Toxicological effects of pet food ingredients on canine bone marrow-derived mesenchymal stem cells and enterocyte-like cells.

We developed an in vitro method to assess pet food ingredients safety. Canine bone marrow-derived mesenchymal stem cells (BMSC) were differentiated into enterocyte-like cells (ELC) to assess toxicity in cells representing similar patterns of exposure in vivo. The toxicological profile of clove leave oil, eugenol, guanosine monophosphate (GMP), GMP + inosine monophosphate, sorbose, ginger root extract, cinnamon bark oil, cinnamaldehyde, thyme oil, thymol and citric acid was assessed in BMSC and ELC. The LC50 for GMP + inosine monophosphate was 59.42 ± 0.90 and 56.7 ± 3.5 mg ml(-1) for BMSC and ELC; 56.84 ± 0.95 and 53.66 ± 1.36 mg ml(-1) for GMP; 0.02 ± 0.001 and 1.25 ± 0.47 mg ml(-1) for citric acid; 0.077 ± 0.002 and 0.037 ± 0.01 mg ml(-1) for cinnamaldehyde; 0.002 ± 0.0001 and 0.002 ± 0.0008 mg ml(-1) for thymol; 0.080 ± 0.003 and 0.059 ± 0.001 mg ml(-1) for thyme oil; 0.111 ± 0.002 and 0.054 ± 0.01 mg ml(-1) for cinnamon bark oil; 0.119 ± 0.0004 and 0.099 ± 0.011 mg ml(-1) for clove leave oil; 0.04 ± 0.001 and 0.028 ± 0.002 mg ml(-1) for eugenol; 2.80 ± 0.11 and 1.75 ± 0.51 mg ml(-1) for ginger root extract; > 200 and 116.78 ± 7.35 mg ml(-1) for sorbose. Lemon grass oil was evaluated at 0.003-0.9 in BMSC and .03-0.9 mg ml(-1) in ELC and its mechanistic effect was investigated. The gene toxicology studies showed regulation of 61% genes in CYP450 pathway, 37% in cholestasis and 33% in immunotoxicity pathways for BMSC. For ELC, 80% for heat shock response, 69% for beta-oxidation and 65% for mitochondrial energy metabolism. In conclusion, these studies provide a baseline against which differential toxicity of dietary feed ingredients can be assessed in vitro for direct effects on canine cells and demonstrate differential toxicity in differentiated cells that represent gastrointestinal epithelial cells.

Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line.

Single-walled carbon nanotubes (SWCNT), fullerenes (C(60)), carbon black (CB), nC(60), and quantum dots (QD) have been studied in vitro to determine their toxicity in a number of cell types. Here, we report that classical dye-based assays such as MTT and neutral red (NR) that determine cell viability produce invalid results with some NM (nanomaterials) due to NM/dye interactions and/or NM adsorption of the dye/dye products. In this study, human epidermal keratinocytes (HEK) were exposed in vitro to CB, SWCNT, C(60), nC(60), and QD to assess viability with calcein AM (CAM), Live/Dead (LD), NR, MTT, Celltiter 96 AQueous One (96 AQ), alamar Blue (aB), Celltiter-Blue (CTB), CytoTox Onetrade mark (CTO), and flow cytometry. In addition, trypan blue (TB) was quantitated by light microscopy. Assay linearity (R(2) value) was determined with HEK plated at concentrations from 0 to 25,000 cells per well in 96-well plates. HEK were treated with serial dilutions of each NM for 24 h and assessed with each of the viability assays. TB, CAM and LD assays, which depend on direct staining of living and/or dead cells, were difficult to interpret due to physical interference of the NM with cells. Results of the dye-based assays varied a great deal, depending on the interactions of the dye/dye product with the carbon nanomaterials (CNM). Results show the optimal high throughput assay for use with carbon and noncarbon NM was 96 AQ. This study shows that, unlike small molecules, CNM interact with assay markers to cause variable results with classical toxicology assays and may not be suitable for assessing nanoparticle cytotoxicity. Therefore, more than one assay may be required when determining nanoparticle toxicity for risk assessment.

Assessment of quantum dot penetration into intact, tape-stripped, abraded and flexed rat skin.

