Tattooing of skin results in transportation and light-induced decomposition of tattoo pigments--a first quantification in vivo using a mouse model.

Millions of people are tattooed with inks that contain azo pigments. The pigments contained in tattoo inks are manufactured for other uses with no established history of safe use in humans and are injected into the skin at high densities (2.5 mg/cm(2)). Tattoo pigments disseminate after tattooing throughout the human body and although some may photodecompose at the injection site by solar or laser light exposure, the extent of transport or photodecomposition under in vivo conditions remains currently unknown. We investigated the transport and photodecomposition of the widely used tattoo Pigment Red 22 (PR 22) following tattooing into SKH-1 mice. The pigment was extracted quantitatively at different times after tattooing. One day after tattooing, the pigment concentration was 186 microg/cm(2) skin. After 42 days, the amount of PR 22 in the skin has decreased by about 32% of the initial value. Exposure of the tattooed skin, 42 days after tattooing, to laser light reduced the amount of PR 22 by about 51% as compared to skin not exposed to laser light. A part of this reduction is as a result of photodecomposition of PR 22 as shown by the detection of corresponding hazardous aromatic amines. Irradiation with solar radiation simulator for 32 days caused a pigment reduction of about 60% and we again assume pigment decomposition in the skin. This study is the first quantitative estimate of the amount of tattoo pigments transported from the skin into the body or decomposed by solar or laser radiation.

Migration of intradermally injected quantum dots to sentinel organs in mice.

Topical exposure to nanoscale materials is likely from a variety of sources including sunscreens and cosmetics. Because the in vivo disposition of nanoscale materials is not well understood, we have evaluated the distribution of quantum dots (QDs) following intradermal injection into female SKH-1 hairless mice as a model system for determining tissue localization following intradermal infiltration. The QD (CdSe core, CdS capped, poly[ethylene glycol] coated, 37 nm diameter, 621 nm fluorescence emission) were injected intradermally (ID) on the right dorsal flank. Within minutes following intradermal injection, the highly UV fluorescent QD could be observed moving from the injection sites apparently through the lymphatic duct system to regional lymph nodes. Residual fluorescent QD remained at the site of injection until necropsy at 24 h. Quantification of cadmium and selenium levels after 0, 4, 8, 12, or 24 h in multiple tissues, using inductively coupled plasma mass spectrometry (ICP-MS), showed a time-dependent loss of cadmium from the injection site, and accumulation in the liver, regional draining lymph nodes, kidney, spleen, and hepatic lymph node. Fluorescence microscopy corroborated the ICP-MS results regarding the tissue distribution of QD. The results indicated that (1) ID injected nanoscale QD remained as a deposit in skin and penetrated the surrounding viable subcutis, (2) QD were distributed to draining lymph nodes through the sc lymphatics and to the liver and other organs, and (3) sentinel organs are effective locations for monitoring transdermal penetration of nanoscale materials into animals.

Prolactin and Dehydroepiandrosterone Levels in Women with Systemic Lupus Erythematosus: The Role of the Extrapituitary Prolactin Promoter Polymorphism at -1149G/T.

Systemic lupus erythematosus (SLE) has shown an association with high levels of prolactin, low levels of dehydroepiandrosterone (DHEA), and induction of inflammatory cytokines in the serum of patients with the disease. This preliminary study examined the relevance of a -1149G/T functional single-nucleotide polymorphism (SNP) (rs1341239) in the promoter of the extrapituitary prolactin gene in a cohort of African American and European American women with lupus. Examination of this SNP revealed that the -1149TT genotype was correlated with higher levels of prolactin in serum and prolactin gene expression (p = 0.0001) in peripheral blood mononuclear cells (PBMCs). Lower levels of DHEA in serum were demonstrated in lupus patients (p = 0.001); those with the -1149TT genotype had the lowest levels of DHEA. Furthermore, a small subset of women who were on DHEA therapy and had a TT genotype showed a significant decrease in prolactin gene expression and lower disease activity scores (SLEDAI). Lupus patients, particularly African Americans, had significantly higher levels of IL-6 (p = 0.0001) and TNF-α (p = 0.042). This study suggests that the -1149TT genotype may be a risk factor for lupus and may predict who could possibly benefit from DHEA therapy; therefore, these results should be validated in a larger cohort with all ethnic groups.

