, keratinocytes, showing BPEI-AuNP having the greatest uptake, although the presence of a PPC did not reduce IPPSF biodistribution as in the cell culture studies. These findings clearly indicate arterial to the venous extraction of AuNP after infusion with the magnitude of extraction being greatest with the BPEI surface coating and provide data and model structure necessary to construct the whole body physiologically based pharmacokinetic models capable of utilizing available in vitro data.

Assessment of penetrant and vehicle mixture properties on transdermal permeability using a mixed effect pharmacokinetic model of ex vivo porcine skin.

The accurate prediction of the rate and extent of transdermal absorption from topical exposure to chemical mixtures would be beneficial in risk assessment and drug delivery applications. The isolated perfused porcine skin flap (IPPSF) has been used as an ex vivo model for assessing transdermal absorption from topical exposures. A mixed effect, pharmacokinetic tissue model was used to model finite dose, transdermal, absorption data from IPPSF experiments for 12 penetrants dosed in up to 10 different vehicles. The model was able to identify permeability constant, while accounting for between and within unit variability, across the entire data set. This approach provides a platform for exploring the relationship between covariates (chemical descriptors and functions thereof) and the model parameters. Successive models were employed that reduced the overall variability in the parameter estimate by modeling the parameters as functions of the covariates. Log kp was initially modeled as a function of LogP and MW of the pure penetrant (adjusted r2 =0.48). The addition of mixture factors to account for the different dosing vehicles further improved the relationship: to r2 =0.56 with Connolly molecular area (CMA) and r2 =0.78 with the further addition of total polar surface area difference (TPSAd). The pharmacokinetic model and quantitative structure property relationship (QSPR) developed for the IPPSF may be relevant to clinical transdermal formulation development. Copyright © 2016 John Wiley & Sons, Ltd.

In vitrobiodistribution of silver nanoparticles in isolated perfused porcine skin flaps

Nanomaterials increasingly are playing a role in society for uses ranging from biomedicine to microelectronics; however, pharmacokinetic studies, which will be necessary for human health risk assessments, are limited. Currently the most widely used nanoparticle in consumer products is silver (Ag). The objective of the present study was to quantify the local biodistribution of two types of Ag nanoparticles, Ag-citrate and Ag-silica, in the isolated perfused porcine skin flap (IPPSF). IPPSFs were perfused for 4 h with 0.84 µg ml(-1) Ag-citrate or 0.48 µg ml(-1) Ag-silica followed by a 4-h perfusion with media only during a washout phase. Arterial and venous concentrations of Ag were measured in the media by inductively coupled plasma optical emission spectrometry (ICP-OES). Venous concentrations of Ag for both types of nanoparticles were best fit with a two compartment model. The normalized volumes of distribution estimated from the noncompartmental analysis of the venous concentrations indicated distribution of Ag greater than the vascular space; however, because total Ag was measured, the extravascular distribution could be attributed to diffusion of Ag ions. The estimated clearance for both types of Ag nanoparticles was 1 ml min(-1) , which was equal to the flap perfusion rate, indicating no detectable elimination of Ag from the system. Four hours after infusion of the Ag nanoparticles, the recovery of Ag in the venous effluent was 90 ± 5.0% and 87 ± 22% of the infused Ag for Ag-citrate and Ag-silica, respectively.

Acute vascular effects of nanoparticle infusion in isolated perfused skin

The majority of studies on the effect of nanomaterials on biological function involves either isolated in vitro cell systems or are concerned with in vivo effects after inhalational or dermal exposure. The current work reports on an intriguing observation of the vascular effects seen in an ex vivo perfused tissue preparation, the isolated perfused porcine skin flap (IPPSF), in studies conducted to assess nanomaterial biodistribution. Compared with a relatively large dataset involving organic chemical infusions (n = 53), infusion of six different nanoparticles of diverse sizes and composition (silica or dextran coated Fe2O3, silica or citrate coated silver, PEG or carboxylated quantum dots [QD]) resulted in statistically significant post-infusion flap weight gain and an increase in arterial perfusion pressure (especially with QD-PEG). In contrast, infusion with nC60 nanoparticles did not produce these effects. These observations suggest certain nanoparticle infusions may be associated with acute vascular physiologic effects that merit further attention. From the Clinical EditorIn this study utilizing a perfused porcine skin flap, specific nanoparticle infusions were demonstrated to be associated with significant acute vascular physiological effects.