Quantum dot (QD) nanoparticles have received attention due to their fluorescent characteristics and potential use in medical applications. Skin penetration is one of the major routes of exposure for nanoparticles to gain access to a biological system. QD655 and QD565 coated with carboxylic acid were studied for 8 and 24 h in flow-through diffusion cells with flexed, tape-stripped and abraded rat skin to determine if these mechanical actions could perturb the barrier and affect penetration. Nonflexed skin did not show QD penetration at 8 or 24 h. Flexed skin showed an increase in QD on the surface of skin but no penetration at 8 and 24 h. Tape-stripped skin depicted QD only on the surface of the viable epidermis. QD655 penetrated into the viable dermal layers of abraded skin at both 8 and 24 h, while QD565 was present only at 24 h. QD were not detected in the perfusate by fluorescence and inductively coupled plasma-optical emission spectroscopy analysis for cadmium at any time point. These results indicate that the rat skin penetration of QD655 and QD565 is primarily limited to the uppermost stratum corneum layers of intact skin. Barrier perturbation by tape stripping did not cause penetration, but abrasion allowed QD to penetrate deeper into the dermal layers.

Nanoceria as Antioxidant: Synthesis and Biomedical Applications.

The therapeutic application of nanomaterials has been a focus of numerous studies in the past decade. Due to its unique redox properties, cerium oxide (ceria) is finding widespread use in the treatment of medical disorders caused by the reactive oxygen intermediates (ROI). The radical-scavenging role of ceria nanoparticles (nanoceria) have been established, as well as the autocatalytic ability of nanoceria to regenerate under various environmental conditions. The synthesis of nanoceria in biocompatible media has also been reported along with cell viability in order to determine the potential use of nanoceria in biomedical applications.

Biological properties of carbon nanotubes.

Carbon nanotubes are novel materials with unique physical and chemical properties, and have been considered for use in numerous technological applications. More recently, attention has turned to the unique biological and medical properties of these materials. In this review, the processing, chemical properties, physical properties, nucleic acid interaction, cell interaction, and toxicologic properties of nanotubes are described. Finally, future directions in this area are discussed.

A system coefficient approach for quantitative assessment of the solvent effects on membrane absorption from chemical mixtures.

A system coefficient approach is proposed for quantitative assessment of the solvent effects on membrane absorption from chemical mixtures. The complicated molecular interactions are dissected into basic molecular interaction forces via Abraham's linear solvation energy relationship (LSER). The molecular interaction strengths of a chemical are represented by a set of solute descriptors, while those of a membrane/chemical mixture system are represented by a set of system coefficients. The system coefficients can be determined by using a set of probe compounds with known solute descriptors. Polydimethylsiloxane (PDMS) membrane-coated fibres and 32 probe compounds were used to demonstrate the proposed approach. When a solvent was added into the chemical mixture, the system coefficients were altered and detected by the system coefficient approach. The system coefficients of the PDMS/water system were (0.09, 0.49, -1.11, -2.36, -3.78, 3.50). When 25% ethanol was added into the PDMS/water system, the system coefficients were altered significantly (0.38, 0.41, -1.18, -2.07, -3.40, 2.81); and the solvent effect was quantitatively described by the changes in the system coefficients (0.29, -0.08, -0.07, 0.29, 0.38, -0.69). The LSER model adequately described the experimental data with a correlation coefficient (r(2)) of 0.995 and F-value of 1056 with p-value less than 0.0001.

Biocorona formation on gold nanoparticles modulates human proximal tubule kidney cell uptake, cytotoxicity and gene expression.

Gold nanoparticles (AuNP) adsorb macromolecules to form a protein corona (PC) after systemic delivery, to which the kidney as the primary excretory organ is constantly exposed. The role of the PC on AuNP cell uptake and toxicity was investigated in vitro in human proximal tubule cells (HPTC) using 40 and 80nm branched polyethylenimine (BPEI), lipoic acid (LA) and polyethylene glycol (PEG) coated AuNP with or without (bare) PCs composed of human plasma (HP) or human serum albumin (HSA) for 0.25 to 24h. Time-dependent intracellular uptake, assessed by ICP-MS showed PC modulated cell uptake and cytotoxicity; with bare 40nm BPEI-AuNP showing the greatest responses. All AuNP showed minimal to no cytokine release. At the nontoxic dose, 40nm bare BPEI-AuNP significantly modified gene expression related to immunotoxicity, steatosis, and mitochondrial metabolism; while at the high dose, pathways of DNA damage and repair, apoptosis, fatty acid metabolism and heat shock response were modulated. HP corona BPEI-AuNP response was comparable to control. These studies clearly showed reduced uptake and cytotoxicity, as well as differentiated gene expression of AuNP with PCs, questioning the utility of in vitro studies using bare NP to assess in vivo effects. Significantly, only cationic bare BPEI-AuNP had HPTC uptake or cytotoxicity suggesting the relative safety of PEG and LA-AuNP as nanomedicine constructs.

