Analysis of nickel distribution by synchrotron radiation X-ray fluorescence in nickel-induced early- and late-phase allergic contact dermatitis in Hartley guinea pigs

Abstract

Background: Nickel-induced allergic contact dermatitis (Ni-ACD) is a global health problem. More detailed knowledge on the skin uptake of haptens is required. This study aimed to investigate the penetration process and distribution of nickel in skin tissues with late phase and early phase of Ni-ACD to understand the mechanisms of metal allergy.

Methods: Forty Hartley guinea pigs were divided into four groups according to the NiSO4 sensitizing concentration and the NiSO4 challenged concentration: the 5% NiSO4-group, 5% to 10% (sensitization-challenge; late phase group); 10% NiSO4-group, 10% to 10% (sensitization-challenge; early-phase group); and the positive and negative controls. Pathological biopsies were performed on each group. The depth profile of nickel element concentration in the skin of guinea pigs was detected by synchrotron radiation micro X-ray fluorescence spectroscopy (SR-μ-XRF) and micro X-ray absorption near-edge spectroscopy (μ-XANES).

Results: In each section, the nickel element concentration in both the 5% NiSO4-group and 10% NiSO4-group was significantly higher than that in the negative control group. In the upper 300-μm section of skin for the early phase group, the nickel element concentration was significantly higher than that in the lower section of skin. In deeper sections (>200 μm) of skin, the concentration of nickel in the early phase group was approximately equal to that in the late phase group. The curve of the late phase group was flat, which means that the nickel element concentration was distributed uniformly by SR-μ-XRF. According to the XANES data for the 10% NiSO4 metal salt solution, structural changes occurred in the skin model sample, indicating that nickel was not present in the Ni aqueous ionic state but in the nickel-binding protein.

Conclusions: This study showed that the distribution of the nickel element concentration in ACD skin tissue was different between the early phase and late phase groups. The nickel element was not present in the Ni aqueous ionic state but bound with certain proteins to form a complex in the stratum corneum in ACD model tissue.

Figure 1

Evaluation of erythema in guinea pigs at different times after challenge. Skin reaction in animal model was correlated with clinical symptom scores with Magnusson and Kligman test's standard. Dot plots show values for average symptom scores in each group (ten Hartley guinea pigs) during 72 h. Data represent at least three independent experiments. The detected data was converted to average value in each group.

Figure 2

Pathological alterations in skin of model after challenge with NiSO₄ at 24 h. Hematoxylin and eosin staining of skin sections from early-phase group (A) and acute phase group (B) showed epidermis, dermis, and peri-vascular hypodermis infiltration of inflammatory symptoms in animal model at 24 h. Stained tissues were observed using a light microscope (original magnification ×40). Scale bars: 500 μm.

Figure 3

The main elements of skin tissues in guinea pigs-model after 48 h treatment. The images showed clear peaks for not only Ni, but also the metal elements Fe and Zn in the skin cross-sections after 48 h. Ar is non-metal element in air, it was not considered in this study. (A) The macroscopic image showed the skin section of animal. (B) Skin cross-section from animals (original magnification ×8, scale bars: 100 μm). The arrow points to direction of scanning before administering SR-XRF. (C) SR-XRF indicated the main elements from the skin cross-sections. The intensity of the element in negative control was regard as the background (pink line). SR-XRF: Synchrotron radiation X-ray fluorescence spectroscopy.

Figure 4

The relative comparison of nickel content in skin tissues of Ni-ACD model. The depth-resolved XRF were acquired to determine the distribution of Ni in the ACD model tissue. XRF scan of first point (0 μm) was determined to be at the position of maximum change point of the profile. Values represent at least three independent experiments and the detected data was converted to average value in each group. Ni-ACD: Nickel induced allergic contact dermatitis; XRF: X-ray fluorescence spectroscopy.

Figure 5

X-ray absorption near-edge spectroscopy of nickel K edge in the skin of Ni-ACD. XANES studies were performed to investigate the nickel binding properties in this model tissue. The peaks will depend on the coordination number and geometry of the metal site. The difference was observed between the ACD model tissue (Skin model) and the 10% NiSO₄ solution (NiSO₄ solution). Ni-ACD: Nickel induced allergic contact dermatitis; XANES: X-ray absorption near-edge spectroscopy.