Sodium selenite-induced apoptosis in murine B-lymphoma cells is associated with inhibition of protein kinase C-delta, nuclear factor kappaB, and inhibitor of apoptosis protein.

Selenium (Se) is an essential trace element possessing anticarcinogenic properties and other biological functions. This study determined the role sodium selenite plays on intracellular signaling, including protein kinase C (PKC), nuclear factor-kappa B (NF-kappaB), and inhibitor of apoptosis protein (IAP) in murine B lymphoma (A20) cells. In vitro supplementation of A20 cells with low concentrations of sodium selenite (0.005-5 microM) caused a significant increase in cellular proliferation exclusively at 72 h. Proliferation and cell viability were decreased in response to selenium concentrations of >/= 25 microM and >/= 5 microM at 72 and 96 h, respectively. Flow cytometric analysis of A20 cells exposed to 5 microM Se at 72 and 96 h indicated G(2)-M phase arrest and increased cell death at higher concentrations. Se-induced cytotoxicity was associated with apoptosis indicated by nuclear fragmentation and DNA laddering. Se concentrations, which induced cell cycle arrest and apoptosis, were associated with inhibition of cytosol to membrane translocation of PKCdelta and PKC activity at 72 h. Coincubation of cultures with 0.5 microM phorbol 12-myristate 13-acetate (PMA) and Se (5 and 25 microM) reversed the Se-induced cell death at 72 h. The nuclear NF-kappaB translocation and NF-kappaB DNA-binding were inhibited by increasing concentrations of Se (5 and 25 microM) at 72 h. After 72 h exposure to 5 and 25 microM Se, cIAP-2 concentration was decreased. Differential inhibition of PKCdelta, NF-kappaB, and cIAP-2 by Se may represent important intracellular signaling processes through which Se induces apoptosis and subsequently exerts its anticarcinogenic potential.

Photodecomposition of Pigment Yellow 74, a pigment used in tattoo inks.

Tattooing has become a popular recreational practice among younger adults over the past decade. Although some of the pigments used in tattooing have been described, very little is known concerning the toxicology, phototoxicology or photochemistry of these pigments. Seven yellow tattoo inks were obtained from commercial sources and their pigments extracted, identified and quantitatively analyzed. The monoazo compound Pigment Yellow 74 (PY74; CI 11741) was found to be the major pigment in several of the tattoo inks. Solutions of commercial PY74 in tetrahydrofuran (THF) were deoxygenated using argon gas, and the photochemical reaction products were determined after exposure to simulated solar light generated by a filtered 6.5 kW xenon arc lamp. Spectrophotometric and high-pressure liquid chromatography (HPLC) analyses indicated that PY74 photodecomposed to multiple products that were isolated using a combination of silica chromatography and reversed-phase HPLC. Three of the major photodecomposition products were identified by nuclear magnetic resonance and mass spectrometry as N-(2-methoxyphenyl)-3-oxobutanamide (o-acetoacetanisidide), 2-(hydroxyimine)-N-(2-methoxyphenyl)-3-oxobutanamide and N,N''-bis(2-methoxyphenyl)urea. These results demonstrate that PY74 is not photostable in THF and that photochemical lysis occurs at several sites in PY74 including the hydrazone and amide groups. The data also suggest that the use of PY74 in tattoo inks could potentially result in the formation of photolysis products, resulting in toxicity at the tattoo site after irradiation with sunlight or more intense light sources.

Sphingoid bases and their phosphates: transient activation and delayed repression of protein kinase C isoforms and their possible involvement in fumonisin B1 cytotoxicity.

