Protective Effect of Metallogluconates Against UVA-Induced Cutaneous Lesion in HR-1 Hairless Mice
302 Biomed Res Trace Elements 16(4) : 302-305, 2005
Short Communications Protective effect of metallogluconates against UVA-induced cutaneous lesion in HR-1 hairless mice
Yuriko Yasuhara, Hiroyuki Yasui, and Hiromu Sakurai*
Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University
Abstract UVA-induced reactive oxygen species (ROS) generation is well known to cause cutaneous lesion in the skin, therefore, it is important to prevent the skin from UV damage due to ROS generation. In 2000, we reported first the in vivo detection and imaging by chemiluminescence (CL) method of the generated ROS in the skin of live mice following UVA-irradiation Ll] . Using this method, we found that superoxide anion radical ( o 02-) was intrinsically generated in the skin of live mice and singlet oxygen (102) was exclusively produced in the skin following UVA-irradiation. In our previously report, we found that topical application and oral administration of zinc compounds to the skin of live mice reduce the formation of UVA-induced ROS L2-3J. Then, in the present study, we examined whether metallogluconates (zinc gluconate (ZnGA ) and copper gluconate (CuGA )) topically applied and orally administered reduce the intrinsic and UVA-induced ROS generations in the skin of live mice or not. We found that topical application and oral supplementation of CUGA reduced CL intensities in terms of the formation of UVA-induced ROS and ZnGA suppressed inflammation caused UVA-exposure.
Keywords : skin, UVA, reactive oxygen species(ROS), inflammation,harrless ' mouse zmc gluconate, copper-gluconate
Introduction In 2000, we found first the in vivo detection and UV rays have been suggested to cause the various imaging by chemiluminescence (CL) method of the cutaneous inflammatory disorders such as the skin generated ROS in the skin of live mice following inflammation, photoaging and skin cancer. The free UVA-exposure LIJ . Using this method, we found that radical theory L4-6] have been proposed to explain eO,- is intrinsically generated in the skin and ~02 is aging in the skin, and received particular attention produced in the skin following UVA-exposure. On the because the skin is constantly exposed to reactive basis of the results, we tried to find potent scavengers oxygen species (ROS) from the environment such as for ~O, with this method. In 2002, we found that air, solar radiation, ozone, and other air-borne pollut- topical application of zinc chloride (ZnCl.) to the skin ants, or from the normal metablism L7] . Accumulated of live mice reduced the formation of UVA-induced ROS have been suggested to play important roles in ROS generation L2] , and in 2003, we revealed that oral the intrinsic aging and photoaging in the skin of administration or intraperitoneal (i.p.) injection of human [8], and to be responsible for various ZnCl, and bis(picolinato)Zn(II) (Zn(pic)2) complex cutaneous inflammatory disorders and skin cancers reduced the formation of UVA-induced ROS genera- [9-10] . tion L3J. Zinc and copper consist of ROS-related enzymes such as superoxide dismutase (SOD), and Address correspondence to : play important roles in growth, the reproductive func- Hiromu Sakurai tion, the skin tissue maintenance in human body. Department of Analytical and Bioinorganic ZnGA and CuGA are supplimented in the breastmilk Chemistry,Kyoto Pharmaceutical University, as food additives for the purpose of the trace metals 5 Nakauchi-cho, Misasagi, Yamashina-ku, strengthening. Recently, it has been revealed that kyoto 607-8414,Japan. these trace elements are deficient in not only youth Accepted : 23 September 2005 but also adult, application of these complexes in ROS scavenging effect of metallogluconate in the skin 303
addition to the breastmilk substitute became possible. trated to female hairless mice at doses of In the present study, we examined whether or not 150 mg Zn/kg body weight/day and 35 mg ZnGA and CuGA given by topically application or oral Cu/kg body weight/day for 14 days. administration reduce both intrinsically generated and Group 5 : GA was orally administrated to female UVA-induced ROS generation in the skin of HR-1 hairless mice at a dose of 1480 mg GA/kg hairless mice. body weight/day for 14 days.