Evaluation of perfused porcine skin as a model system to quantitate tissue distribution of fullerene nanoparticles

Nanomaterials are increasingly playing a role in society for uses ranging from biomedicine to microelectronics, however pharmacokinetic studies, which will be necessary for human health risk assessments, are limited. Tissue distribution, one component of pharmacokinetics, can be assessed by quantifying arterial extraction of materials in an isolated perfused porcine skin flap (IPPSF). The objective of this study was to assess the IPPSF as a model system to quantitate the distribution of fullerene nanoparticles (nC 60) from the vascular space into tissues. IPPSFs were perfused for 4 h with 0.885 μg/mL nC 60 in media with immunoglobulin G present (IgG +) or absent (IgG −) followed by a 4 h perfusion with media only during a washout phase. Arterial and venous concentrations of nC 60 were measured in the media by HPLC–UV/vis chromatography. Steady state differences in the arterial and venous nC 60 concentrations were compared to determine extraction from the vascular space of the IPPSF, and the venous nC 60 concentration versus time profiles were used to calculate compartmental pharmacokinetic parameters. The steady state differences in the arterial and venous concentrations in the IPPSF were small with extraction percentages (mean ± sd) of 8.2 ± 5.7% and 4.2 ± 6.7% for IgG + and IgG − media, respectively, and were not significantly different between the types of media. The venous concentrations of nC 60 in both types of media were best fit with a 2 compartment model with terminal half lives (harmonic mean) of 17.5 and 28.0 min for IgG + and IgG − media, respectively. The apparent volumes of distribution at steady state were 0.12 ± 0.047 and 0.10 ± 0.034 L/kg, for IgG + and IgG − media, respectively. By 4 h following infusion of nC 60, the recovery of nC 60 in the venous effluent was 94 ± 5.5% and 97 ± 6.8% of the infused nC 60 for IgG + and IgG − media, respectively. Based on the apparent volume of distribution, the low extraction during the perfusion, and the high percentage recovery following the washout phase, there was limited distribution of nC 60 from the vascular space into the extracellular space and negligible intracellular uptake of nC 60 in this system.

Dermatotoxicity of Cutting Fluid Mixtures:In Vitro and In Vivo Studies

Cutting fluids are widely used in the metal-machining industry to lubricate and reduce heat generation when metals are cut by a metal-cutting tool. These cutting fluids have caused occupational irritant contact dermatitis (OICD), and many of the additives used in these cutting fluid mixtures are thought to be responsible for OICD in workers. The purpose of this study was to assess single or various combinations of these additives in initiating the OICD response following an acute 8-hour exposure in porcine skin in vivo and in vitro using the isolated perfused porcine skin flap (IPPSF) and human epidermal keratinocytes (HEK). Pigs (n = 4) were exposed to 5% mineral oil (MO) or 5% polyethylene glycol (PEG) aqueous mixtures containing various combinations of 2% triazine (TRI), 5% triethanolamine (TEA), 5% linear alkylbenzene sulfonate (LAS), or 5% sulfurized ricinoleic acid (SRA). Erythema and edema were evaluated and skin biopsies for histopathology were obtained at 4 and 8 hours. IPPSFs (n = 4) were exposed to control MO or PEG mixtures and complete MO or PEG mixtures, and perfusate samples were collected hourly to determine interleukin- (IL-) 8 release. The only significant (p < 0.05) mixture effects observed in IPPSFs were with SRA + MO that caused an increase in IL-8 release after 1 or 2 hours' exposure. In vivo exposure to TRI alone appeared to increase erythema, edema, and dermal inflammation compared to the other additives, while SRA alone was least likely to initiate a dermal inflammatory response. In 2-component mixture exposures, the presence of TRI appeared to increase the dermal inflammatory response at 4 and 8 hours especially with the PEG mixtures. In the 3- and 4-component mixtures, MO mixtures are more likely to incite an inflammatory response than PEG mixtures. TRI exhibited the highest toxicity toward HEK, which correlates well to the in vivo irritation and morphology results. In summary, these preliminary studies suggest that the biocide, TRI, is the more potent of the 4 performance additives in causing dermal irritation, and this may vary depending on whether the worker is exposed to a synthetic (PEG)- or MO-based fluid. These findings will however require further clinical studies to validate these acute dermal effects as well as human cumulative irritation following exposure to similar cutting fluid formulations in the workplace.