A physiologically based pharmacokinetic model of organophosphate dermal absorption.

The rate and extent of dermal absorption are important in the analysis of risk from dermal exposure to toxic chemicals and for the development of topically applied drugs, barriers, insect repellents, and cosmetics. In vitro flow-through cells offer a convenient method for the study of dermal absorption that is relevant to the initial processes of dermal absorption. This study describes a physiologically based pharmacokinetic (PBPK) model developed to simulate the absorption of organophosphate pesticides, such as parathion, fenthion, and methyl parathion through porcine skin with flow-through cells. Parameters related to the structure of the stratum corneum and solvent evaporation rates were independently estimated. Three parameters were optimized based on experimental dermal absorption data, including solvent evaporation rate, diffusivity, and a mass transfer factor. Diffusion cell studies were conducted to validate the model under a variety of conditions, including different dose ranges (6.3-106.9 microg/cm2 for parathion; 0.8-23.6 microg/cm2 for fenthion; 1.6-39.3 microg/cm2 for methyl parathion), different solvents (ethanol, 2-propanol and acetone), different solvent volumes (5-120 microl for ethanol; 20-80 microl for 2-propanol and acetone), occlusion versus open to atmosphere dosing, and corneocyte removal by tape-stripping. The study demonstrated the utility of PBPK models for studying dermal absorption, which can be useful as explanatory and predictive tools that may be used for in silico hypotheses generation and limited hypotheses testing. The similarity between the overall shapes of the experimental and model-predicted flux/time curves and the successful simulation of altered system conditions for this series of small, lipophilic compounds indicated that the absorption processes that were described in the model successfully simulated important aspects of dermal absorption in flow-through cells. These data have direct relevance to topical organophosphate pesticide risk assessments.

Differential down-regulation of epidermal protein kinase C by 12-O-tetradecanoylphorbol-13-acetate and diacylglycerol: association with epidermal hyperplasia and tumor promotion.

A single topical application of 2 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) to CD-1 mouse skin resulted in a rapid decrease in cytosolic, particulate, and total epidermal protein kinase C (PKC) activity at 6 h, which remained decreased by 70% at 96 h. This dose of TPA produced epidermal hyperplasia as determined by an increase in the number of nucleated epidermal cell layers. A single application of 10 mumol sn-1,2-didecanoylglycerol, a model sn-1,2-diacylglycerol and complete tumor promoter, induced ornithine decarboxylase to an extent similar to that of 2 nmol TPA. However, sn-1,2-didecanoylglycerol produced an 80% increase in particulate PKC activity that was accompanied by a 45% decrease in cytosolic PKC activity, resulting in no net change in total PKC activity. Unlike TPA, this dose of sn-1,2-didecanoylglycerol did not produce a hyperplastic response. Additional dosing regimens were examined to determine whether the down-regulation of particulate PKC activity was associated with hyperplasia and tumor promotion. A tumor-promoting dosing regimen consisting of multiple applications of 5 or 10 mumol sn-1,2-didecanoylglycerol twice daily for 1 week resulted in more than a 60% decrease in cytosolic and particulate PKC activity and a marked epidermal hyperplasia. Twice-weekly application of 10 mumol sn-1,2-didecanoylglycerol, a nonpromoting dosing rate, for 1 week decreased cytosolic PKC activity but increased particulate PKC activity and did not produce hyperplasia. Dosing regimens utilizing multiple applications of TPA decreased both particulate and cytosolic PKC activity and were also hyperplastic. PKC activity was also measured in epidermal papillomas from mice initiated with 7,12-dimethylbenz[a]- anthracene and promoted with either sn-1,2-didecanoylglycerol or TPA. Cytosolic- and particulate-associated PKC activity in these papillomas was decreased by at least 70% and 40%, respectively, when compared with epidermis and whole skin. After 2 months without promoter treatment, both cytosolic and particulate PKC activity remained decreased in the papillomas, whereas epidermal PKC activity returned to control values by 2 to 3 weeks following cessation of several weeks of TPA treatment. Collectively, these data demonstrate that the down-regulation of epidermal PKC is associated with and may be a permissive event for epidermal hyperplasia and tumor promotion.

