Pigmentation in intrinsically aged skin of A1 guinea pigs

It is known that skin often shows irregular pigmentation during aging which is frequently associated with hyperpigmentation. Many studies have utilized brownish A1 guinea pigs to investigate the pathogenesis of ultraviolet (UV)-induced skin pigmentation, however, changes associated with intrinsic aging in A1 guinea pig skin have not been documented. To characterize such changes, skin from the dorsal and neck areas of 20-week, 1-, 2-, 3- and 5-yr-old guinea pigs was examined. Skin color was measured using a colorimeter, and biopsy specimens were stained with Masson-Fontana, L-3,4-dihydroxyphenylalanine (DOPA), and antibodies against KIT (ACK-45), gp100 (HMB-45) and S-100 proteins. The L* value of skin color decreased with aging and melanin deposits increased in the epidermis. Further, DOPA+, gp100+ and S-100+ melanocytes increased, indicating that the number of melanocytes had increased with age, whereas KIT+ melanocytes did not increase in dorsal skin and actually decreased in neck skin with aging. Further, rippled pigmented areas appeared in the neck skin of the 3-yr-old animals, and in the dorsal and neck skin of 5-yr-old guinea pigs in the absence of UV irradiation. Melanocytes were distributed uniformly in younger skin, whereas they were clustered in older skin. UV irradiation caused an increase in the number of melanocytes, although they were not clustered. These results are the first to provide evidence that pigmentation is induced in the skin of intrinsically aged A1 guinea pigs in the absence of UV irradiation, a process that differs from that elicited by UV irradiation.     

Involvement of Nitric Oxide in UVB‐Induced Pigmentation in Guinea Pig Skin

Ultraviolet (UV) B irradiation evokes erythema and delayed pigmentation in skin, where a variety of toxic and modulating events are known to be involved. Nitric oxide (NO) is generated from l ‐arginine by NO synthases (NOS). Production of NO is enhanced in response to UVB‐stimulation and has an important role in the development of erythema. NO has recently been demonstrated as a melanogen which stimulates melanocytes in vitro, however, no known in vivo data has been reported to support this finding. In this study, we investigated the contribution of NO with UV‐induced pigmentation in an animal model using an NOS inhibitor. UVB‐induced erythema in guinea pig skin was reduced when an NOS inhibitor, l ‐NAME (N‐nitro‐ l ‐arginine methylester hydrochloride), was topically applied to the skin daily, beginning 3 days before UVB‐irradiation. Delayed pigmentation and an increased number of DOPA‐positive melanocytes in the skin were markedly suppressed by sequential daily treatment with l ‐NAME. Furthermore, melanin content 13 days after UVB‐irradiation was significantly lower in skin treated with l ‐NAME than in the controls. In contrast, d ‐NAME (N‐nitro‐ d ‐arginine methylester hydrochloride), an ineffective isomer of l ‐NAME, demonstrated no effect on these UV‐induced skin responses. These results suggest that NO production may contribute to the regulation of UVB‐induced pigmentation.     

Hairless pigmented guinea pigs: a new model for the study of mammalian pigmentation

A stock of hairless pigmented guinea pigs was developed to facilitate studies of mammalian pigmentation. This stock combines the convenience of a hairless animal with a pigmentary system that is similar to human skin. In both human and guinea pig skin, active melanocytes are located in the basal layer of the interfollicular epidermis. Hairless albino guinea pigs on an outbred Hartley background (CrI:IAF/HA(hr/hr)BR; designated hr/hr) were mated with red-haired guinea pigs (designated Hr/Hr). Red-haired heterozygotes from the F1 generation (Hr/hr) were then mated with each other or with hairless albino guinea pigs. The F2 generation included hairless pigmented guinea pigs that retained their interfollicular epidermal melanocytes and whose skin was red-brown in color. Following UV irradiation, there was an increase in cutaneous pigmentation as well as an increase in the number of active epidermal melanocytes. An additional strain of black hairless guinea pigs was developed using black Hr/Hr animals and a similar breeding scheme. These two strains should serve as useful models for studies of the mammalian pigment system.     

