HIV +/AIDS RELATED BIOETHICAL ISSUES IN JAPAN

Annual and cumulative incidences of HIV + and AIDS in patients reported by the AIDS Surveillance Committee of the Ministry of Health and Welfare are cited to illustrate some characteristics in Japan: nearly 59% of either HIV + or AIDS patients were infected through injection of blood products or by blood transfusion. A number of plaintiffs have sued the Japanese government and pharmaceutical companies since 1989, but no judicial decisions have yet been made. The incidence of HIV decreases for each of the following routes of infection: the second highest route of transmission being heterosexual, followed by bisexual and unconfirmed or unknown cases, and less frequently homosexual, drug use and lastly via vertical transmission. The most serious bioethical problems with HIV + and AIDs patients in Japan is the social segregation of these patients. There are many emotional fears among the general public and medical personnel about AIDS because of their inadequate scientific knowledge of this disease. The Japan Hospital Association is doing The Stop AIDS Campaign and made several surveys. Japan has become increasingly aware of the importance of AIDS education and a great deal of effort is being made to enhance bioethical considerations.     

Spectrophotometric Characterization of Eumelanin and Pheomelanin in Hair

Mammalian melanins exist in two chemically distinct forms: the brown to black eumelanins and the yellow to reddish-brown pheomelanins. They can be quantified by HPLC analysis of pyrrole-2,3,5-tricarboxylic acid (PTCA) and aminohydroxyphenylalanine (AHP). We recently developed a spectrophotometric method for assaying the total amount of eu- and pheomelanins by dissolving melanins in Soluene-350 plus water. In this study, we examined whether absorbance at 500 nm (A₅₀₀) of the Soluene-350 solution reflects the total amount of melanins obtained by the HPLC methods, and whether the ratio of absorbances between 650 and 500 nm reflects the eumelanin/total melanin ratio in mouse hair, sheep wool, and human hair. Our findings were as follows: (1) Total melanin levels calculated from A₅₀₀ values correlate well with those obtained from PTCA and AHP values by multiplying with the following factors: for mice, PTCA × 45 + AHP × 2.5; for sheep, PTCA × 40 + AHP × 15; and for humans, PTCA × 160 + AHP × 10. (2) The A₆₅₀/A₅₀₀ ratios were higher (0.25–0.33) in black to brown hair while they were significantly lower (0.10–0.14) in yellow to red hair. These results indicate that (1) the A₅₀₀ value can be used to quantify the total combined amount of eu- and pheomelanins, and (2) the A₆₅₀/A₅₀₀ ratio can serve as a parameter to estimate the eumelanin/total melanin ratio. The present method provides a convenient way to qualitatively characterize eu- and pheomelanins in melanins produced in follicular melanocytes.     

Antimelanoma Activity of Chloroquine,an Antimalarial Agent With High Affinity for Melanin

The antimalarial agent chloroquine is known for high affinity for melanin. This 4-aminoquinoline derivative was examined for anti-melanoma activity and uptake into melanoma cells. Chloroquine inhibited growth of cultured melanoma cells; the effect was much greater to a moderately pigmented cell line HMV-II than to a nonpigmented HMV-I. Treatment with chloroquine at a dose of 62 mg/kg i.p. for 12 days prolonged by 71% the life span of mice bearing B16 melanoma, while 24-day treatment at 31 mg/kg resulted in a 81% increase in life span. HMV-II cells showed a two-fold increase in up-take of chloroquine as compared with HMV-I cells. Chloroquine, 24 hr after administration to mice implanted s.c. with B16 melanoma, was selectively accumulated in the pigmented tissues, melanoma and eyes. Other nonpigmented tissues such as the liver, lung, and kidney showed rapid uptake (within 1 hr) and release. These results suggest that chloroquine is toxic to pigmented melanoma cells, the process being partly mediated by binding to melanin     

Chemical Analysis of Melanin Pigments in Feather Germs of Japanese Quail Bh (Black at Hatch) Mutants