Materials and Methods Ultra-Low chemiluminescene detecting system Materials A Iuminograph, NightOWL Molecular Light Imager The chemiluminescent probe, CLA (2-methyl-6- (Luminograph LB 981, Wallack Berthold, Germany) phenyl-3,7-dihydroimidazo- L1,2-a]pyrazin-3-0ne), was was used as a high-performance low-1ight imaging purchased from Tokyo Kasei Organic Chemicals system for detection of luminescent emission (400-600 (Tokyo Japan). ZnGA and CUGA were gift from Fuso nm) L1-2].The NightOWL possesses a peltier-air- Chemical Co. (Tokyo, Japan) All another chemicals cooled CCD camera for ultra-low light imaging with a used were of the highest analytical grade. high sensitivity and is designed for macro imaging such as the tissue or whole organisms. Its analytical Animals performance is sufficient to evaluate the quantitative Female HR-1 hairless mice ( 4 and 6 weeks old ) detection of ultra-low light chemiluminescence L13]. were purchased from Simizu Experiment Materials The luminescent signals on a photocathode in the (Kyoto, Japan) and were maintained in a light/dark image intensifier were detected as photons. The sys- cycle in the central animal facility of Kyoto Pher- tem was controlled by a DOS/V PC provided with maceutical University for experimental periods. The software for quantitative image analysis (WinLight animals were given free access to standard mice chow 32). and water for these periods. Measurements of chemiluminescence in the skin of Application methods hairless mice The hairless mice was anesthetized by i.p. injection The skin surface on abdomen of a hairless mouse of pentobarbital (50 mglkg body weight), fixed on a was carefully cleaned three times with 50% ethanol. heating pad. The skin surface on the abdomen of The hairless mice was anesthetized by i.p. injection of hairless mice was carefully cleaned three times with pentobarbital (50 mglkg body weight), fixed on a 50% ethanol. ZnGA 100pL ( 1.0 and 2.0 I/mol/ 5 cm2) heating pad, covered with a black cloth in which two or CuGA( 0.5 and 1.0 and 2.0 pmol/ 5 cm2) was applied holes of the same area (10 mm diameter, 78.5 mm2) on the skin surface of abdomen for 30 min and then were cut. The measured area were divided into two was removed with 50% ethanol. The mice was applied parts (1eft and right side in the abdomen skin) for 50% ethanol as control. comparative investigation of intrinsic and UVA- induced ROS generation. One area was covered with Administration methods black tape not to be exposured by UVA, and the other The hairless mice were devided into 5 groups, and was exposured to UVA Iight (18 J/cm2). After UVA each group had 5 mice. irradiation, CLA was immediately applied to both two Group I : Physiological saline was orally adminis- areas on the skin of mice, and the CL was measured. trated to female hairless mice for 14 days. The quantity of ROS generated in the skin of mice (control) was determined, as reported previously L1-2] . Group 2 : ZnGA was orally administrated to female hairless mice at a dose of 150 mg Zn/kg Statistical analysis body weight/day for 14 days. All experimental results are presented as the mean Group 3 : CUGA was orally administrated to female values i standard deviations. Statistical treatment hairless mice at a dose of 50 mg Culkg was performed by analysis of variance (ANOVA) at a body weight/day for 14 days. 0.1%, l% or 5% significance of difference. Group 4 : Both ZnGA and CUGA were orally adminis- 304 Biomed Res Trace Elements 16(4) : 302-305 2005
Results ZnGA, CuGA and ZnGA+CUGA, CL intensity due to Topical application ROS generation under UVA irradiation was signifi- Metallogluconates were topically applied for 30 cantly reduced, but after 3 and 7 days the suppressive min, and CL due to ROS generation was measured and effects disappeared (Fig.2). quantified with a NightOWL. On topical application of CuGA, the CL intensity due to UVA-induced ROS Discussion generation in the skin of mice was significantly In our previous research L3] , we reported that on i. reduced, but topical application of ZnGA was un- p. and oral administration of ZnC12 and Zn(pic)2, the changed (Fig.1). CL intensity due to UVA-induced ROS generation in the skin of mice was significantly reduced. Further- 30 (a) 2o more, Zn(pic)2 COmplex exhibited a persistent suppres- (b) sion of UVA-induced ROS generation for longer-term .a S 15c ~ 20 periods rather than the ionic form (ZnC12) did. Zn has o ~o o o = ~: CL CL 10 been used as an anti-inflammatory or antioxidative "o "o ~ OE agent for many years L15] . In this research, we found - 10 O :, O that metallogluconates significantly reduced the CL ~
~~~ee the last administration. Metallogluconates are soluble ~,,o in water, therefore, it is suggested that the excretion ~:o o of metallogluconates is earlier than that of Zn(pic)2, ~ 20 indicating the importance of daily intake of ZnGA. "O At present, it is difficult to explain the reason why L~o CL orally administration Zn and Cu compounds reduce = CO ROS in the skin of mice under UVA exposure, where o many factors contribute in the experimental systems. Time (day) Zinc and copper has been known to induce the synthe- Fig. 2 Suppressive effect of oral metallogluconates sis of metallothioneine (MT), which is a cystein-rich- and GA supprementations for 14 days in the protein, as a Zn-dependent antioxidant in several skin of HR-1 hairless mice under UVA irradia- tion (n=3-5) organs involving the skin of animals [19]. We try to IZnGA, ACuGA, <~ZnGA+CuGA, ･ GA Sig- clarify the mechanism for the antioxidative properties of nificance : *p<0.05 vs control Zn and Cu including the induction of MT in the skin. ROS scavenging effect of metallogluconate in the skin 305
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