Chikage Mitoma

An environmental contaminant, benzo(a)pyrene, induces oxidative stress-mediated interleukin-8 production in human keratinocytes via the aryl hydrocarbon receptor signaling pathway

BACKGROUND Benzo(a)pyrene (BaP) is an environmental contaminant found in cigarette smoke. It is well known that cigarette smoking exacerbates interleukin-8 (IL-8)-related inflammatory skin diseases such as psoriasis, palmoplantar pustulosis and acne. Although BaP has been shown to exert its biological effects via the aryl hydrocarbon receptor (AhR) signaling pathway, the mechanism of its inflammatory effects on skin remains unanswered. OBJECTIVE To elucidate whether or not BaP cause AhR activation and subsequent oxidative stress leading to IL-8 production in normal human epidermal keratinocytes (NHEKs). METHODS NHEKs exposed to BaP were analyzed. Immunofluorescence, real-time PCR, Western blotting, ELISA, reactive oxygen species (ROS) detection using H2DCFDA and RNA interference using si (small interfering) RNA were employed. RESULTS Immunofluorescence analysis clearly demonstrated that BaP induced nuclear translocation of AhR from cytoplasm. The AhR activation subsequently induced CYP1A1 mRNA and protein expression in a dose-dependent manner. In addition, ROS and IL-8 production were coordinately augmented by BaP, whereas this was not the case in IL-1α, IL-6, TNF-α or GM-CSF production. Knockdown of AhR expression using siRNA transfection inhibited BaP-induced-ROS and IL-8 production, suggesting that these responses are strongly dependent on the AhR signaling pathway. Furthermore, the addition of N-acetyl cystein or catalase cancelled the IL-8 production by BaP, indicating that ROS production is essential for IL-8 production. RESULTS This data highlights AhR-ROS-dependent regulation of IL-8 in NHEKs by BaP, providing a plausible explanation, at least in part, for why cigarette smoking exacerbates IL-8-related skin diseases such as psoriasis, palmoplantar pustulosis and acne.

Maternal exposure to high levels of dioxins in relation to birth weight in women affected by Yusho disease

BACKGROUND Studies on the association of maternal exposure to polychlorinated dibenzo-p-dioxin (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) with decreased birth weight in humans have produced conflicting results. In Japan in 1968, an accidental human exposure to rice oil contaminated with PCDDs, PCDFs, and PCBs, led to the development of Yusho disease. OBJECTIVE The Yusho cohort was used to evaluate the effect of maternal exposure to PCDDs, PCDFs, and PCBs on birth weight. METHODS Blood samples, obtained from 101 Yusho women (190 births) who gave birth after exposure, were analyzed for congeners of seven PCDDs, ten PCDFs, and four non-ortho PCBs. RESULTS Total PCDD TEQ (adjusted beta=-161.9g; 95% CI, -265.3 to -58.6), total PCDF TEQ (adjusted beta=-105.9g; 95% CI, -179.5 to -32.2), and total non-ortho PCBs (adjusted beta=-178.4g; 95% CI, -318.3 to -38.5) levels were inversely associated with birth weight. Significant inverse associations with birth weight were also found for total PCDD TEQ, total PCDF TEQ, and total non-ortho PCB TEQ levels among male, but not female, infants. Significant inverse associations with birth weight were also found for nine congeners among all infants; the adjusted beta coefficients were largest for 1,2,3,6,7,8-HxCDD and smallest for 2,3,4,7,8-PeCDF. CONCLUSION In the setting of exposure to high levels of dioxins, maternal blood levels of PCDDs, PCDFs and PCBs are associated with lower birth weight in Yusho patients. The association exhibited gender-specific differences, as male infants are more susceptible than females to growth restriction induced by in utero dioxin exposures.

Gene regulation of filaggrin and other skin barrier proteins via aryl hydrocarbon receptor.

Aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that binds to structurally diverse chemicals including dioxins, coal tar, flavonoids and tryptophan photoproducts. Upon ligation, cytoplasmic AHR translocates to the nucleus, heterodimerizes with aryl hydrocarbon receptor nuclear translocator and mediates numerous biological effects by inducing the transcription of various AHR-responsive genes such as epidermal barrier proteins. The activation of AHR usually generates oxidative stress. However, AHR also mediates antioxidant signaling by a plethora of ligands via nuclear factor-erythroid 2-related factor-2. Both oxidative and antioxidant ligands upregulate the expression of the filaggrin gene. We review the role of AHR signaling in the gene regulation of epidermal barrier proteins.