Effect of JP-8 jet fuel exposure on protein expression in human keratinocyte cells in culture.

Dermal exposure to jet fuel is a significant occupational hazard. Previous studies have investigated its absorption and disposition in skin, and the systemic biochemical and immunotoxicological sequelae to exposure. Despite studies of JP-8 jet fuel components in murine, porcine or human keratinocyte cell cultures, proteomic analysis of JP-8 exposure has not been investigated. This study was conducted to examine the effect of JP-8 administration on the human epidermal keratinocyte (HEK) proteome. Using a two-dimensional electrophoretic approach combined with mass spectrometric-based protein identification, we analyzed protein expression in HEK exposed to 0.1% JP-8 in culture medium for 24 h. JP-8 exposure resulted in significant expression differences (p<0.02) in 35 of the 929 proteins matched and analyzed. Approximately, a third of these alterations were increased in protein expression, two-thirds declined with JP-8 exposure. Peptide mass fingerprint identification of effected proteins revealed a variety of functional implications. In general, altered proteins involved endocytotic/exocytotic mechanisms and their cytoskeletal components, cell stress, and those involved in vesicular function.

Effect of in vivo jet fuel exposure on subsequent in vitro dermal absorption of individual aromatic and aliphatic hydrocarbon fuel constituents.

The percutaneous absorption of topically applied jet fuel hydrocarbons (HC) through skin previously exposed to jet fuel has not been investigated, although this exposure scenario is the occupational norm. Pigs were exposed to JP-8 jet fuel-soaked cotton fabrics for 1 and 4 d with repeated daily exposures. Preexposed and unexposed skin was then dermatomed and placed in flow-through in vitro diffusion cells. Five cells with exposed skin and four cells with unexposed skin were dosed with a mixture of 14 different HC consisting of nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, ethyl benzene, o-xylene, trimethyl benzene (TMB), cyclohexyl benzene (CHB), naphthalene, and dimethyl naphthalene (DMN) in water + ethanol (50:50) as diluent. Another five cells containing only JP-8-exposed skin were dosed solely with diluent in order to determine the skin retention of jet fuel HC. The absorption parameters of flux, diffusivity, and permeability were calculated for the studied HC. The data indicated that there was a two-fold and four-fold increase in absorption of specific aromatic HC like ethyl benzene, o-xylene, and TMB through 1- and 4-dJP-8 preexposed skin, respectively. Similarly, dodecane and tridecane were absorbed more in 4-d than 1-dJP-8 preexposed skin experiments. The absorption of naphthalene and DMN was 1.5 times greater than the controls in both 1- and 4-d preexposures. CHB, naphthalene, and DMN had significant persistent skin retention in 4-d preexposures as compared to 1-d exposures that might leave skin capable of further absorption several days postexposure. The possible mechanism of an increase in HC absorption in fuel preexposed skin may be via lipid extraction from the stratum corneum as indicated by Fourier transform infrared (FTIR) spectroscopy. This study suggests that the preexposure of skin to jet fuel enhances the subsequent in vitro percutaneous absorption of HC, so single-dose absorption data for jet fuel HC from naive skin may not be optimal to predict the toxic potential for repeated exposures. For certain compounds, persistent absorption may occur days after the initial exposure.

In vitro safety assessment of food ingredients in canine renal proximal tubule cells.

