- Email: [email protected]
Oxidation of protein and reduction of catalase activity in mouse skin homogenate by UVB irradiation
Symposium IV: Photobiology Photoimmunology Masahlro Takigawa Dep.Dermatology.
-Where
are we now?EPIDERMIS
Hamamatsu
Univ. Sch. Med., Hamamatsu.
Japan.
Photolmmunology is the study of the effects of light on the immune system. The skin is equipped with immunoconpetent cells such as Langerhans cells. lymphocytes. and draining lymph nodes. and a site In which an immunologic reaction first occurs following the invasion of an antigen. Exposure of the skin to solar radiation alters the cutaneous reactivity to various stimuli by affecting functions of the circulating and non-circulating components of the immune system. The altered reactlcn may be in the form of an accentuation of the normal responses or development of an abnormal response. Solar urtlcaria and photoallergic eczema are the example in which immune mechanisms Immunosuppression seems to be participate in the development, associated with an increased risk of skin cancer on the sun-exposed sites. Whether it is given locally or systenlcally. UV-B affects certain elements of the immune system. and perturbation of antigen presentation by UV-B leads to antigen-specific unresponsiveness that is associated with the generation of suppressorT cells. Although the significance of these findings in the pathogenesis of photorelated diseases i's ill-defined, further experfments in both animals and man vlll reveal the way in which we successfully deal with these diseases. On the other hand. the beneficial effect of UV radiation 1s demonstrated in the treatment of various skin diseases. Psoriasis and early mycosis fungoides aretreated with psoralen plus UV-A or Once again. it is also possible that some of the adverse uv-B alone. effects fallow such treatments because of an Interaction between rad~atian and the immune system.
ACCUMULATION OF CELLS BY REPEATED PUVA
S. Kawara and T. Hirone Department of Dermatology, Kanazawa, Japan
DIMERS
IN THE GENE
Y. Fu~lwara' and Y. Mlsh1m.a M. Ogosh~. M. Ichihash,. A. Matsumoto'. Department of Dermatology. and *Department of Rad,at,on Blophys,cs. Kobe Umvers~ty School of Medic,ne. Kobe. Japan Cultured slrln f~broblasts der,ved from pat,ents v~th Cockayne's syndrome (CS) have been shown to present a high sens,t,v,ty to the lethal effect of 254 nm UV. despite apparently normal removal of cyclobutane pyrim,d,ne dimers from the whole genom,c DNA, and a defective recovery of post-UV DNA and RNA synthesis. The latter may be a molecular bas,s of UV hypersens,tiv,ty of CS cells, although the precise molecular mechanism IS as yet unknown. In this study. we have been attempt,ng to eluc,date the ex~l~lon reParr of T4 endonuclease V-susceptible sates (T4 endo Sites: cyclobutane d~mers) ?n the act.,ve d,hydrofolate reductase (dhfr) gene of normal human and camplementatlon group A CS cells. Fibroblastl grown to confluence and further ,n low serum were exposed to 20 J/m 254 nm UV and High-molecular-we,ght DNA was extracted and incubated for 6 and 24 hr. restncted to completion ulth3ylnd III. wh,ch gave a 23 kb dhfr fragment after hybrid,rat,on w,th the P-labeled 1.8 kb dhfr DNA probe. Restncted DNA was treated with the T4 enzyme or not, electrophresed 1" d denatunng gel and subJected to Saurthern blot hybnd,rat,on. Removal of d~mers in the 23 kb dhfr fragment was assessed by dens,tometry. Results showed that normal human cells exc,sed approx,mately 60% as T4 endo Sites from the dhfr gene in 24 hr after Irrad,at,on 20 J/m but CSZDS group A cells d,d not. The CS cells showed a greater decrease I" post-UV colon,es than d,d normal cells. Those results suggest that CS may be defect~ue ln the exl~l~lon repa,r ,n only act,ve gene.
,
Kanazawa
University
AND
School
PROLIFERATING
of Medicine,
Effects of repeated PUVA on kinetic parameters of the normal and nhexadecane(n-HD)-induced proliferating epidermis of guinea pig were studied. Skin biopsies were obtained at various times following three treatments with topical PUVA (0.5% B-MOP + 1, 2 J/cm* of UV-A). In the epidermis treated with PUVA alone, the relative numbers of cells in 5 and G2+M phase showed respectively moderate and marked increase of 120hr duration than those of the non-treated nonal epidermis (controls). The labeling index showed an increase in varying degrees until 120 hr. The mitotic index was below or near the control level until 96 hr, and there In the epidermis after slightly increased than that of the controls. treated with n-HD and PUVA, the relative number of cells in 5 phase showed an increase in varying degrees until 120 hr than that of the epidermis treated with n-HO alone (controls). The relative number of cells in GZ+M phase showed a moderate increase until 120 hr. with the maximam at 48 hr. The labeling index showed a bimodal increase with low peaks at 12 and 72 hr. The mitotic index showed d markedly decrease until 72 hr, and thereafter approached control level. The results indicate that repeated PUVA suppresses the mitosis resulting 1" a subsequent accumulation of cells in G2 phase.