Fumonisin B(1), a potent inhibitor of ceramide synthase, leads to accumulation of sphinganine, and later sphingosine, in vivo and in vitro. Fumonisin B(1) modulates the activity of protein kinase C (PKC), however, which metabolite of disrupted sphingolipid metabolism is involved, has not been ascertained. In the present study, we evaluated the modulation of PKC by sphingolipid bases and their metabolites using exogenous sphingolipid analogues in porcine renal epithelial (LLC-PK(1)) cells. In preliminary studies we found that fumonisin B(1) (1 microM) selectively and transiently activated PKCalpha, whereas fumonisin B(1) concentrations of 1-50 microM at 48 h repressed PKC-alpha, -delta, - epsilon and -zeta isoforms in a concentration-dependent manner. Addition of exogenous sphinganine-1-phosphate (1 microM for 5 min) alone stimulated cytosolic to membrane translocation of PKCalpha. Co-exposure of fumonisin B(1) with N,N-dimethylsphingosine, an inhibitor of sphingosine/sphinganine kinase, prevented the effects of fumonisin B(1) on PKCalpha. Sphinganine, sphingosine, sphingosine-1-phosphate and ceramide (all at 1 microM) added exogenously, did not alter PKCalpha cytosolic to membrane translocation at 5 min. Fumonisin B(1) (10 microM), sphinganine, sphingosine and ceramide (1 microM each) significantly repressed PKC-alpha and -delta isoforms at 48 h, whereas all the exogenously added sphingolipids significantly repressed PKC- epsilon and zeta similar to fumonisin B(1). Co-exposure of myriocin with fumonisin B(1) prevented the delayed inhibitory effects of fumonisin B(1) on PKC isoforms in LLC-PK(1) cells. This study demonstrated that selective and transient activation of PKCalpha may be due to the fumonisin B(1)-induced accumulation of the bioactive sphinganine-1-phosphate, whereas the long-term repression of PKC isoforms may be predominantly due to the accumulation of sphinganine or its metabolite, and to a lesser extent sphingosine or its metabolite in LLC-PK(1) cells. These findings suggest that the direct or indirect modulation of PKC by these sphingolipids is involved at least in part in the action of fumonisin B(1).

Selective and transient activation of protein kinase C alpha by fumonisin B1, a ceramide synthase inhibitor mycotoxin, in cultured porcine renal cells.

Fumonisin B(1) (FB(1)), a potent and naturally occurring mycotoxin produced by the fungus Fusarium verticillioides, has been implicated in fatal and debilitating diseases in animals and humans. FB(1) affects a variety of cell signaling proteins including protein kinase C (PKC); a serine/threonine kinase, involved in a number of signal transduction pathways that include cytokine induction, carcinogenesis and apoptosis. The aim of this study was to investigate the short-term temporal and concentration-dependent effects of FB(1) on PKC isoforms present in LLC-PK(1) cells in relation to the FB(1)-induced accumulation of sphinganine and sphingosine utilizing various inhibitors and activators. Our studies demonstrated that FB(1) (0.1-1 microM) selectively and transiently activated PKCalpha at 5 min, without affecting PKC-delta, -epsilon and -zeta isoforms. At higher FB(1) concentrations and later time points (15-120 min), PKCalpha membrane concentrations declined to untreated levels. The observed increase in cytosol PKCalpha protein expression at 15 min was not associated with an increase in its activity or protein biosynthesis. Calphostin C, a PKC inhibitor, abrogated the FB(1)-induced translocation of PKCalpha. Pre-incubation with the PKC activator, phorbol 12-myristate 13-acetate, resulted in an additive effect on membrane translocation of PKCalpha. Intracellular sphinganine and sphingosine concentrations were unaltered at the time points tested. Myriocin, a specific inhibitor of serine palmitoyltransferase, the first enzyme in de novo sphingolipid biosynthesis, did not prevent the FB(1)-induced PKCalpha cytosol to membrane redistribution. Altering PKCalpha and its signal transduction pathways may be of importance in the ability of FB(1) to exert its toxicity via apoptosis and/or carcinogenesis.

Fumonisin B1-induced apoptosis is associated with delayed inhibition of protein kinase C, nuclear factor-kappaB and tumor necrosis factor alpha in LLC-PK1 cells.