Comparative mixture effects of JP-8(100) additives on the dermal absorption and disposition of jet fuel hydrocarbons in different membrane model systems

Jet fuel are complex mixtures of hydrocarbon fuel components and performance additives. Three different membrane systems, silastic, porcine skin and the isolated perfused porcine skin flap (IPPSF) were used to gain insight into the possible mechanism for additive interactions on hydrocarbon component absorption. Influence of JP-8(100) additives on the dermal kinetics of 14 C -naphthalene and 14 C/ 3 H -dodecane as markers of hydrocarbon absorption, were evaluated using analysis of means (ANOM) and analysis of variance (ANOVA). This study indicated that the naphthalene absorption through silastic membrane was significantly different with JP-8 plus individual additives as compared to controls, i.e. JP-8 and JP-8(100). The porcine skin data indicated that neither individual nor combinations of additives affected naphthalene absorption. The third membrane system (IPPSF) showed that only MDA and BHT were important additives altering naphthalene absorption. MDA was a significant suppressor while BHT was a significant enhancer of naphthalene absorption. MDA significantly decreased dodecane absorption in skin flaps. All individual and combinations of two additives with JP-8 affected naphthalene and dodecane surface retention in silastic membrane. The IPPSF indicated that only 8Q405 is a significant modulator of surface retention for both marker hydrocarbons. The 8Q405 significantly reduced naphthalene contents in dosed silastic and skin indicating a direct interaction between additive and marker hydrocarbons. The MDA and BHT, which significantly retained naphthalene in the stratum corneum of porcine skin individually, led to a statistical decrease in its retention in the stratum corneum when in combination (MDA+BHT) suggesting a potential biological interaction. These observations demonstrate that the single membrane system may not be suitable for the final prediction of complex additive interactions in jet fuels. Rather a combination of different membrane systems may provide the insight to elucidate the possible mechanism for additive interactions. Finally, it is important to assess all components of a chemical mixture since the effects of single components administered alone or as pairs may be confounded when all are present in the complete mixture.

Percutaneous absorption of 2,6-di- tert-butyl-4-nitrophenol (DBNP) in isolated perfused porcine skin

DBNP (2,6-di- tert-butyl-4-nitrophenol) has been reported as a potential contaminant in submarines. This yellow substance forms when lubrication oil mist containing the antioxidant additive 2,6-di- tert-butylphenol passes through an electrostatic precipitator and is nitrated. Percutaneous absorption of 14C-DBNP was assessed in the isolated perfused porcine skin flap (IPPSF). Four treatments were studied ( n=4 flaps/treatment): 40.0 μg/cm 2 in 100% ethanol; 40.0 μg/cm 2 in 85% ethanol/15% H 2O; 4.0 μg/cm 2 in 100% ethanol; and 4.0 μg/cm 2 in 85% ethanol/15% water. DBNP absorption was minimal across all treatment groups, with the highest absorption detected being only 1.08% applied dose in an aqueous ethanol group. The highest mass of 14C-DBNP absorbed was only 0.5 μg. The majority of the applied dose remained on the surface of the skin. This suggests that there is minimal dermal exposure of DBNP when exposed topically to skin.