Antioxidant soybean tar Glyteer rescues T‐helper‐mediated downregulation of filaggrin expression via aryl hydrocarbon receptor

Soybean tar Glyteer (Gly) has been widely used for the treatment of various inflammatory skin diseases in Japan since 1924 as an alternative to coal tar remedy. Recently, coal tar has been shown to induce barrier repair in atopic dermatitis via aryl hydrocarbon receptor (AhR). In this study, we demonstrated that Gly activated AhR by inducing its cytoplasmic to nuclear translocation in keratinocytes. The AhR ligation by Gly was biologically active, with significant and dose‐dependent upregulation of CYP1A1 expression, which is a specific marker for AhR activation. Gly upregulated the expression of filaggrin in an AhR‐dependent manner because its enhancing effect was completely abrogated in AhR‐knockdown keratinocytes. T‐helper (Th)2 cytokines inhibited the expression of filaggrin; however, Gly completely restored the Th2‐mediated inhibition of filaggrin expression. Furthermore, Gly coordinately upregulated a series of epidermal differentiation complex genes, including involucrin, loricrin and hornerin. In addition, Gly exhibited potent antioxidant activity through the activation of nuclear factor‐erythroid 2‐related factor‐2 (Nrf2) and downstream antioxidant enzymes such as NAD(P)H:quinone oxidoreductase 1 (Nqo1), which actually inhibited the generation of reactive oxygen species in keratinocytes treated with tumor necrosis factor‐α or benzo[α]pyrene. In conclusion, antioxidant Gly rescues the downregulated expression of filaggrin (and plausibly other barrier proteins) in a Th2‐skewed milieu via AhR activation, which may partly explain its empirical anti‐inflammatory therapeutic effects.

Variation in half-life of penta-chlorodibenzofuran (PeCDF) blood level among Yusho patients

Yusho (oil disease) is the name given to a food poisoning incident caused by rice bran oil that occurred in west Japan in 1968. The causative agents of Yusho are currently considered to be polychlorinated biphenyls (PCBs) and their by-products, such as dioxin like compounds. The levels of 2,3,4,7,8-penta-cholorodibenzofuran (PeCDF) are measured in the blood of Yusho patients who attend medical check-ups. The objectives of this study were to determine the half-life of PeCDF in these patients. Linear regression analysis was performed with the binary logarithm of PeCDF blood levels in Yusho patients as the dependent variable and the measurement year as the independent variable. The linear coefficient determined in this analysis is the reciprocal of the half-life. The half-life of PeCDF varied among patients. Among patients with PeCDF blood levels of 50pgg(-1) or higher, there were two groups: one showing a half-life of approximately 7 years and the other showing no reduction in PeCDF levels over time. The results indicate that there is a group of patients whose PeCDF levels are maintained at a high level. Our study suggests that a more complicated model is required to explain PeCDF excretion in humans.

Antioxidants for Healthy Skin: The Emerging Role of Aryl Hydrocarbon Receptors and Nuclear Factor-Erythroid 2-Related Factor-2

Skin is the outermost part of the body and is, thus, inevitably exposed to UV rays and environmental pollutants. Oxidative stress by these hazardous factors accelerates skin aging and induces skin inflammation and carcinogenesis. Aryl hydrocarbon receptors (AHRs) are chemical sensors that are abundantly expressed in epidermal keratinocytes and mediate the production of reactive oxygen species. To neutralize or minimize oxidative stress, the keratinocytes also express nuclear factor-erythroid 2-related factor-2 (NRF2), which is a master switch for antioxidant signaling. Notably, there is fine-tuned crosstalk between AHR and NRF2, which mutually increase or decrease their activation states. Many NRF2-mediated antioxidant phytochemicals are capable of up- and downmodulating AHR signaling. The precise mechanisms by which these phytochemicals differentially affect the AHR and NRF2 system remain largely unknown and warrant future investigation.

Antioxidant Opuntia ficus-indica Extract Activates AHR-NRF2 Signaling and Upregulates Filaggrin and Loricrin Expression in Human Keratinocytes

UNLABELLED Opuntia ficus-indica (OFI) is a cactus species widely used as an anti-inflammatory, antilipidemic, and hypoglycemic agent. It has been shown that OFI extract (OFIE) inhibits oxidative stress in animal models of diabetes and hepatic disease; however, its antioxidant mechanism remains largely unknown. In this study, we demonstrated that OFIE exhibited potent antioxidant activity through the activation of nuclear factor erythroid 2-related factor 2 (NRF2) and the downstream antioxidant enzyme NAD(P)H quinone oxidoreductase 1 (NQO1), which inhibited the generation of reactive oxygen species in keratinocytes challenged with tumor necrosis factor α or benzo[α]pyrene. The antioxidant capacity of OFIE was canceled in NRF2 knockdown keratinocytes. OFIE exerted this NRF2-NQO1 upregulation through activation of the aryl hydrocarbon receptor (AHR). Moreover, the ligation of AHR by OFIE upregulated the expression of epidermal barrier proteins: filaggrin and loricrin. OFIE also prevented TH2 cytokine-mediated downregulation of filaggrin and loricrin expression in an AHR-dependent manner because it was canceled in AHR knockdown keratinocytes. Antioxidant OFIE is a potent activator of AHR-NRF2-NQO1 signaling and may be beneficial in treating barrier-disrupted skin disorders.