Bh (black at hatch) is a mutation of Japanese quails which causes darkening or lightening of the plumage in heterozygotes or homozygotes, respectively. We chemically analyzed melanin pigments in feather germs of Bh mutant embryos and in feathers of adult animals. Dark brown dorsal feathers of wild-type adult animals had white barrings, but heterozygous ones lacked clear barrings. The feathers of wild-type and heterozygote animals contained both eumelanins and pheomelanins, the latter being more pheomelanic. On the dorsal skin of 10-day old wild-type embryos, longitudinal stripes from black and yellow rows of feather germs developed; two or three longitudinal rows of black feather germs and then two or three rows of yellow feather germs next to the short central feather germs. Heterozygous embryos appeared black in plumage pigmentation, due to the presence of‘gray’feather germs in rows of dorsal feather germs that corresponded to yellow rows in wild-type embryos. Homozygous dorsal feather germs did not develop the black and yellow longitudinal stripes, but were brown. Chemical analysis showed that embryos of each genotype contained both eumelanins and pheomelanins in the feather germs; however, the eumelanin content in ho-mozygous feather germs was very low. These results suggest that the Bh mutation causes pheomelanic changes in feathers of quails.     

Eumelanin and Phaeomelanin Contents of Human Epidermis and Cultured Melanocytes

There are two chemically distinct types of melanin: the red-yellow phaeomelanins and the brown-black eumelanins. While both melanins have been detected in human epidermis and cultured melanocytes, it is unknown how the phaeomelanin/eumelanin ratio in human melanocytes maintained in vitro relates to that in the epidermis from which they were isolated. This study uses high-performance liquid chromatography to quantify the eumelanin and phaeomelanin contents of epidermis and/or cultured melanocytes from 12 Europeans with lightly pigmented skin and 9 non-Europeans with more deeply pigmented skin. Epidermis from non-Europeans contained the highest levels of both eumelanin and phaeomelanin and had the lowest phaeomelanin/eumelanin ratios. In contrast, while cultured melanocytes from non-Europeans also had higher levels of eumelanin and phaeomelanin than melanocytes from Europeans, there was no difference in the phaeomelanin/eumelanin ratios in the two groups. However, the phaeomelanin/eumelanin ratios were higher in the cultured melanocytes than in the corresponding epidermis so that while eumelanin was the predominant melanin in the epidermis, phaeomelanin was the major melanin in the cultured melanocytes. These observations may have important implications for the use of cultured human melanocytes in the study of melanogenesis in man.     

The Expression of Tyrosinase,Tyrosinase-Related Proteins 1 and 2 (TRP1 and TRP2), the Silver Protein,and a Melanogenic Inhibitor in Human Melanoma Cells of Differing Melanogenic Activities

The expression of various melanogenic proteins, including tyrosinase, the tyrosinase-related proteins 1 (TRP1) and 2 (TRP2/DOPAchrome tautomerase), and the silver protein in human melanocytes was studied in six different human melanoma cell lines and compared to a mouse derived melanoma cell line. Analysis of the expression of tyrosinase, TRP1, TRP2, and the silver protein using flow cytometry revealed that in general there was a positive correlation between melanin formation and the expression of those melanogenic enzymes. Although several of the melanoma cell lines possessed significant activities of TRP2, the levels of DOPAchrome tautomerase in extracts of human cells were relatively low compared to those in murine melanocytes. Melanins derived from melanotic murine JB/MS cells, from melanotic human Ihara cells and HM-IY cells, from sepia melanin, and from C57BL/6 mouse hair were chemically analyzed. JB/MS cells, as well as Ihara cells and HM-TY cells, possessed significant amounts of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derived melanins, this being dependent on the activity of TRP2. Kinetic HPLC assays showed that 5,6-dihydroxyindole (DHI) produced during melanogenesis was metabolized quickly to melanin in pigmented KHm-1/4 cells, whereas DHI was stable in amelanotic human SK-MEL-24 cells. A melanogenic inhibitor that has been purified from SK-MEL-24 cells that suppressed oxidation of DHI in the presence or absence of tyrosinase, but had no effect on DHICA oxidation. The sum of these results suggest that the expression of melanogenic enzymes as well as the activity of a melanogenic inhibitor are critical to the production of melanin synthesis in humans.     