Protective effects of topical alpha-tocopherol acetate on UVB irradiation in guinea pigs: importance of free radicals

Reactive oxygen species can be generated by daily exposure of the skin to ultraviolet light and may cause some subchronic and chronic skin disorders. The aim of this study was to investigate a possible preventive role of alpha-tocopherol acetate (ATA) on ultraviolet B (UVB) induced peroxidation by assessing lipid peroxide (LPO) levels and activity of reactive oxygen scavenging enzymes including glutathione peroxidase and superoxide dismutase (SOD) in guinea pigs. ATA was topically applied to the skin for three weeks before a single dose of 0.9 J/cm2 UVB irradiation on the skin and lipid peroxide levels and antioxidants in plasma, skin and liver and erythrocytes were determined after decapitation. Topical application of ATA prevented the UVB irradiation-induced reduction of scavenging enzyme activities in skin and erythrocytes. In conclusion, we suggest that topical applications of ATA before UVB irradiation is effective in protecting the skin from unwanted effects of UVB irradiation.     

Protective effects of amentoflavone on Lamin A-dependent UVB-induced nuclear aberration in normal human fibroblasts

Lamin A-dependent nuclear aberration and DNA damage was found in premature aging disease or normally old individuals. In this study, UVB irradiation was used as a cellular senescence inducer, and it was found that Lamin A and phospho-H2AX protein was increased by UVB treatment on normal human fibroblast. Lamin A-dependent morphological nuclear defect was observed in UVB treated fibroblast. Amentoflavone, a well known biflavonoid, inhibited the increase of Lamin A or phospho-H2AX protein in dose dependent manner which was induced by UVB irradiation, and also protected nuclear aberration dramatically. These results indicated that amentoflavone is an anti-aging candidate for UVB related skin aging process.     

Lightening Effect on Ultraviolet‐Induced Pigmentation of Guinea Pig Skin by Oral Administration of a Proanthocyanidin‐Rich Extract from Grape Seeds

Antioxidants such as vitamins C and E have been reported to inhibit the progression of ultraviolet (UV) radiation‐induced pigmentation in the skin of hairless mice. However, little is known of the lightening effect of proanthocyanidin, a powerful polyphenolic antioxidant, on UV‐induced pigmentation of the skin. We investigated the lightening effect of oral administration of a proanthocyanidin‐rich grape seed extract (GSE) using guinea pigs with UV‐induced pigmentation. These pigmented guinea pigs were fed diets containing 1% GSE or 1% vitamin C (w/w) for 8 weeks. GSE‐feeding had an apparent lightening effect on the guinea pigs’ pigmented skin. Histologic evaluation demonstrated a decrease in the number of 3,4‐dihydroxyphenylalanine (DOPA)‐positive melanocytes as well as 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG)‐positive, Ki‐67‐positive, proliferating cell nuclear antigen (PCNA)‐positive melanin‐containing cells in the basal epidermal layer of the UV‐irradiated skin in GSE‐fed guinea pigs. In contrast, these parameters did not change in the skin of vitamin C‐fed or control guinea pigs. GSE inhibited the activity of mushroom tyrosinase and also inhibited melanogenesis without inhibiting the growth of cultured B16 mouse melanoma cells. In conclusion, we demonstrated that oral administration of GSE is effective in lightening the UV‐induced pigmentation of guinea pig skin. This effect may be related to the inhibition of melanin synthesis by tyrosinase in melanocytes and the reactive oxygen species (ROS)‐related proliferation of melanocytes.     

Lightening effect on ultraviolet-induced pigmentation of guinea pig skin by oral administration of a proanthocyanidin-rich extract from grape seeds

Antioxidants such as vitamins C and E have been reported to inhibit the progression of ultraviolet (UV) radiation-induced pigmentation in the skin of hairless mice. However, little is known of the lightening effect of proanthocyanidin, a powerful polyphenolic antioxidant, on UV-induced pigmentation of the skin. We investigated the lightening effect of oral administration of a proanthocyanidin-rich grape seed extract (GSE) using guinea pigs with UV-induced pigmentation. These pigmented guinea pigs were fed diets containing 1% GSE or 1% vitamin C (w/w) for 8 weeks. GSE-feeding had an apparent lightening effect on the guinea pigs' pigmented skin. Histologic evaluation demonstrated a decrease in the number of 3,4-dihydroxyphenylalanine (DOPA)-positive melanocytes as well as 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive, Ki-67-positive, proliferating cell nuclear antigen (PCNA)-positive melanin-containing cells in the basal epidermal layer of the UV-irradiated skin in GSE-fed guinea pigs. In contrast, these parameters did not change in the skin of vitamin C-fed or control guinea pigs. GSE inhibited the activity of mushroom tyrosinase and also inhibited melanogenesis without inhibiting the growth of cultured B16 mouse melanoma cells. In conclusion, we demonstrated that oral administration of GSE is effective in lightening the UV-induced pigmentation of guinea pig skin. This effect may be related to the inhibition of melanin synthesis by tyrosinase in melanocytes and the reactive oxygen species (ROS)-related proliferation of melanocytes.     