Fumonisin B1 (FB1), the most potent of the fumonisin mycotoxins, is a carcinogen and causes a wide range of species-specific toxicoses. FB1 modulates the activity of protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinases that play important role in modulating a variety of biologic responses ranging from regulation of cell growth to cell death. Although it has been demonstrated that FB1 induces apoptosis in many cell lines, the precise mechanism of apoptosis is not fully understood. In this study, we investigated the membrane localization of various PKC isoforms, PKC enzyme activity, and its downstream targets, namely nuclear factor-kappa B (NF-kappaB), tumor necrosis factor alpha (TNFalpha), and caspase 3, in porcine renal epithelial (LLC-PK1) cells. FB1 repressed cytosol to membrane translocation of PKC-alpha, -delta, -epsilon, and -zeta isoforms over 24-72 h. The FB1-induced membrane PKC repression was corroborated by a concentration-dependent decrease in total PKC activity. Exposure of cells to phorbol 12-myristate 13-acetate (PMA) for this duration also resulted in repressed PKC membrane localization and activity comparable to FB1. Exposure of cells to FB1 (10 microM) was associated with inhibition of cytosol to nuclear translocation of NF-kappaB and NF-kappaB-DNA binding at 72 h. The expression of TNFalpha was significantly inhibited at 24 and 48 h in response to 1 and 10 microM FB1. Increased caspase 3 activity was observed in LLC-PK1 cells exposed to > or =1 microM FB1 at 48 h. PMA also increased the caspase 3 activity at 24 and 48 h. Results suggest that FB1-induced apoptosis involves the activation of caspase 3, which is associated with the repression of PKC and possibly its down-stream effectors, NF-kappaB and TNFalpha.

Proteomic analysis of early response lymph node proteins in mice treated with titanium dioxide nanoparticles.

Human exposure to nanoparticles is inevitable from natural and anthropogenic sources. Titanium dioxide (TiO2) nanoparticles are increasingly being used in pharmaceutical and cosmetic products. Previous studies revealed that TiO2 levels were significantly increased in tissues (e.g., lymph nodes) after mice were injected with nanosized TiO2. To identify early response lymph node proteins to TiO2 nanoparticles, groups of mice were intradermally injected with a low dose of DeGussa P25 TiO2 nanoparticles or vehicle alone. The proteomes of lymph nodes at 24 h were quantitatively analyzed using trypsin-catalyzed 16O/18O labeling in conjunction with two-dimensional liquid chromatography separation and tandem mass spectrometry (2DLC-MS/MS). A total of 33 proteins were significantly changed (over 1.3-fold, p<0.05) in the mice treated with TiO2 nanoparticles, which accounted for approximately 1% of the total proteins identified. The differentially expressed proteins mainly involve the immune response (e.g., inflammation), lipid and fatty acid metabolism, mRNA processing, and nucleosome assembly. Regulation of functionally distinct classes of proteins could be mediated by estrogen receptor (ESR1), PPARγ, and c-Myc signalings, etc. The differentially expressed proteins identified in this experiment could represent early response proteins to TiO2 nanoparticle treatment in mouse lymph nodes.

Lack of significant dermal penetration of titanium dioxide from sunscreen formulations containing nano- and submicron-size TiO2 particles.

Titanium dioxide (TiO(2)) is included in some sunscreen formulations to physically block ultraviolet radiation. A dermal penetration study was conducted in minipigs with three TiO(2) particles (uncoated submicron sized, uncoated nano-sized, and dimethicone/methicone copolymer-coated nanosized) applied 5% by weight in a sunscreen. These and control formulations were topically applied to minipigs at 2 mg cream/cm(2) skin (4 applications/day, 5 days/week, 4 weeks). Skin (multiple sites), lymph nodes, liver, spleen, and kidneys were removed, and the TiO(2) content was determined (as titanium) using inductively coupled plasma mass spectroscopy. Titanium levels in lymph nodes and liver from treated animals were not increased over the values in control animals. The epidermis from minipigs treated with sunscreens containing TiO(2) showed elevated titanium. Increased titanium was detected in abdominal and neck dermis of minipigs treated with uncoated and coated nanoscale TiO(2). Using electron microscopy-energy dispersive x-ray analysis, all three types of TiO(2) particles were found in the stratum corneum and upper follicular lumens in all treated skin samples (more particles visible with coated nanoscale TiO(2)). Isolated titanium particles were also present at various locations in the dermis of animals treated with all three types of TiO(2)-containing sunscreens; however, there was no pattern of distribution or pathology suggesting the particles could be the result of contamination. At most, the few isolated particles represent a tiny fraction of the total amount of applied TiO(2). These findings indicate that there is no significant penetration of TiO(2) nanoparticles through the intact normal epidermis.