OXIDATION IN MOUSE SKIN REPAIR OF ULTRAVIOLT(UV)-INDUCED PYRIMIDINE OF NORMAL AND COCKAYNE'S SYNDROME FIBROBLASTS
IN 62 PHASE IN NORMAL
OF PROTEIN AND REDUCTION OF CATALASE HOMOGENATE BY UVB IRRADIATION
Seung Churl Lee and Young Pio Km Department of Dermatology, Chonnam University Korea
MedIcal
School.
ACTIVITY
Kwangju.
It 1s well known that ultraviolet light accelerates the aging process 1” the skin. Since the oxldative changes in tissue proteins have been demonstrated to take place I” the aging process. the present study was undertaken to fmd out whether UVB xradiatmn causes the oxidatxon of protans in mouse skin. The oxidation of protems was assayed by the determinatm” of protan carbonyl content with 2.4 - dlnltro phenylhvdrazlne. and catalase activzty in the skin was measured in relation to the protein oxldatio”. The carbonyl content !n the control group was 3.5 * 0.17 nmoles/mg protein (Mean 2 SD). When the tissue homogenate prepared from mouse s!an was lrradmted with UVB for I hr. 2 and 3 hrs (I mJ/cd,‘sec). the carbonyl contents increased to 6.4 f 0.75. 10.6 i 1.29, and 15.5? 2.54, respectively. The oxldatlo” of skin pr”tems was also wsualized by the reactlo” of protein carbonyl groups with fluorescent reagent (Fluorescein thlosemicarbazide) under anaerobic condition and then being subjected to lithium dodecyl sulfate polyacrylamlde gel electrophoresls. The cat&se actlvitles of the skin after UVB rrradlatlon were 1.0 t 0.16 LA A,,/mg protel”,‘mm) (Mean f SD). 0.6 t 0.02, and 0.3 2 0.15 I” each group from I to 3 hrs Irradiation. respectively. The actlvitles of cat&se I” the UVB lrradmted groups were decreased as the exposure time increased compared with that of control group (1.2 f 0.10). These results suggest that UVB irradiatmn enhances the oxldatm” of skin proteins. and that the acceleration of ski” agrng by UV light may be at least 1” part due to the increased oxidation of the skin “roteIns by UV light
THE INFLUENCE OF URSODEOXYCHOLIC ACID (GF)-INDUCED PROTOPORPHYRIA H. Irlfune. Department Nagasaki.
S. Nonaka. N. Tsukazaki. T. Ohgamx, of Dermatology, Nagasaki University Japan
(UDCA)
ON GRISEOFULVIN
H. Yoshlda School
of
Medicine,
“DCA, a chollc ac1d derivative I” the bile. facllltates absorption of GF from the gut, resulting in a” elevation of GF levels in the blood. In this study, we investigated whether or not UDCA assists in the induction of GF-Induced protoporphyria and porphyrx photosensitivxty. Materials and Methods: Forty dd-y strain male mice were used for this Study. The mice were divided into 4 roups treated vxth 0.5% GF, group B with 0.5% GF and 0.5 .? UDCA and group C with 0.51: “DCA. Group D was a control recaving no treatment. Liver tissue and blood were take” as samples for the analysis of porphyrins. The porphyrins in these samples were analyzed using the chromatographic method combined with the extraction method. Hisroparhological study of the liver was 11 -n n0rfnrmnll Results: Although erythrocytic protoporphyrin levels m roup 6 (0.5% GF and 0.5% UDCA) were higher than those in group A (0.5% GB1. there was no difference in hepatic and fecal porphyrin levels between both groups. On the other hand. there was no abnormality in the porphyrin metabolism Histopathologically, I” group c. small necrotic lesions were see” ln the liver of group B mice, but there were no abnormal fIndings in the other groups. Discussion: UDCA caused a different patter” of porphyrin metabolism when compared with the erythrocytic, hepatic and fecal protoporphyri” levels I” the mice treated with cholic acid. I” a previous study. we observed that cholic acid, which is one of the bile acids. suppressed a deposition of protoporphyrl” in the liver by large amounts of GF. resulting in a lover level of erythrocytic protoporphyri” end a hyperexcretion of fecal porphyrins. UDCA may act dzfferently an GFInduced protoporphyria.