Antiradiation effects of leucynferon on dogs and guinea pigs

In experiments with two species of animals (dogs, guinea pigs) irradiated with sublethal and lethal doses of gamma-rays, it was observed, that leucynferon had antiradiation effect. Course of injections: dogs--8 injections subcutaneus: 2.0 ml (1, 3, 5, 8, 12, 14, 21, 34 days after irradiation); guinea pigs--14 injections subcutaneus, 0.2 ml (1-14 days after irradiation). Therapeutical effect was explained by capacity of the preparation to defend the hemopoietic organs from the radiation and to stimulate hemopoiesis. Leucynferon hindered the development of acute radiation sickness symptoms. Immunoreactivity of dogs and guinea pigs in experimental group was more complete and restored faster. The growth of the automicroflora on the skin was restrained. Production of interferon-gamma (which is a function of T-lymphocytes) was restored faster.     

Evaluating the Clinical and Physiological Effects of Long Term Ultraviolet B Radiation on Guinea Pigs (Cavia porcellus)

Vitamin D is an important hormone in vertebrates. Most animals acquire this hormone through their diet, secondary to exposure to ultraviolet B (UVB) radiation, or a combination thereof. The objectives for this research were to evaluate the clinical and physiologic effects of artificial UVB light supplementation on guinea pigs (Cavia porcellus) and to evaluate the long-term safety of artificial UVB light supplementation over the course of six months. Twelve juvenile acromelanic Hartley guinea pigs were randomly assigned to one of two treatment groups: Group A was exposed to 12 hours of artificial UVB radiation daily and Group B received only ambient fluorescent light for 12 hours daily. Animals in both groups were offered the same diet and housed under the same conditions. Blood samples were collected every three weeks to measure blood chemistry values, parathyroid hormone, ionized calcium, and serum 25-hydroxyvitamin D₃ (25-OHD₃) levels. Serial ophthalmologic examinations, computed tomography scans, and dual energy x-ray absorptiometry scans were performed during the course of the study. At the end of the study the animals were euthanized and necropsied. Mean ± SD serum 25-OHD₃ concentrations differed significantly in the guinea pigs (p<0.0001) between the UVB supplementation group (101.49±21.81 nmol/L) and the control group (36.33±24.42 nmol/L). An increased corneal thickness in both eyes was also found in the UVB supplementation compared to the control group (right eye [OD]: p<0.0001; left eye [OS]: p<0.0001). There were no apparent negative clinical or pathologic side effects noted between the groups. This study found that exposing guinea pigs to UVB radiation long term significantly increased their circulating serum 25-OHD₃ levels, and that this increase was sustainable over time. Providing guinea pigs exposure to UVB may be an important husbandry consideration that is not currently recommended.     

Comparison of apoptosis and terminal differentiation: the mammalian aging process.

Apoptosis is the ordered chain of events that lead to cell destruction. Terminal differentiation (denucleation) is the process in which cells lose their nuclei but remain functional. Our group examined cell death in three tissues using two different fixatives and a postfixation procedure, involving young (5 months) and old (2 years) guinea pigs. The data reveal that B-DNA and Z-DNA content decreases, whereas single-stranded (ss-) DNA increases, in older tissues undergoing apoptosis (skin and cornea) and terminal differentiation (ocular lens). We speculate that some of the factors that contribute to the aging process might also be responsible for the enhanced amount of damaged DNA in older tissues undergoing cell death. (J Histochem 49:929-930, 2001)     

白藜芦醇对紫外线照射后人角质形成细胞AQP3 表达的影响

目的:探讨白藜芦醇对紫外线照射后人皮肤角质形成细胞水通道蛋白3(AQP3)表达的影响及意义。方法:原代培养人皮肤角质形成细胞,采用UVB(20mJ/cm2,40mJ/cm2)照射角质形成细胞后,立即加入0.1mmol/L的白藜芦醇进行干预。RT-PCR检测照射前后角质形成细胞中AQP3 mRNA的表达量,并用羟胺法、比色法、TBA法检测照射前后细胞超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)含量。结果:1.UVB照射后角质形成细胞AQP3 mRNA的表达量下降(P<0.05),且UVB照射剂量越大,AQP3 mRNA下降越显著(P<0.05)。2.白藜芦醇能显著增加UVB照射后角质形成细胞SOD和GSH-Px活性,并降低细胞MDA含量(P<0.05)。3.白藜芦醇能显著抑制UVB导致的角质形成细胞AQP3 mRNA下降(P<0.05)。结论:白藜芦醇可能通过抑制UVB导致的AQP3 mRNA下降,及提高氧化酶活性、清除自由基的功能,从而延缓皮肤衰老。     