In Vitro Percutaneous Absorption of Nonylphenol (NP) and Nonylphenol Ethoxylates (NPE-4 and NPE-9) in Isolated Perfused Skin

Skin contact with nonylphenol ethoxylates (NPE), a group of widely used surfactants, is the primary source of human exposure. Previous studies have shown that the absorption of NPE through human and animal skin in vitro is limited (<1% over 8 hr) [Monteiro-Riviere et al. Toxicol Indust Health 2000; 16:49–57]. The purpose of this study was to examine the percutaneous absorption of NPE and the chemical precursor, nonylphenol (NP), in the isolated perfused porcine skin flap (IPPSF) model for comparison to the in vitro porcine skin flow through (PSFT) diffusion studies. The IPPSF model is considered to accurately predict absorption of chemicals through human skin. The IPPSF was dosed with 100 μl of 1% 14C ring-labeled NP, 14C ring-labeled NPE-4, or 14C ring-labeled NPE-9 in aqueous polyethylene glycol (PEG-400) solution and perfused for 8 hr. All three chemicals were minimally absorbed, with only approximately 0.1% of the applied dose found in the perfusate over the 8-hr collection. This absorbed material rep...

Percutaneous Absorption of Topical N , N -Diethyl- m -Toluamide (Deet): Effects of Exposure Variables and Coadministered Toxicants

Exposure to N,N-diethyl-m-toluamide (DEET) commonly occurs in the general population and has been implicated as a contributory factor to the Gulf War Illness. The focus of the present studies was to determine the effect of coexposure factors, potentially encountered in a military environment, that could modulate transdermal flux of topically applied DEET. Factors investigated were vehicle, dose, coexposure to permethrin, low-level sulfur mustard, occlusion, and simultaneous systemic exposure to pyridostigmine bromide and the nerve agent simulant diisopropylfluorophosphate (DFP). Studies were conducted using the isolated perfused porcine skin flap (IPPSF), with a few mechanistically oriented studies conducted using in vitro porcine skin and silastic membrane diffusion cells. DEET was quantitated using high-performance liquid chromatography. The vehicle-control transdermal DEET flux in the IPPSF was approximately 2 w g/cm 2 /h for both 7.5 and 75% DEET concentrations, a value similar to that reported in humans. DEET absorption was enhanced by coinfusion of pyridostigmine bromide and DFP, by the presence of sulfur mustard, or by dosing under complete occlusion. The greatest increase in baseline flux was fivefold. In vitro diffusion cell studies indicated that silastic membranes had two orders of magnitude greater permeability than porcine skin, and showed vehicle effects on flux that were not detected in the IPPSF. These results suggest that coexposure to a number of chemicals that potentially could be encountered in a military environment may modulate the percutaneous absorption of topically applied DEET beyond that seen for normal vehicles at typically applied concentrations.

Pyridostigmine bromide modulates topical irritant-induced cytokine release from human epidermal keratinocytes and isolated perfused porcine skin

Gulf War personnel were given pyridostigmine bromide (PB) as a prophylactic treatment against organophosphate nerve agent exposure, and were exposed to the insecticide permethrin and the insect repellent N, N-diethyl- m-toluamide (DEET). The purpose of this study was to assess the effects of PB to modulate release of inflammatory biomarkers after topical chemical exposure to chemical mixtures containing permethrin and DEET applied in ethanol or water vehicles. Treatments were topically applied to isolated perfused porcine skin flaps (IPPSFs). Concentrations of interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α) and prostaglandin E 2 (PGE 2) were assayed in perfusate to probe for potential inflammatory effects after complex mixture application. IPPSFs ( n=4/treatment) were topically dosed with mixtures of permethrin, DEET, and permethrin/DEET, in ethanol. Each treatment was repeated with perfusate spiked with 50 ng/ml of PB. Perfusate was also spiked with 30 ng/ml diisopropylfluorophosphate to simulate low level organophosphate nerve agent exposure. Timed IPPSF venous effluent samples (0.5,1,2,4, and 8 h) were assayed by ELISA for IL-8 and TNF-α and by EIA for PGE 2. Overall, PB infusion caused a decrease or IL-8 and PGE 2 release. Effects on TNF-α were vehicle dependent. To probe the potential mechanism of this PB effect, human epidermal keratinocyte HEK cell cultures were exposed to permethrin DEET permethrin/DEET, with and without PB in DMSO. IL-8 was assayed at 1, 2, 4, 8, 12 and 24 h. PB suppressed IL-8 in permethrin and ethanol treatment from 4 to 24 h confirming the IPPSF results. In conclusion, these studies suggest that systemic exposure to PB suppressed IL-8 release at multiple time points in two skin model systems. This interaction merits further study.