Female mini-pig performance of temporal response differentiation, incremental repeated acquisition, and progressive ratio operant tasks.

Increased knowledge of the cognitive abilities of mini-pigs is needed due to their increasing use in behavioral neuroscience research. Here, six female Yucatan mini-pigs performed tasks thought to measure timing behavior (temporal response differentiation, TRD), learning (incremental repeated acquisition, IRA), and motivation (progressive ratio, PR). Daily 30-min sessions for food reinforcers required a lever press be maintained for at least 10 but no longer than 14s (TRD), learning a new sequence of lever presses each test day (IRA) or an escalating number of presses for subsequent reinforcers (PR). All animals performed PR two days/week while three performed TRD five days/week and the other three performed IRA five days/week. Over the four test weeks, no animal completed TRD training and only one appeared to progress. For this task, lever press maintenance appeared difficult since by choice, the pigs used a front hoof, rather than the snout, to press the lever. IRA subjects showed gradually increasing performance with response rates comparable to those of rats but below those of children and monkeys and accuracy below that for rats. PR response rates were higher than those typically reported for rats, but lower than for adult rhesus monkeys or children. Physical differences in the way that each species responds likely account for these differences.

Preferences of minipigs for environmental enrichment objects.

The minipig is an increasingly popular species for use in behavioral and toxicologic studies. As a result, quantification of environmental enrichment preferences for this species is especially important. We exposed 6 individually housed prepubertal female Yucatan minipigs to 1 of 3 different objects on a rotating schedule: 2 sessions with a hard plastic ball (diameter, 21.0 cm), and 3 sessions each with a large plastic apple (diameter, 22.5 cm) and a soft rubber cone (height, 48.0 cm). Objects were changed every 4 to 5 d. The initial 15 min after each object change was recorded, and duration of object interaction and other behaviors (activity and interaction with the food bowl) were measured. Results indicated significantly longer interactions with the cone (mean +/- SE, 282 +/- 54 s) than the ball (14 +/- 3 s). Interactions with the apple (66 +/- 18 s) and ball did not differ significantly. Interactions with the apple decreased across the 3 sessions, whereas interaction with the cone remained high for most minipigs over the 3 sessions. Duration of activity appeared to be inversely correlated with duration of object interaction (that is, the longer the subject interacted with the object, the less it engaged in nonobject activity). These results provide valuable and practical information on the features of objects that minipigs appear to prefer and offer suggestions for future studies evaluating environmental enrichment paradigms with individually housed minipigs.

Quantitative determination of skin penetration of PEG-coated CdSe quantum dots in dermabraded but not intact SKH-1 hairless mouse skin.

Many cosmetics, sunscreens, and other consumer products are reported to contain nanoscale materials. The possible transdermal absorption of nanoscale materials and the long-term consequences of the absorption have not been determined. We used polyethylene glycol coated cadmium selenide (CdSe) core quantum dots (QD; 37 nm diameter) to evaluate the penetration of nanoscale material into intact, tape stripped, acetone treated, or dermabraded mouse skin. QD were suspended in an oil-in-water emulsion (approximately 9 microM) and the emulsion was applied at 2 mg/cm(2) to mouse dorsal skin pretreated as follows: intact; tape stripped to remove the stratum corneum; acetone pretreated; dermabraded to remove stratum corneum and epidermis. QD penetration into the skin was monitored in sentinel organs (liver and regional draining lymph nodes) using inductively coupled plasma mass spectrometry analysis of cadmium (from the CdSe QD). No consistent cadmium elevation was detected in the sentinel organs of mice with intact, acetone pretreated, or tape-stripped skin at 24- and 48-h post-QD application; however, in dermabraded mice, cadmium elevations were detected in the lymph nodes and liver. QD accumulation (as cadmium) in the liver was approximately 2.0% of the applied dose. The passing of QD through the dermabraded skin was confirmed using confocal fluorescence microscopy. These results suggest that transdermal absorption of nanoscale materials depends on skin barrier quality, and that the lack of an epidermis provided access to QD penetration. Future dermal risk assessments of nanoscale materials should consider key barrier aspects of skin and its overall physiologic integrity.

A time course study demonstrating RNA stability in postmortem skin.