Effects of a turmeric extract (Curcuma longa) on chronic ultraviolet B irradiation-induced skin damage in melanin-possessing hairless mice

Turmeric (the rhizomes of Curcuma longa L., Zingiberacease) is widely used as a dietary pigment and spice, and has been traditionally used for the treatment of inflammation, skin wounds and hepatic disorders in Ayurvedic, Unani and Chinese medicine. Although the topical application or oral administration of turmeric is used to improve skin trouble, there is no evidence to support this effect. The aim of this study was to clarify whether turmeric prevents chronic ultraviolet B (UVB)-irradiated skin damage. We examined the effects of a turmeric extract on skin damage including changes in skin thickness and elasticity, pigmentation and wrinkling caused by long-term, low-dose ultraviolet B irradiation in melanin-possessing hairless mice. The extract (at 300 or 1000 mg/kg, twice daily) prevented an increase in skin thickness and a reduction in skin elasticity induced by chronic UVB exposure. It also prevented the formation of wrinkles and melanin (at 1000 mg/kg, twice daily) as well as increases in the diameter and length of skin blood vessels and in the expression of matrix metalloproteinase-2 (MMP-2). Prevention of UVB-induced skin aging by turmeric may be due to the inhibition of increases in MMP-2 expression caused by chronic irradiation.     

Age-dependent changes in particulate and soluble guanylyl cyclase activities in urinary tract smooth muscle

Regional and age specific differences are observed in the sodium nitroprusside induced relaxation responses in the urinary tract. To clarify these differences, guanylyl cyclase activity is assayed in particulate and soluble fractions from the ureter, bladder dome, and urethra of young (11-18 days), adult (90-100 days), and old adult (2-3 years) guinea pigs. The rank order of soluble guanylyl cyclase activities is urethra = ureter > bladder dome with the largest decreases with aging occurring in the bladder. Atrial natriuretic factor (10-7 M) increases particulate guanylyl cyclase activity in the three tissues at all ages tested, with the activity being highest in the ureter. ATP (0.5 mM) activates particulate guanylyl cyclase in the ureter, bladder and urethra of old adult guinea pigs, and enhances atrial natriuretic factor induced activation of particulate guanylyl cyclase in all tissues and at all ages tested. The higher levels of soluble guanylyl cyclase activity in the urethra and ureter compared to the bladder parallel sodium nitroprusside induced relaxation in these tissues.     

Distinct melanogenic response of human melanocytes in mono-culture, in co-culture with keratinocytes and in reconstructed epidermis, to UV exposure

Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co-cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm2), an increase in melanin synthesis whereas in melanocyte mono-cultures, UVB doses up to 50 mJ/cm2 had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono- and co-cultures. Removing certain keratinocyte growth factors from the co-culture medium abolished the melanogenic response to UVB, but not to UVA exposure. When integrated into the basal layer of a reconstructed human epidermis, human melanocytes similarly reacted to UVA and UVB irradiation as in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. The presence of a dense stratum corneum, known to scatter and absorb UV light, is responsible for higher minimal UVB and UVA doses required to trigger a melanogenic response in the reconstructed epidermis compared to keratinocyte-melanocyte co-cultures. Furthermore, an immediate tanning response was observed in the pigmented epidermis following UVA irradiation. From these results we conclude that: (i) keratinocytes play an important role in mediating UVB-induced pigmentation, (ii) UVA-induced pigmentation is the result of a rather direct effect on melanocytes and (iii) reconstructed pigmented epidermis is the most appropriate model to study UV-induced pigmentation in vitro.     

Photo-Oxidation Products of Skin Surface Squalene Mediate Metabolic and Inflammatory Responses to Solar UV in Human Keratinocytes