Gulf War related exposure factors influencing topical absorption of 14C-permethrin

Topical exposure to permethrin has often been implicated as a mitigating factor in the illnesses reported in Gulf War veterans. These studies were designed to assess the effect of co-exposure to low level sulfur mustard, JP-8 jet fuel, N, N-diethyl- m-toluamide (DEET) and fabric occlusion on the percutaneous absorption and skin disposition of topically applied 14C-permethrin (40 μg/cm 2) in the isolated perfused porcine skin flap (IPPSF) model. Extent of dermal absorption in vehicle controls in the IPPSF was comparable to literature values for humans. These studies demonstrated a two-fold increased 14C-permethrin percutaneous absorption and almost three-fold increased penetration when JP-8 was present, compared to a one-third decreased permethrin flux in the presence of sulfur mustard. Complete occlusion slightly increased 14C-permethrin absorption, while occlusion with fabric showed no significant effect. A previously noted effect of DEET to inhibit permethrin absorption was still seen in the presence of sulfur mustard exposure. These studies suggest that co-exposure to JP-8 or sulfur mustard may modulate transdermal flux of 14C-permethrin. However, the JP-8 increase in absorption and penetration was less than the five-fold increase previously seen with arterial infusion of pyridostigmine bromide and diisopropylfluorophosphate in the IPPSF. The toxicologic significance of this moderate increase in permethrin absorption remains unclear.

Pyridostigmine Bromide Modulates the Dermal Disposition of [14C]Permethrin

The cause of the Gulf War Syndrome may be related to soldiers being exposed to insecticides (e.g., permethrin (P)), insect repellents (e.g., N,N-diethyl-m-toluamide (DEET)), an organophosphate nerve agent simulant (e.g., diisopropyl fluorpohosphate (DFP)), and/or prophylactic treatment (e.g., pyridostigmine bromide (PB)) against potential nerve gas attacks. The purpose of this study was to assess the dermal disposition of [14C]permethrin in ethanol or ethanol:water (3:2) in the isolated perfused porcine skin flap (IPPSF) model with simultaneous dermal exposure to DEET or DFP. These IPPSFs were also simultaneously perfused arterially with or without PB, DFP, or DFP + PB. The results indicated that DFP + PB significantly increased [14C]permethrin absorption compared to controls (1.06% dose vs 0.14% dose). PB significantly increased [14C]permethrin disposition in the stratum corneum (SC) in aqueous mixtures only (9.40 vs 3.35% dose), while topical DEET or topical DFP reduced [14C]permethrin levels in the SC especially in nonaqueous mixtures. PB also significantly enhanced [14C]permethrin penetration into all skin tissues and perfusate in aqueous mixtures, while DEET reversed this effect. PB appeared to influence [14C]permethrin disposition in flowthrough diffusion cells, suggesting that the mechanism of this interaction may be associated predominantly with epidermal permeability, although muscarinic effects in the vasculature in IPPSFs should not be ruled out and requires further investigation. These experiments suggest that intraarterial perfusion of PB and/or DFP and topical application of DFP or DEET can alter the disposition of [14C]permethrin in skin and possibly its bioavailability in soldiers simultaneously exposed to these chemicals.