Knowledge of the factors regulating the rate of mRNA degradation, including postmortem delay, is important in determining the reliability of gene expression patterns in dermal tissue. Since RNA stability can be tissue dependent, this study evaluates the effect of postmortem interval on the integrity of total RNA or the levels of representative mRNA species in murine cutaneous tissue. Pieces of fresh skin tissue were excised for periods of 0-60 min from SKH-1 female hairless mice that were maintained at room temperature post-sacrifice. Total RNA was subsequently isolated and RNA integrity from each specimen was evaluated. Bioanalyzer profiles showed no apparent change in 28S/18S rRNA ratio or RNA integrity number at time points up to 60 min. Changes in mRNA expression levels of five selected genes were determined by real-time quantitative PCR. There were no statistical differences in the relative gene expressions of Ccnd1, Hif1alpha, cMyc and Cyr61 as a function of postmortem interval. Our data suggest that the molecular quality of cutaneous tissue is well preserved for at least 60 min after death, which can be regarded as important information for consideration of the order for tissue procurement in in vivo studies and acute ex vivo dermal studies.

Establishment of an extraction method for the recovery of tattoo pigments from human skin using HPLC diode array detector technology.

Tattooing is a widespread process of puncturing pigments into skin, whereas the resulting concentration inside the skin remains unknown. Many tattoo colorants are organic pigments, such as azo pigments, manufactured for other uses. To remove tattoos from skin, laser pulses at very high intensities are applied to the skin to destroy the tattoo pigments. Recent investigations have shown that several azo compounds are cleaved by laser light leading to potentially toxic or carcinogenic compounds. To assess the risk of tattooing and laser treatment of tattoos, the concentration of the pigments and their decomposition products in the skin must be determined. Therefore, an extraction method was established to determine the concentration of tattoo pigments and decomposition products quantitatively. The extraction of two widely used azo compounds, Pigment Red 22 and Pigment Red 9, and their laser-induced decomposition products, 2-methyl-5-nitroaniline, 4-nitrotoluene, 2,5-dichloraniline, and 1,4-dichlorobenzene, was accomplished using recovery experiments and HPLC-DAD technology. Despite the poor solubility of the pigments, a nearly complete recovery from aqueous suspension (> 92%) or lysed skin (> 94%) was achieved. The decomposition products were extracted from aqueous suspension or skin showing a recovery of up to 100%, except for the very volatile 1,4-DCB.

Response of mouse skin to tattooing: use of SKH-1 mice as a surrogate model for human tattooing.

Tattooing is a popular cosmetic practice involving more than 45 million US citizens. Since the toxicology of tattoo inks and pigments used to formulate tattoo inks has not been reported, we studied the immunological impact of tattooing and determined recovery time from this trauma. SKH-1 hairless mice were tattooed using commercial tattoo inks or suspensions of titanium dioxide, cadmium sulfide, or iron oxide, and sacrificed at 0.5, 1, 3, 4, 7, or 14 days post-tattooing. Histological evaluation revealed dermal hemorrhage at 0.5 and 1 day. Acute inflammation and epidermal necrosis were initiated at 0.5 day decreasing in incidence by day 14. Dermal necrosis and epidermal hyperplasia were prominent by day 3, reducing in severity by day 14. Chronic active inflammation persisted in all tattooed mice from day 3 to 14 post-tattooing. Inguinal and axillary lymph nodes were pigmented, the inguinal being most reactive as evidenced by lymphoid hyperplasia and polymorphonuclear infiltration. Cutaneous nuclear protein concentrations of nuclear factor-kappa B were elevated between 0.5 and 4 days. Inflammatory and proliferative biomarkers, cyclooxygenase-1, cyclooxygenase-2, and ornithine decarboxylase protein levels were elevated between 0.5 and 4 days in the skin and decreased to control levels by day 14. Interleukin-1 beta and interleukin-10 were elevated in the lymph nodes but suppressed in the tattooed skin, with maximal suppression occurring between days 0.5 and 4. These data demonstrate that mice substantially recover from the tattooing insult by 14 days, leaving behind pigment in the dermis and the regional lymph nodes. The response seen in mice is similar to acute injury seen in humans, suggesting that the murine model might be a suitable surrogate for investigating the toxicological and phototoxicological properties of ingredients used in tattooing.