Yusho and its latest findings—A review in studies conducted by the Yusho Group

The Yusho incident is an unprecedented mass food poisoning that occurred in Japan in 1968. It was caused by the ingestion of rice bran oil contaminated with polychlorinated biphenyls (PCBs) and various dioxins and dioxin-like compounds, such as polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). The victims of Yusho have suffered from characteristic skin manifestations associated with systemic, ophthalmological, and mucosal symptoms for a long period of time. The Study Group of Yusho (the Yusho Group) has been conducting annual medical check-ups on Yusho victims for more than 45years. Since 2002, when concentrations of dioxins in the blood of Yusho patients started to be measured, the pharmacokinetics of dioxins, relationship between blood levels of dioxins and symptoms/signs in patients directly exposed to dioxins, and the adverse effects on the next generation have become dramatically clear. Herein we review recent findings of studies conducted by the Yusho Group to evaluate chronic dioxin-induced toxicity to the next generation as well as Yusho patients in comparison with a similar food mass poisoning, the Yucheng incident. Additionally, we summarized basic studies carried out by the Yusho Group to re-evaluate the mechanisms of dioxin toxicities in experimental models and various functions of the aryl hydrocarbon receptor (AhR), known as the dioxin receptor, pathway.

Comparison of the concentrations of polychlorinated biphenyls and dioxins in mothers affected by the Yusho incident and their children

Accumulated maternal dioxins are passed onto the fetus and neonate via the placenta and maternal milk. In Japan in 1968, an accidental human exposure to rice oil contaminated with polychlorinated biphenyls (PCBs) and other dioxin-related compounds, such as polychlorinated dibenzofurans (PCDFs), led to development of Yusho oil disease. We investigated differences in blood dioxin concentrations in mother-children pairs affected by the Yusho incident. From 2002 to 2008, blood samples were collected from 26 pairs of Yusho mothers and their children (19 mothers, 26 children). Specific congeners of seven polychlorinated dibenzo-p-dioxins (PCDDs), ten PCDFs, and four non-ortho PCBs were analyzed. The children had significantly lower TEQ concentrations of PCDDs, PCDFs, and coplanar PCBs compared to their mothers. The mother-child difference in blood concentrations varied with the congeners; the largest for 2,3,4,7,8-pentaCDF and the smallest for 1,2,3,4,6,7,8-heptaCDD. The level for 2,3,4,7,8-pentaCDF, which characterizes Yusho oil disease, was approximately 17-30 times higher in the mothers than in the general population, whereas there were no significant differences between children in the formula-fed group and the general population. In contrast, the mean level for 2,3,4,7,8-pentaCDF in the breast-fed group was approximately 1.5 times, (range 0.5-6.5 times) higher than that in the general population. Over 30 years after the Yusho incident, the mean blood dioxin levels in the offspring were only a fraction of the levels in their mothers. This is more consistent with exposure via breast milk than via transplacental transfer in the Yusho incident.

Inhibition of aryl hydrocarbon receptor signaling and induction of NRF2-mediated antioxidant activity by cinnamaldehyde in human keratinocytes.

BACKGROUND Dioxins and other environmental pollutants are toxic and remain in biological tissues for a long time leading to various levels of oxidative stress. Although the toxicity of these agents has been linked to activation of the aryl hydrocarbon receptor (AHR), no effective treatment has been developed. OBJECTIVE To explore novel phytochemicals that inhibit AHR activation in keratinocytes. METHODS Keratinocytes were used in this study because the skin is one of the organs most affected by dioxin and other environmental pollutants. HaCaT cells, which are a human keratinocyte cell line, and normal human epidermal keratinocytes were stimulated with benzo[a]pyrene to induce AHR activation, and the effects of traditional Japanese Kampo herbal formulae were analyzed. Quantification of mRNA, western blotting, immunofluorescence localization of molecules, siRNA silencing, and visualization of oxidative stress were performed. RESULTS Cinnamomum cassia extract and its major constituent cinnamaldehyde significantly inhibited the activation of AHR. Cinnamaldehyde also activated the NRF2/HO1 pathway and significantly alleviated the production of reactive oxygen species in keratinocytes. The inhibition of AHR signaling and the activation of antioxidant activity by cinnamaldehyde operated in a mutually independent manner as assessed by siRNA methods In addition, AHR signaling was effectively inhibited by traditional Kampo formulae containing C. cassia. CONCLUSION Cinnamaldehyde has two independent biological activities; namely, an inhibitory action on AHR activation and an antioxidant effect mediated by NRF2/HO1 signaling. Through these dual functions, cinnamaldehyde may be beneficial for the treatment of disorders related to oxidative stress such as dioxin intoxication, acne, and vitiligo.