Mixture Effects of JP-8 Additives on the Dermal Disposition of Jet Fuel Components

Aliphatic and aromatic components in formulated jet fuels can cause occupational dermatitis. However, the influence of JP-8 performance additives (DIEGME, 8Q21, and Stadis450) on the dermal disposition of fuel components is not well understood. These additives are formulated with commercial Jet-A to form military JP-8 fuel. The purpose of this study is to assess the influence of these additives on the dermal disposition of marker aromatic and aliphatic components, naphthalene and dodecane, respectively. Porcine skin sections in an in vitro system were used to characterize chemical–biological interactions that modulate diffusion of jet fuel components and isolated perfused porcine skin flaps (IPPSFs) were used to evaluate diffusion in a viable skin model with an intact microvasculature. In these 5-h studies, Jet-A, Jet-A + DIEGME, Jet-A + 8Q21, and Jet-A + Stadis450, Jet-A + DIEGME + 8Q21, Jet-A + DIEGME + Stadis450, Jet-A + 8Q21 + Stadis450, and JP-8 mixtures were tested. In general, naphthalene absorption (0.76–2.39% dose) was greater than dodecane absorption (0.10–0.84% dose), while the IPPSFs alone demonstrated that dodecane absorption was significantly greater in JP-8 than in Jet-A. Synergistic interactions with 8Q21 + Stadis450 appear to enhance systemic absorption of either naphthalene or dodecane, while DIEGME + Stadis450 increased naphthalene (1.88% dose) and dodecane (2.02% dose) penetration into the skin and fat tissues of IPPSFs. These findings were supported by the fact that 8Q21 + Stadis450 significantly increased dodecane flux and permeability in porcine skin sections, but 8Q21 alone reduced marker diffusion in both membrane systems. Furthermore, dodecane is more likely than naphthalene to remain in the stratum corneum and skin surface at 5 h, and DIEGME mixtures played a significant role in skin and surface retention of both markers. In summary, the data suggest that various combinations of these three performance additives in JP-8 can potentially alter the dermal disposition of aromatic and aliphatic fuel components in skin. More importantly, products of two-factor interactions were not predictable from single-factor exposures and, by extension, cannot be extrapolated to three-factor interactions.

Efficacy of topical phenol decontamination strategies on severity of acute phenol chemical burns and dermal absorption: in vitro and in vivo studies in pig skin.

Pure phenol is colorless and used in the manufacture of phenolic resins, plastics, explosives, fertilizers, paints, rubber, textiles, adhesives, pharmaceuticals, paper, soap, and wood preservatives. The purpose of this study was to compare the efficacy of several phenol decontamination strategies following dermal exposure using the pig as a model for human exposure, and then assess the effect of the two best treatments on phenol absorption in the isolated perfused porcine skin flap (IPPSF). Six anesthetized Yorkshire pigs were exposed to 89% aqueous phenol for 1 min using Hilltop chambers (10 skin sites/pig; 400 microl/site). Exposure to phenol was followed by one of 10 different decontamination procedures: 1-, 5-, 15-, and 30-min water wash; Ivory soap solution; polyethylene glycol (PEG 400); PEG 400/industrial methylated spirits (IMS); PEG 400/ethanol (EtOH); polyvinyl pyrrolidone (PVP)/70% isopropanol (IPA); and 70% IPA. For each of the last five strategies, 1-min treatment washes were repeatedly alternated with 1-min water washes for a total of 15 min. Evaluation was based on scoring of erythema, edema, and histological parameters such as intracellular and intercellular epidermal edema, papillary dermal edema, perivascular infiltrates, pyknotic stratum basale cells, and epidermal-dermal separation. It was concluded that PEG 400 and 70% IPA were superior to the other treatments investigated and equally efficacious in the reduction of phenol-induced skin damage. In addition, phenol absorption was assessed utilizing the two most effective in vivo treatments in the IPPSF. The assessment of percutaneous absorption of phenol found the PEG 400, 70% IPA, and 15-min water treatments significantly (P < 0.05) reduced phenol absorption relative to no treatment.

Use of methyl salicylate as a simulant to predict the percutaneous absorption of sulfur mustard.