The study aimed to identify endogenous lipid mediators of metabolic and inflammatory responses of human keratinocytes to solar UV irradiation. Physiologically relevant doses of solar simulated UVA+UVB were applied to human skin surface lipids (SSL) or to primary cultures of normal human epidermal keratinocytes (NHEK). The decay of photo-sensitive lipid-soluble components, alpha-tocopherol, squalene (Sq), and cholesterol in SSL was analysed and products of squalene photo-oxidation (SqPx) were quantitatively isolated from irradiated SSL. When administered directly to NHEK, low-dose solar UVA+UVB induced time-dependent inflammatory and metabolic responses. To mimic UVA+UVB action, NHEK were exposed to intact or photo-oxidised SSL, Sq or SqPx, 4-hydroxy-2-nonenal (4-HNE), and the product of tryptophan photo-oxidation 6-formylindolo[3,2-b]carbazole (FICZ). FICZ activated exclusively metabolic responses characteristic for UV, i.e. the aryl hydrocarbon receptor (AhR) machinery and downstream CYP1A1/CYP1B1 gene expression, while 4-HNE slightly stimulated inflammatory UV markers IL-6, COX-2, and iNOS genes. On contrast, SqPx induced the majority of metabolic and inflammatory responses characteristic for UVA+UVB, acting via AhR, EGFR, and G-protein-coupled arachidonic acid receptor (G2A). CONCLUSIONS/SIGNIFICANCE: Our findings indicate that Sq could be a primary sensor of solar UV irradiation in human SSL, and products of its photo-oxidation mediate/induce metabolic and inflammatory responses of keratinocytes to UVA+UVB, which could be relevant for skin inflammation in the sun-exposed oily skin.     

Distinct Melanogenic Response of Human Melanocytes in Mono‐culture,in Co‐Culture with Keratinocytes and in Reconstructed Epidermis,to UV Exposure

Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co‐cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm²), an increase in melanin synthesis whereas in melanocyte mono‐cultures, UVB doses up to 50 mJ/cm² had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono‐ and co‐cultures. Removing certain keratinocyte growth factors from the co‐culture medium abolished the melanogenic response to UVB, but not to UVA exposure. When integrated into the basal layer of a reconstructed human epidermis, human melanocytes similarly reacted to UVA and UVB irradiation as in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. The presence of a dense stratum corneum, known to scatter and absorb UV light, is responsible for higher minimal UVB and UVA doses required to trigger a melanogenic response in the reconstructed epidermis compared to keratinocyte–melanocyte co‐cultures. Furthermore, an immediate tanning response was observed in the pigmented epidermis following UVA irradiation. From these results we conclude that: (i) keratinocytes play an important role in mediating UVB‐induced pigmentation, (ii) UVA‐induced pigmentation is the result of a rather direct effect on melanocytes and (iii) reconstructed pigmented epidermis is the most appropriate model to study UV‐induced pigmentation in vitro.     

Depigmenting Action of Phenylhydroquinone,an o‐Phenylphenol Metabolite,on the Skin of JY‐4 Black Guinea‐Pigs

The effects of o‐phenylphenol (OPP) and its metabolite, phenylhydroquinone (PHQ) on the skin of JY‐4 black guinea‐pigs were studied. Topical application of 1 or 5% PHQ on the black skin of the back caused marked depigmentation and hypopigmentation of the skin after 5 weeks, whereas OPP applied at the same concentrations had little effect. Depigmented skin had an increased L* (lightness) value in the CIE‐L*a*b* color system. This corresponded with a decreased number of melanocytes and melanosomes in the melanocytes and keratinocytes, the disruption of melanosomes in the melanocytes, and destruction of the membranous organelles of the melanocytes. These morphological and numerical changes in epidermal melanocytes indicate that selective melanocyte toxicity occurred. Furthermore, application of PHQ to the skin of white guinea‐pigs caused skin irritation, as shown by a colorimetric increase in a* value (redness) and by histological observation of inflammation. This study confirmed that OPP, which is a reported depigmenter, has little depigmenting action, while its metabolite, PHQ, is a potent depigmenter preferentially affecting melanocytes.     

Effects of marrow donor and recipient age on immune responses

This report describes treatments to restore diminished splenic immune responses of old mice. Lethal irradiation, followed by young bone marrow and infant thymus transplants, restored the T cell mitogen response and the antibody-forming cell response against sheep red blood cells in the old mice. Although old bone marrow cells restore these immune responses in young recipients, as well as do young bone marrow cells, old bone marrow in old recipients did not improve their levels of response. Longevities of old recipients with rejuvenated responses were not increased, and aging of tail tendon collagen was not affected. The effect of lethal irradiation before the marrow transplant was shown to be minimal, by the use of unirradiated old W-anemic recipients. Parabiosing young mice with old partners caused impairment of these two immune responses in the young partners without enhancing them in the old partners. The old partners did not have increased longevities. To explain these results, we suggest the following hypothesis: old bone marrow contains precursors that produce suppressive factors or cells when in an old environment but not when in a young environment. However, these factors, if allowed to develop in an old environment, can function in a young parabiosed partner.