In vitro models are useful tools to initially assess the toxicological safety hazards of food ingredients. Toxicities of cinnamaldehyde (CINA), cinnamon bark oil, lemongrass oil (LGO), thymol, thyme oil (TO), clove leaf oil, eugenol, ginger root extract (GRE), citric acid, guanosine monophosphate, inosine monophosphate and sorbose (SORB) were assessed in canine renal proximal tubule cells (CPTC) using viability assay and renal injury markers. At LC50, CINA was the most toxic (0.012mg/ml), while SORB the least toxic (>100mg/ml). Toxicities (LC50) of positive controls were as follows: 4-aminophenol (0.15mg/ml in CPTC and 0.083mg/ml in human PTC), neomycin (28.6mg/ml in CPTC and 27.1mg/ml in human PTC). XYL displayed lowest cytotoxic potency (LC50=82.7mg/ml in CPTC). In vivo renal injury markers in CPTC were not significantly different from controls. The LGO toxicity mechanism was analyzed using qPCR and electron microscopy. Out of 370 genes, 57 genes (15.4%) were significantly up (34, 9.1%) or down (23, 6.2%) regulated, with the most upregulated gene gsta3 (∼200-fold) and the most affected pathway being oxidative stress. LGO induced damage of mitochondria, phospholipid accumulation and lack of a brush border. Viability assays along with mechanistic studies in the CPTC model may serve as a valuable in vitro toxicity screening tool.

Interspecies and interregional analysis of the comparative histologic thickness and laser Doppler blood flow measurements at five cutaneous sites in nine species.

Studies in dermatology, cutaneous pharmacology, and toxicology utilize skin from different animal species and body sites. However, regional differences exist in topical chemical percutaneous absorption studies in man and in animal. The objective of this study was to compare epidermal thickness and number of cell layers across species and body sites using both formalin-fixed paraffin and frozen sections. Cutaneous blood flow determined by laser Doppler velocimetry (LDV) was compared to histologic data. Six animals of each of the following species were used: monkeys, pigs, dogs, cats, cows, horses, rabbits, rats, and mice. Cutaneous blood flow was determined and 6-mm skin biopsies were taken directly from the following sites: buttocks, ear, humeroscapular joint, thoracolumbar junction, and abdominal area. When the two histologic methods were compared across all species and body sites, the thickness of the epidermis was significantly greater, and the thickness of the stratum corneum significantly less, in paraffin sections versus frozen sections (p less than 0.05). There were no differences in the number of viable cell layers determined by both methods. The values for LDV-determined blood flow did not significantly correlate (p greater than 0.05) to epidermal or stratum corneum thickness. However, regional and species differences were noted in all these parameters. In conclusion, these data indicate that thickness and LDV blood flow are independent and must be evaluated separately when comparisons are made between species and body sites. This work provides a data base for future comparative studies in which a knowledge of skin thickness or blood flow might be important variables.

Minimal role of enhanced cell proliferation in skin tumor promotion by mirex: a nonphorbol ester-type promoter.

Mirex, a chlorinated hydrocarbon previously used as a systemic insecticide and flame retardant, is a nongenotoxic hepatocarcinogen in both rats and mice. In liver, mirex induced biochemical responses and hyperplasia characteristic of increased cell proliferation, which is consistent with its role as a liver tumor promoter. We have recently shown that mirex is a potent nonphorbol ester-type skin tumor promoter in 7, 12-dimethylbenz[a]anthracene (DMBA)-initiated mice. However, unlike its effect in liver, a single topical application of mirex to skin does not induce the acute biochemical responses, such as increased epidermal DNA synthesis and ornithine decarboxylase activity, indicative of increased cell proliferation. Multiple topical applications of mirex over a 1 month period induced only a minimal increase in the number of epidermal nucleated cell layers, which contrasts with definitive hyperplasia induced by a comparable tumor-promoting dose of 12-O-tetradecanoylphorbol-13-acetate (TPA). Collectively, these data indicated that mirex is promoting through a novel mechanism. Further evidence that mirex promotes tumors through a mechanism distinct from that of the prototypical skin tumor promoter, TPA, was obtained by examining the effect of their simultaneous co-treatment. The co-application of mirex and TPA yielded a tumor multiplicity greater than the sum of the responses of each promoter individually. In summary, our results demonstrate that mirex, a carcinogenic and hyperplastic agent in liver, is also a very effective tumor promoter in mouse skin, but suggest that mirex operates via a novel mechanism in skin that may involve only a minimal role for enhanced cell proliferation.