Exposure to chemical vesicants such as sulfur mustard (HD) continues to be a threat to military forces requiring protectant strategies to exposure to be evaluated. Methyl salicylate (MS) has historically been the simulant of choice to assess HD exposure. The purpose of this study was to compare the percutaneous absorption and skin deposition of MS to HD in the isolated perfused porcine skin flap (IPPSF). The HD data were obtained from a previously published study in this model wherein 400 microg cm(-2) of ](14)C[-MS or ](14)C[-HD in ethanol were topically applied to 16 IPPSFs and experiments were terminated at 2, 4 or 8 h. Perfusate was collected at increasing time intervals throughout perfusion. Radioactivity was determined in perfusate and skin samples. Perfusate flux profiles were fitted to a bi-exponential model Y(t) = A(e(-bt) - e(-dt)) and the area under the curve (AUC), peak flux and time to peak flux were determined. Sulfur mustard had more pronounced and rapid initial flux parameters (P < 0.05). The AUCs determined from observed and model-predicted parameters were not statistically different, although the mean HD AUC was 40--50% greater than MS. The HD skin and fat levels were up to twice those seen with MS, but had lower stratum corneum and residual skin surface concentrations (P < 0.05). Compared with other chemicals studied in this model, HD and MS cutaneous disposition were very similar, supporting the use of MS as a dermal simulant for HD exposure.

CHARACTERIZATION OF SULFUR MUSTARD–INDUCED TOXICITY BY ENZYME HISTOCHEMISTRY IN PORCINE SKIN

The isolated perfused porcine skin flap (IPPSF) has been validated as an invitro model to study the cutaneous toxicology of numerous compounds, including sulfur mustard (bis(2-chloroethyl)sulfide; HD). Enzyme histochemistry of alkaline phosphatase (ALP), acid phosphatase (ACP), and nonspecific esterase (NSE) was performed on skin dosed with HD from the IPPSF. Flaps from the dose response study were treated with 10.0 (n = 5), 5.0 (n = 4), 2.5 (n = 4), 1.25 (n = 3), 0.5 (n = 3), and 0.2 (n = 4) mg/mL HD in absolute ethanol (EtOH) and perfused for 8 h. Flaps from the time response study were treated with 10.0 mg/mL HD in EtOH or the EtOH vehicle and perfused for 1 (n = 4), 3 (n = 4), 5 (n = 4), and 8 h (n = 3). In the time response study, significant differences (SD) (p <. 05) were found in the stratum basale with ALP (3 h HD SD from the 3 h EtOH; 3 h HD SD from the 8 h HD), ACP (3 h HD SD from the 3 h EtOH), and NSE (8 h HD SD from the 8 h EtOH); in the stratum spinosum with NSE (1 h HD SD from the 1 h EtOH...

METHODS OF ASSESSING THE PERCUTANEOUS ABSORPTION OF VOLATILE CHEMICALS IN ISOLATED PERFUSED SKIN: STUDIES WITH CHLOROPENT AFLUOROBENZENE AND DICHLOROBENZENE

The experimental determination of dermal absorption of volatile chemicals is fraught with difficulties. The isolated perfused porcine skin flap (IPPSF) is a biologically intact, perfused skin preparation that has been employed to predict dermal absorption of chemicals in humans. The purpose of this work was to explore various experimental dosing strategies for volatile chemicals using dichlorobenzene (DCB) and chloropentafluorobenzene (CPFB) as model compounds. Effects of complete occlusion and various strategies of vapor trapping, vapor dosing, and solvent effects were explored. The results suggest that dosing methodology is a major determinant of dermal absorption and could easily skew results obtained from different systems. A biologically sensitive system such as the IPPSF is particularly sensitive to the manipulations required to ensure precise dosing of these compounds. An interesting finding was that the effects of solvents on compound absorption that are routinely described in liquid dosing scenar...

