Endogenous Interleukin-1α Is Associated with Skin Irritation Induced by Tributyltin

doi:10.1006/taap.1996.0125

Toxicology and Applied Pharmacology

Volume 138, Issue 2, June 1996, Pages 268-274

https://doi.org/10.1006/taap.1996.0125 Get rights and content

Abstract

Tributyltin (TBT) salts are well-known skin irritants in both human and rodents. This study investigated the role of interleukin-1α (IL-1α) in the process in mice and in murine keratinocytes. The ears of Balb/c mice were painted with different amounts of TBT (67–536 nmol in acetone) or with acetone alone. Two hours later there was dose-related production of IL-1α along with ear swelling and accumulation of skin water, all of which were partially prevented by intraperitoneal injection of antibody against murine IL-1α. By reverse transcription–polymerase chain reaction we were able to show that the neutralizing antibody also partially prevented TBT-inducedin vivoIL-6 expression but no TBT-induced TNF-α expression, suggesting a paracrine effect of IL-1α on IL-6 production but not TNF-α expression and indicating that other inflammatory mediators are involved. TBT induced both intracellular production of IL-1α and its release into culture medium in a murine keratinocyte cell line (HEL30). IL-1α production was inhibited by addition of a neutralizing antibody against IL-1α, which suggests an autocrine effect of IL-1α on its own production. The intracellular production of IL-1α could be significantly inhibited by prior treatment with antioxidants, which strongly suggests a role for oxidative species in the mechanism of action of TBT in IL-1α induction. The complex-1 inhibitor rotenone also significantly inhibits IL-1α production. Since TBT causes disturbances in the respiratory chain in mitochondria, the mechanism of its action may be the production of reactive oxygen intermediates at the ubiquinone site, which activate transcription factors and promote IL-1α synthesis.

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Cited by (56)