: groupA was
“, ____r_.__...._
-Where
are we now?EPIDERMIS
Hamamatsu
Univ. Sch. Med., Hamamatsu.
Japan.
Photolmmunology is the study of the effects of light on the immune system. The skin is equipped with immunoconpetent cells such as Langerhans cells. lymphocytes. and draining lymph nodes. and a site In which an immunologic reaction first occurs following the invasion of an antigen. Exposure of the skin to solar radiation alters the cutaneous reactivity to various stimuli by affecting functions of the circulating and non-circulating components of the immune system. The altered reactlcn may be in the form of an accentuation of the normal responses or development of an abnormal response. Solar urtlcaria and photoallergic eczema are the example in which immune mechanisms Immunosuppression seems to be participate in the development, associated with an increased risk of skin cancer on the sun-exposed sites. Whether it is given locally or systenlcally. UV-B affects certain elements of the immune system. and perturbation of antigen presentation by UV-B leads to antigen-specific unresponsiveness that is associated with the generation of suppressorT cells. Although the significance of these findings in the pathogenesis of photorelated diseases i's ill-defined, further experfments in both animals and man vlll reveal the way in which we successfully deal with these diseases. On the other hand. the beneficial effect of UV radiation 1s demonstrated in the treatment of various skin diseases. Psoriasis and early mycosis fungoides aretreated with psoralen plus UV-A or Once again. it is also possible that some of the adverse uv-B alone. effects fallow such treatments because of an Interaction between rad~atian and the immune system.
ACCUMULATION OF CELLS BY REPEATED PUVA
S. Kawara and T. Hirone Department of Dermatology, Kanazawa, Japan
DIMERS
IN THE GENE
Y. Fu~lwara' and Y. Mlsh1m.a M. Ogosh~. M. Ichihash,. A. Matsumoto'. Department of Dermatology. and *Department of Rad,at,on Blophys,cs. Kobe Umvers~ty School of Medic,ne. Kobe. Japan Cultured slrln f~broblasts der,ved from pat,ents v~th Cockayne's syndrome (CS) have been shown to present a high sens,t,v,ty to the lethal effect of 254 nm UV. despite apparently normal removal of cyclobutane pyrim,d,ne dimers from the whole genom,c DNA, and a defective recovery of post-UV DNA and RNA synthesis. The latter may be a molecular bas,s of UV hypersens,tiv,ty of CS cells, although the precise molecular mechanism IS as yet unknown. In this study. we have been attempt,ng to eluc,date the ex~l~lon reParr of T4 endonuclease V-susceptible sates (T4 endo Sites: cyclobutane d~mers) ?n the act.,ve d,hydrofolate reductase (dhfr) gene of normal human and camplementatlon group A CS cells. Fibroblastl grown to confluence and further ,n low serum were exposed to 20 J/m 254 nm UV and High-molecular-we,ght DNA was extracted and incubated for 6 and 24 hr. restncted to completion ulth3ylnd III. wh,ch gave a 23 kb dhfr fragment after hybrid,rat,on w,th the P-labeled 1.8 kb dhfr DNA probe. Restncted DNA was treated with the T4 enzyme or not, electrophresed 1" d denatunng gel and subJected to Saurthern blot hybnd,rat,on. Removal of d~mers in the 23 kb dhfr fragment was assessed by dens,tometry. Results showed that normal human cells exc,sed approx,mately 60% as T4 endo Sites from the dhfr gene in 24 hr after Irrad,at,on 20 J/m but CSZDS group A cells d,d not. The CS cells showed a greater decrease I" post-UV colon,es than d,d normal cells. Those results suggest that CS may be defect~ue ln the exl~l~lon repa,r ,n only act,ve gene.
,
Kanazawa
University
AND
School
PROLIFERATING
of Medicine,
Effects of repeated PUVA on kinetic parameters of the normal and nhexadecane(n-HD)-induced proliferating epidermis of guinea pig were studied. Skin biopsies were obtained at various times following three treatments with topical PUVA (0.5% B-MOP + 1, 2 J/cm* of UV-A). In the epidermis treated with PUVA alone, the relative numbers of cells in 5 and G2+M phase showed respectively moderate and marked increase of 120hr duration than those of the non-treated nonal epidermis (controls). The labeling index showed an increase in varying degrees until 120 hr. The mitotic index was below or near the control level until 96 hr, and there In the epidermis after slightly increased than that of the controls. treated with n-HD and PUVA, the relative number of cells in 5 phase showed an increase in varying degrees until 120 hr than that of the epidermis treated with n-HO alone (controls). The relative number of cells in GZ+M phase showed a moderate increase until 120 hr. with the maximam at 48 hr. The labeling index showed a bimodal increase with low peaks at 12 and 72 hr. The mitotic index showed d markedly decrease until 72 hr, and thereafter approached control level. The results indicate that repeated PUVA suppresses the mitosis resulting 1" a subsequent accumulation of cells in G2 phase.