Ultrastructural characterization of sulfur mustard-induced vesication in isolated perfused porcine skin.

The isolated perfused porcine skin flap (IPPSF) is a novel alternative, humane in vitro model consisting of a viable epidermis and dermis with a functional microvasculature. For this study, 200 microliters of either 10.0, 5.0, 2.5, 1.25, 0.50, or 0.20 mg/ml of bis (2-chloroethyl) sulfide (HD) in ethanol or ethanol control was topically applied to a 5.0 cm2 dosing area of the IPPSF and perfused for 8 h with recirculating media. HD dermatotoxicity was assessed in the flap by cumulative glucose utilization (CGU), vascular resistance (VR), light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). HD produced a statistically significant dose relationship for gross blisters and microvesicles. The HD-treated IPPSFs were also characterized by a decrease in CGU and an increase in VR. Light microscopic changes included mild intracellular and slight intracellular epidermal edema, multifocal epidermal-dermal separation, and dark basal cells. Ultrastructural alterations consisted of cytoplasmic vacuoles, pyknotic basal cells, nucleolar segregation, and epidermal-dermal separation occurring between the lamina lucida and lamina densa of the basement membrane. The severity of these changes increased in a dose-dependent manner. Morphologically, the IPPSF appeared similar to human skin exposed to HD with the formation of macroscopic blisters and microscopic vesicles. In conclusion, the IPPSF appears to be an appropriate in vitro model with which to study the pathogenesis of vesicant-induced toxicity.

Comparison of an in vitro skin model to normal human skin for dermatological research.

EpiDerm, an in vitro human skin equivalent (HSE), was compared to normal human breast skin (NHS) to morphologically and biochemically assess its feasibility for dermatological research. Intralot and interlot variability was studied in day 0, 1, 2, and 3 in vitro cultures and in day 0, 3, 5, and 7 NHS. For NHS, light microscopy (LM) at day 0 showed stratified epidermis which exhibited an increase in vacuoles and dark basal cells as storage increased to 3, 5, and 7 days. Transmission electron microscopy (TEM) revealed typical organelles in the epidermis and a convoluted basement membrane at day 0. With increased storage, vacuoles and paranuclear clefts became numerous, necrosis increased, tonofilaments became less organized, and overall cellular integrity decreased. Biochemical data showed consistent MTT and glucose utilization (GU) through day 5, while lactate production decreased to 75% by day 3. By LM, day 0 HSE consisted of a thick, compact, stratum corneum that sent projections between the stratum granulosum cells. By TEM, the configuration organization, differentiation, distribution, and frequency of the organelles differed slightly from NHS. In addition, the basement membrane of the HSE was not completely differentiated, and the dermis was thin and acellular. Although day 1 and 2 cultures showed little change, day 3 exhibited an overall degeneration. Biochemical analysis showed GU and the lactate production decreased through day 3. In conclusion, the EpiDerm HSE, although exhibiting slight differences, was morphologically and biochemically similar to normal human epidermis and may be a valuable model in assessing the toxicology, metabolism, or pharmacology of nonvesicating compounds.

Effects of organic solvent vehicles on the viability and morphology of isolated perfused porcine skin.

Although many organic solvents are known to be cutaneous irritants, they are commonly utilized as vehicles in percutaneous absorption and toxicity studies. The isolated perfused porcine skin flap (IPPSF) is an alternative animal model that has been used to study percutaneous absorption and cutaneous toxicity. The purpose of this study was to evaluate the effect of five organic solvents (ethanol, acetone, dimethyl sulfoxide (DMSO), toluene, and cyclohexane) on biochemical viability parameters, vascular response, and epidermal morphology of the IPPSF. Cumulative glucose utilization (CGU), the ratio of lactate production/glucose utilization (L/CGU ratio), and the leakage of lactate dehydrogenase (LDH) were used as biochemical indicators of alterations in glucose metabolism and flap viability. Only ethanol resulted in a statistically significant decrease in the average rate of CGU over the perfusion period. All of the solvent treatments resulted in slight increases in LDH release versus the controls. Vascular resistance (VR) was measured to examine the response of the cutaneous vasculature to these solvents, and most treatments resulted in a decreased VR in the terminal phases of perfusion. Ethanol was the only solvent to cause an apparent increase in terminal VR. Light microscopy demonstrated a moderate increase in intracellular edema in the DMSO, toluene, and acetone flaps. Ultrastructural evaluation showed focal blebbing of the nuclear envelope and vesiculation of the rough endoplasmic reticulum in cells of the stratum basale and stratum spinosum layers with DMSO treatment. The IPPSF allowed the evaluation of subtle biochemical, vascular, and morphological changes associated with non-occlusive topical exposure to these organic solvents. These findings support the necessity of documenting vehicle effects which might mask or otherwise alter subtle, but potentially important, compound-specific responses.