Establishment and evaluation of immortalized human epidermal keratinocytes for an alternative skin irritation test
2017, Journal of Pharmacological and Toxicological Methods
Citation Excerpt :
In another previous study, the intercellular production of IL-1α was significantly upregulated by tributyltin, a well-known skin irritant in both humans and rodents, in the HEL30 murine keratinocyte cell line (Corsini et al., 1996). The aforementioned previous studies have shown that cytokines in these keratinocytes have excellent responses to inflammatory responses (Corsini et al., 1996; Wilmer et al., 1994). Similar to previous studies, SV-HEK2 cells also showed a tendency to quantitatively secrete cytokines such as IL-1a and IL-8 in response to irritants.
Human skin is located at the outermost part of the body, and various cosmetics and chemicals that may come in contact with human skin need to be evaluated for their potential to cause irritation. Until recently, the Draize test was considered the standard method for skin irritation; however, this technique has disadvantages such as the need to sacrifice many rabbits and subjective scoring. Thus, to contribute to the development of an animal-free alternative skin irritation test, we investigated the cytotoxicity and inflammatory response to standard skin irritants in SV40 large T antigen-transformed human epidermal keratinocyte 2 cells (SV-HEK2 cells). In this study, we established an SV-HEK2 cell line immortalized by SV40 large T antigen (SV40 T) and characterized the inherent morphological and cytological properties. We next used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or neutral red uptake (NRU) assays of cell viability to investigate the optimal experimental conditions for determining SV-HEK2 cell viability after exposure to sodium dodecyl sulfate at 6.25 × 10^− 3% to 1 × 10^− 1% as a standard skin irritant. We then examined the viability of SV-HEK2 cells in response to five skin irritants (benzalkonium chloride, isopropanol, sodium dodecyl sulfate, Triton X-100 and Tween20) at 5 × 10^− 3% to 1 × 10^− 1% by MTT or NRU assay. Finally, we estimated the level of cytokines secretion in response to stimulation by skin irritants in SV-HEK2 cells. The results revealed that SV-HEK2 cells responded well to skin irritants in a concentration-dependent manner and that there was good correlation between irritant concentration and cytotoxicity (or cytokine secretion) when cells were exposed to skin irritants for 10 min at room temperature (RT) using an MTT assay. Overall, these findings suggest that SV-HEK2 cells could be a good alternative in vitro model for skin irritation tests.
Differentiation of skin sensitizers from irritant chemicals by interleukin-1α and macrophage inflammatory protein-2 in murine keratinocytes
2013, Toxicology Letters
The development of novel alternative testing methods is required to identify the sensitizing capacity of chemicals as a replacement for animal experimentation. We aimed to evaluate in vitro assays as screening tools for detecting skin sensitizers. The murine epidermal keratinocyte cell line HEL-30 was exposed to 16 relevant skin sensitizers and 6 skin irritants. The dose causing 75% cell viability (CV₇₅) measured by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was chosen as a highest dose and three more doses (0.5×, 0.1×, and 0.01× of CV₇₅) were tested. As parameters, levels of interleukin 1α (IL-1α), macrophage inflammatory protein 2 (MIP-2), IL-6, and IL-18 production were measured using 4 different doses. The accuracy of detecting sensitizers or irritants by IL-1α or MIP-2 alone was exactly same: 75% (12 out of 16) for sensitizers, 83% (5 out of 6) for irritants, and overall 77% (17 out of 22). However, combination of IL-1α and MIP-2 showed better accuracy: 94% (15 out of 16), 67% (4 out of 6), and overall 86% (19 out of 22). IL-6 and IL-18 could not differentiate sensitizers from irritants. This study suggests that the combination of pro-inflammatory cytokines IL-1α and MIP-2 in murine HEL-30 cells can be a reliable in vitro method for identifying chemicals that may act as skin sensitizers.
Tributyltin induces oxidative stress and neuronal injury by inhibiting glutathione S-transferase in rat organotypic hippocampal slice cultures
2012, Neurochemistry International
Citation Excerpt :
These data confirmed that neuronal injury was induced by TBT in hippocampal slices, as previously reported by Mizuhashi et al. (2000). Because the cell death induced by TBT was reportedly attenuated in the presence of antioxidants, oxidative stress was considered to be involved in TBT-induced cellular injury (Corsini et al., 1996; Mizuhashi et al., 2000; Nakatsu et al., 2007). However, a detailed analysis of the oxidative stress induced by TBT has never been conducted.
Tributyltin (TBT) has been used as a heat stabilizer, agricultural pesticide and antifouling agents on ships, boats and fish-farming nets; however, the neurotoxicity of TBT has recently become a concern. TBT is suggested to stimulate the generation of reactive oxygen species (ROS) inside cells. The aim of this study was to determine the mechanism of neuronal oxidative injury induced by TBT using rat organotypic hippocampal slice cultures. The treatment of rat hippocampal slices with TBT induced ROS production, lipid peroxidation and cell death. Pretreatment with antioxidants such as superoxide dismutase, catalase or trolox, suppressed the above phenomena induced by TBT, indicating that TBT elicits oxidative stress in hippocampal slices, which causes neuronal cell death. TBT dose-dependently inhibited glutathione S-transferase (GST), but not glutathione peroxidase or glutathione reductase in the cytosol of rat hippocampus. The treatment of hippocampal slices with TBT decreased the GST activity. Pretreatment with reduced glutathione attenuated the reduction of GST activity and cell death induced by TBT, indicating that the decrease in GST activity by TBT is involved in hippocampal cell death. When hippocampal slices were treated with sulforaphane, the expression and activity of GST were increased. Notably, TBT-induced oxidative stress and cell death were significantly suppressed by pretreatment with sulforaphane. These results indicate that GST inhibition could contribute, at least in part, to the neuronal cell death induced by TBT in hippocampal slices. This study is the first report to show the link between neuronal oxidative injury and the GST inhibition elicited by TBT.