Dermal Absorption and Distribution of Topically Dosed Jet Fuels Jet-A, JP-8, and JP-8(100)

Dermal exposure to jet fuels has received increased attention with the recent release of newer fuels with novel performance additives. The purpose of these studies was to assess the percutaneous absorption and cutaneous disposition of topically applied (25 μl/5 cm2) neat Jet-A, JP-8, and JP-8(100) jet fuels by monitoring the absorptive flux of the marker components 14C naphthalene and 3H dodecane simultaneously applied nonoccluded to isolated perfused porcine skin flaps (IPPSF) (n = 4). Absorption of 14C hexadecane was estimated from JP-8 fuel. Absorption and disposition of naphthalene and dodecane were also monitored using a nonvolatile JP-8 fraction reflecting exposure to residual fuel that might occur 24 h after a jet fuel spill. In all studies, perfusate, stratum corneum, and skin concentrations were measured over 5 h. Naphthalene absorption had a clear peak absorptive flux at less than 1 h, while dodecane and hexadecane had prolonged, albeit significantly lower, absorption flux profiles. Within JP-8, the rank order of absorption for all marker components was (mean ± SEM % dose) naphthalene (1.17 ± 0.07) > dodecane (0.63 ± 0.04) > hexadecane (0.18 ± 0.08). In contrast, deposition within dosed skin showed the reverse pattern. Naphthalene absorption into perfusate was similar across all fuel types, however total penetration into and through skin was highest with JP-8(100). Dodecane absorption and total penetration was greatest from JP-8. Absorption of both markers from aged JP-8 was lower than other fuels, yet the ratio of skin deposition to absorption was greatest for this treatment group. In most exposure scenarios, absorption into perfusate did not directly correlate to residual skin concentrations. These studies demonstrated different absorption profiles for the three marker compounds, differential effects of jet fuel types on naphthalene and dodecane absorption, and uncoupling of perfusate absorption from skin disposition.

Percutaneous Absorption of Salicylic Acid, Theophylline, 2,4-Dimethylamine, Diethyl Hexyl Phthalic Acid, andp-Aminobenzoic Acid in the Isolated Perfused Porcine Skin Flap Compared to Manin Vivo

Human risk assessment for topical exposure requires percutaneous absorption data to link environmental contamination to potential systemic dose. Human absorption data are not readily available, so absorption models are used.In vitrodiffusion systems are easy to use but have proved to be somewhat unreliable and are not validated to man. This study compares percutaneous absorption in the isolated perfused porcine skin flap (IPPSF) system with that in manin vivo.The study design utilized the same compounds and the same dose concentration and vehicle in both systems. Methodology for each system was that which is routinely used ineach system. The skin surface was not protected during the absorption dosing period. Percutaneous absorption values were, for man and the IPPSF system, respectively: salicylic acid (6.5 ± 5.0%; 7.5 ± 2.6%), theophylline (16.9 ± 11.3%; 11.8 ± 3.8%), 2,4-dimethylamine (1.1 ± 0.3%; 3.8 ± 0.6%), diethyl hexyl phthalic acid (1.8 ± 0.5%; 3.9 ± 2.4%), andp-aminobenzoic acid (11.5 ± 6.3%; 5.9 ± 3.7%) (correlation coefficient was 0.78;p< 0.04).The skin surface wash recovery postapplication was similar for salicylic acid in man (53.4 ± 6.3%) and the IPPSF system (48.2 ± 4.9%). With the other compounds the majority of surface chemical was recovered in the surface wash and skin tape strip in the IPPSF system. With man, other than salicylic acid, only a few percent applied dose was recovered with surface washing and tape stripping. Since the wash procedure was effective with pig skin, we can assume that these chemicals in man were lost to adsorption to any clothing or bedding with the volunteers. The absorption in man was not less than that in the IPPSF. Assuming the dose was lost in man, it seems plausible that whatever compound was to penetrate human skin in solvent vehicle did so in the period of time before the chemical was removed.The IPPSF system appears to be a good model for predicting percutaneous absorption relative to man. This study design should be used to validate other systems to humansin vivo.