Detection of sulfur mustard bis (2-chloroethyl) sulfide and metabolites after topical application in the isolated perfused porcine skin flap.

The purpose of this study was to develop an assay to study the flux of sulfur mustard (HD) through the skin and determine if metabolites are formed due to the epidermal metabolism of HD after topical exposure of the isolated perfused porcine skin flap (IPPSF) to 14C-HD. Four IPPSFs were topically dosed with 2.85 mg of 14C-HD in ethanol. Venous perfusate samples were collected and added to a 34% solution of NaCl and snap-frozen to inhibit the metabolism of HD until time for assay. Perfusate samples were extracted using a solid-phase extraction cartridge with ethyl acetate and then assayed using gas chromatography. Two of the 4 IPPSFs showed detectable levels of HD in the venous perfusate 15 min after dosing, with 1 of these 2 IPPSFs showing detectable levels of HD in the perfusate 2 hours after dosing. All 4 IPPSFS had no more than 3 metabolites of HD appearing in the perfusate throughout the 2 hr experiment, with one of the these metabolites identified as thiodiglycol. These experiments showed that little, if any, HD appears in the venous perfusate intact after percutaneous absorption and that epidermal metabolism of HD does occur to a significant degree in the IPPSF.

Evaluation of protective effects of sodium thiosulfate, cysteine, niacinamide and indomethacin on sulfur mustard-treated isolated perfused porcine skin.

Sulfur mustard (bis(2-chloroethyl)sulfide, HD), a bifunctional alkylating agent, causes severe cutaneous injury, including cell death, edema and vesication. However, the mechanisms underlying HD-induced cutaneous toxicity remain undefined. The isolated perfused porcine skin flap (IPPSF) has been utilized to investigate dermal toxic compounds and pharmacological intervention. In this study, 4 compounds with different pharmacological mechanisms were tested for their ability to prevent the dark basal cell formation, vesication and vascular response charcteristic of exposure to HD in the IPPSF. Reduction of HD-induced dark basal cells was observed in IPPSFs perfused with sodium thiosulfate and cysteine, which are HD scavengers; niacinamide, a possible NAD+ stabilizer and an inhibitor of poly (ADP-ribose) polymerase; or indomethacin, a cyclooxygenase inhibitor, respectively. Treatments with niacinamide and indomethacin, but not sodium thiosulfate or cysteine, resulted in an inhibition of the vascular response in IPPSF exposed to HD. Microvesicles caused by HD were only partially prevented in the indomethacin-perfused IPPSFs. These data suggest that none of these agents alone would be successful antivesicant agents and different mechanisms are involved in production of HD-induced dark basal cells, microvesicles and the vascular response; unfortunately, blocking of the cellular toxicity as evidenced by dark basal cell formation did not prevent vesication, suggesting that other mechanisms must be operative and that there is a multistep, biochemical process that leads to a final lesion.

In vitro penetration of pesticides through human newborn foreskin.

The in vitro dermal penetration of 14C-labelled parathion, fenvalerate, carbofuran, and lindane through fresh full-thickness human newborn foreskin was determined at 1, 6, 24, and 48 h. The pesticides were applied to a constant dosing area (0.031 cm2), and a fixed dose (1.18 microgram), for each of the compounds studied. 90%, or greater, of the labelled pesticides were recovered in all cases. Carbofuran showed the greatest mean penetration of 82% followed by parathion and lindane with mean penetrations of 79 and 66%, respectively. Fenvalerate exhibited a mean penetration of 9% which is significantly lower than that of the other three compounds. No difference was noted in the penetration of pesticides through human skin from blacks and whites.