Tributyltin (TBT) and mitochondrial respiration in mussel digestive gland
2011, Toxicology in Vitro
Citation Excerpt :
Inhibition of mitochondrial respiration by drugs with known organ toxicity is increasingly documented (Dykens et al., 2008). Even if field and laboratory effects of TBT cannot be easily matched (Salazar, 1989), the mitochondrial damage caused by TBT was related to skin irritation (Corsini et al., 1996), neurotoxicity (Kotake, 2007) and overall cytotoxicity (Hoth et al., 2005) in mammals, as well as embryotoxicity (Dolcemascolo et al., 2005) and reproductive failure (Villa et al., 2003) in tunicates. Different pathways which link the inhibition of mitochondrial respiratory chain to toxic effects can be perceived.
The toxicity of organotins and especially tri-n-butyltin (TBT) on mitochondria is well known. However as far as we are aware, effects on mitochondrial respiration are unexplored in mollusks. In this work mitochondria isolated from the digestive gland of Mytilus galloprovincialis and susceptive to the classical respiratory chain inhibitors, were assayed in the presence of micromolar TBT concentrations to investigate mitochondrial respiratory activities. Intact and freeze–thawed mitochondria were used. TBT significantly inhibited oxygen consumption in the presence of glutamate/malate or succinate as substrates. Conversely cytochrome c oxidase activity (complex IV), assayed both polarographically and spectrophotometrically, was unaffected. The addition of 1,4-dithioerythritol (DTE) decreased the TBT-driven inhibition of complexes I and III. The TBT capability of covalent binding to thiol groups of mitochondrial proteins in a dose-dependent manner was confirmed by the aid of Ellman’s reagent. Data strongly suggests that TBT may prevent the electron transfer from complexes I and III to downhill respiratory chain complexes by binding to critical SH residues.
A catch-up validation study on reconstructed human epidermis (SkinEthic™ RHE) for full replacement of the Draize skin irritation test
2010, Toxicology in Vitro
Efforts to fully replace the in vivo Draize skin irritation test, according to the Directive 67/548/ECC or OECD TG 404, were reinforced with the seventh Amendment of the Cosmetic Directive and the REACh regulation. In 2007, the EpiSkin™ test method was scientifically validated and recognized as the stand alone method to discriminate skin irritants (R38) from non-irritants (no label) according to the definition of the EU risk phrases. An ECVAM performance standards (PS) document was defined to evaluate the accuracy and reliability of other analogous test methods (ECVAM SIVS, May 2007). The present test was designed to determine the reliability and relevance of the Reconstructed Human Epidermis (RHE) model commercialized by SkinEthic™. The RHE skin irritation test method consisted to topically apply topically the test substances for 42 min followed by a 42 h post-incubation. The main selected endpoint was the cell viability (MTT reduction), with a threshold of 50% viability. The RHE test method showed a good intra and inter-laboratory reproducibilities in a multicentric study involving three independent laboratories. The SkinEthic™ RHE test method showed to be relevant and reliable with a sensitivity of 90% and a specificity of 80% (MTT only) and was not improved by integrating another endpoint such as IL-1α. The overall accuracy was 85% resulting in the recognition of the SkinEthic™ RHE test method, by the ECVAM Scientific Advisory Committee in November 2008, as a stand alone replacement test method for the Draize rabbit in vivo test, as a screen, or as part of a sequential testing strategy in a weight of evidence approach, for classifying non-irritant and irritant test substances, depending on country requirements.
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2009, Journal of Controlled Release
Transcutaneous immunization, topical application of vaccines on skin, provides several advantages over needle based immunization. However, simple topical application of vaccines does not generate sufficient immune response due to limited transport of vaccines across the stratum corneum of skin. Here we report that chemicals used in common skin products can enhance the immunogenicity of topically applied antigens. Six hundred formulations of commonly used chemicals were screened systematically for their potency (delivery of antigen) in vitro. A selected subset of these formulations was subsequently tested for their adjuvanticity (activation of immune response) in vitro. Lead formulations were tested in vivo for their ability to generate antibody titers against topically applied ovalbumin, a model antigen. Lead formulations were significantly more effective in generating anti-ovalbumin IgG titers. Our results demonstrate that chemical formulations can be successfully used to deliver antigens and that such formulations can be rationally designed by combinatorial screening of individual chemical components.

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Regular ArticleEndogenous Interleukin-1α Is Associated with Skin Irritation Induced by Tributyltin

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Endogenous Interleukin-1α Is Associated with Skin Irritation Induced by Tributyltin