EARLY HISTOGENESIS OF TRANSPLANTED MOUSE MAMMARY GLANDS III. PARENCHYMAL-STROMAL INTERACTIONS *

Mammary isografts dissociated in gland-free fat pads in 24 hr but regenerated single glands. Isografts in the anterior chamber of the eye did not dissociate but regenerated only after collagenase treatment and retransplantation to gland-free fat pads.     

TWO TYPES OF MELANOMAS IN A NEW EXPERIMENTAL SYSTEM OF PLATYFISHSWORDTAIL HYBRIDS

A new experimental system for investigating platyfish-swordtail hybrid melanomas was developed using ornamental platyfish ( Xiphophorus maculatus ) and swordtails ( Xiphophorus helleri ). The incidence of melanomas in the hybrids was studied in relation to individual age factors. Based on the age of the onset of the melanomas, two types of the melanomas were discerned and were designated as the fry and the adult melanomas, respectively. The fry melanomas were characterized by early onset, wide-ranged lesions, an invasive nature owing to densely pigmented cells, and a tendency toward regression. The adult melanomas were characterized by occurrence in the later stage of life, a small number of primary sites, and rapid, expanding, and invasive growth due to sparsely pigmented cells.     

Agouti Protein Inhibits the Production of Eumelanin and Phaeomelanin in the Presence and Absence of α-Melanocyte Stimulating Hormone

Melanocytes synthesise two types of melanin: the brown-black eumelanin and the red-yellow phaeomelanin. In mice, the relative proportions of these two melanins are regulated by α-MSH, which preferentially increases the synthesis of eumelanin and by the Agouti protein (AP), the expression of which correlates with the growth of yellow phaeomelanin-containing hair. It has been proposed that AP acts by antagonizing the action of α-MSH at the MCI receptor, although it has been suggested that it may also act independently of α-MSH. In the present study we show that AP inhibits melanogenesis in B16F1 melanoma cells in the presence and absence of α-MSH and also causes dose-related decreases in the synthesis of both eumelanin and phaeomelanin. In the presence of α-MSH AP had a greater effect on eumelanin production and this is consistent with an antagonistic action at the MCI receptor. In the absence of α-MSH however, AP produced similar reductions in the synthesis of both melanins. These changes were not seen in B16G4F cells which lack the MCI receptor, suggesting that even in the absence of α-MSH AP acts at the MCI receptor. How this action is mediated at the intracellular level is not yet clear, although it appears to be associated with a decrease in tyrosinase activity.     

Chemical Characterization of Melanins in Sheep Wool and Human Hair

The color of hair and wool in mammals and feathers in birds is mostly determined by the quantity and quality of melanins that are synthesized in follicular melanocytes and transferred to keratinocytes. There are two chemically distinct types of melanin pigments: the black to brown eumelanins and the yellow to reddish pheomelanins. Melanins in sheep wool and human hair of various colors were characterized by HPLC methods to estimate 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-derived units in eumelanins and benzothiazine units in pheomelanins. Melanins were also characterized by spectrophotometric methods after differential solubilization in alkalies. It was demonstrated that 1) black wool in Asiatic sheep contains eumelanin with the DHICA content similar to black mouse melanin, while black to brown melanins from human hair contain much lower ratios of DHICA-derived units, comparable to the slaty mutation in mice, 2) dark brown to brown hair in human contains eumelanin whose chemical properties are indistinguishable from those of black hair, 3) dark red wool and red human hair contain pheomelanic pigments whose chemical properties are rather different from those of yellow pheomelanins in mice, and 4) light brown, blonde, and red hairs in human can be differentiated from each other with this methodology.