OXIDATION IN MOUSE SKIN REPAIR OF ULTRAVIOLT(UV)-INDUCED PYRIMIDINE OF NORMAL AND COCKAYNE'S SYNDROME FIBROBLASTS
IN 62 PHASE IN NORMAL
OF PROTEIN AND REDUCTION OF CATALASE HOMOGENATE BY UVB IRRADIATION
Seung Churl Lee and Young Pio Km Department of Dermatology, Chonnam University Korea
MedIcal
School.
ACTIVITY
Kwangju.
It 1s well known that ultraviolet light accelerates the aging process 1” the skin. Since the oxldative changes in tissue proteins have been demonstrated to take place I” the aging process. the present study was undertaken to fmd out whether UVB xradiatmn causes the oxidatxon of protans in mouse skin. The oxidation of protems was assayed by the determinatm” of protan carbonyl content with 2.4 - dlnltro phenylhvdrazlne. and catalase activzty in the skin was measured in relation to the protein oxldatio”. The carbonyl content !n the control group was 3.5 * 0.17 nmoles/mg protein (Mean 2 SD). When the tissue homogenate prepared from mouse s!an was lrradmted with UVB for I hr. 2 and 3 hrs (I mJ/cd,‘sec). the carbonyl contents increased to 6.4 f 0.75. 10.6 i 1.29, and 15.5? 2.54, respectively. The oxldatlo” of skin pr”tems was also wsualized by the reactlo” of protein carbonyl groups with fluorescent reagent (Fluorescein thlosemicarbazide) under anaerobic condition and then being subjected to lithium dodecyl sulfate polyacrylamlde gel electrophoresls. The cat&se actlvitles of the skin after UVB rrradlatlon were 1.0 t 0.16 LA A,,/mg protel”,‘mm) (Mean f SD). 0.6 t 0.02, and 0.3 2 0.15 I” each group from I to 3 hrs Irradiation. respectively. The actlvitles of cat&se I” the UVB lrradmted groups were decreased as the exposure time increased compared with that of control group (1.2 f 0.10). These results suggest that UVB irradiatmn enhances the oxldatm” of skin proteins. and that the acceleration of ski” agrng by UV light may be at least 1” part due to the increased oxidation of the skin “roteIns by UV light
THE INFLUENCE OF URSODEOXYCHOLIC ACID (GF)-INDUCED PROTOPORPHYRIA H. Irlfune. Department Nagasaki.
S. Nonaka. N. Tsukazaki. T. Ohgamx, of Dermatology, Nagasaki University Japan
(UDCA)
ON GRISEOFULVIN
H. Yoshlda School
of
Medicine,
“DCA, a chollc ac1d derivative I” the bile. facllltates absorption of GF from the gut, resulting in a” elevation of GF levels in the blood. In this study, we investigated whether or not UDCA assists in the induction of GF-Induced protoporphyria and porphyrx photosensitivxty. Materials and Methods: Forty dd-y strain male mice were used for this Study. The mice were divided into 4 roups treated vxth 0.5% GF, group B with 0.5% GF and 0.5 .? UDCA and group C with 0.51: “DCA. Group D was a control recaving no treatment. Liver tissue and blood were take” as samples for the analysis of porphyrins. The porphyrins in these samples were analyzed using the chromatographic method combined with the extraction method. Hisroparhological study of the liver was 11 -n n0rfnrmnll Results: Although erythrocytic protoporphyrin levels m roup 6 (0.5% GF and 0.5% UDCA) were higher than those in group A (0.5% GB1. there was no difference in hepatic and fecal porphyrin levels between both groups. On the other hand. there was no abnormality in the porphyrin metabolism Histopathologically, I” group c. small necrotic lesions were see” ln the liver of group B mice, but there were no abnormal fIndings in the other groups. Discussion: UDCA caused a different patter” of porphyrin metabolism when compared with the erythrocytic, hepatic and fecal protoporphyri” levels I” the mice treated with cholic acid. I” a previous study. we observed that cholic acid, which is one of the bile acids. suppressed a deposition of protoporphyrl” in the liver by large amounts of GF. resulting in a lover level of erythrocytic protoporphyri” end a hyperexcretion of fecal porphyrins. UDCA may act dzfferently an GFInduced protoporphyria.
: groupA was
“, ____r_.__...._