Gender differences of enzymatic activity and distribution of 17beta-hydroxysteroid dehydrogenase in human skin in vitro.

The interconversion of estrone (E1) and 17beta-estradiol (E2) is catalyzed by 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in peripheral steroidogenic organs such as the skin. To investigate gender differences of activity and skin distribution of 17beta-HSD in human skin, enzymatic activity was measured in skin homogenates and skin horizontally sliced by 10 microm thickness in vitro. Reductive 17beta-HSD (E2 formation from E1) in female skin has a lower substrate affinity than in male skin; Km (Michaelis-Menten constant) of female and male skin is 11.8 +/- 6.5 and 2.0 +/- 2.0 microM, respectively. Female skin had a tendency to activate estrogen; Vmax (maximum rate) for E2 formation, 5.8 +/- 4.0 pmol/min/mg protein, is 1.7 times larger than E1 formation, 3.5 +/- 1.5 pmol/min/mg protein, and, on the other hand, male skin tends to deactivate estrogen; Vmax for E1 and E2 is 10.5 +/- 6.1 and 4.2 +/- 3.7 pmol/min/mg protein, respectively. The concentration of metabolite had a peak value at 80-120 microm from the skin surface. Therefore, these in vitro results suggest that the enzymatic activities of 17beta-HSDs have a gender difference in estrogen formation/metabolism and are distributed around the basement layer of the epidermis irrespective of sex. 17Beta-HSDs distributed around the basement epidermis may be effectively supplied with circulating estrogen from the papillary plexus to maintain the estrogen level in skin. This distribution pattern having a peak surrounding 100 microm from the skin surface indicates the importance for defense from noxae (e.g. detoxication) and maintenance of the internal environment (e.g. biosynthesis of hormones). Future studies should increase sample size and confirm these results by stricter statistical analysis.

Skin metabolism in transdermal therapeutic systems.

Skin has at least two barriers with protective functions: the stratum corneum physical barrier and a biochemical barrier in the epidermis and dermis. Numerous chemical and physical enhancers exist for transdermal therapeutic systems; some cause irritation, and possibly influence enzyme deactivation. Knowledge of enzymatic skin reactions is important for developing safe and efficacious transdermal systems for treatment not only of skin diseases but also for systemic application. This paper overviews the effects of (a) chemical enhancers and additives, (b) drug structure, and (c) physical enhancement on skin metabolism.

Crystallographic studies on damaged DNAs IV. N( 4)-methoxycytosine shows a second face for Watson-Crick base-pairing, leading to purine transition mutagenesis.

To investigate the mutation mechanism of purine transitions in DNA damaged with methoxyamine, a DNA dodecamer with the sequence d(CGCAAATTmo(4)CGCG), where mo(4)C is 2'-deoxy-N(4)-methoxycytidine, has been synthesized and the crystal structure determined by X-ray analysis. The duplex structure is similar to that of the original undamaged B-form dodecamer, indicating that the methoxylation does not affect the overall DNA conformation. Electron density maps clearly show that the two mo(4)C residues form Watson-Crick-type base pairs with the adenine residues of the opposite strand and that the methoxy groups of mo(4)C adopt the anti conformation to N(3) around the C(4)-N(4) bond. For the pair formation through hydrogen bonds the mo(4)C residues are in the imino tautomeric state. Together with previous work, the present work establishes that the methoxylated cytosine residue can present two alternate faces for Watson-Crick base-pairing, thanks to the amino<-->imino tautomerism allowed by methoxylation. Based on this property, two gene transition routes are proposed.

Crystallographic studies on damaged DNAs: III. N(4)-methoxycytosine can form both Watson-Crick type and wobbled base pairs in a B-form duplex.

To investigate the mutation mechanism of purine transition in DNA damaged with methoxyamine, a DNA dodecamer with the sequence d(CGCGAATTmo(4)CGCG), where mo(4)C is 2'-deoxy-N(4)-methoxycytidine, has been synthesized and its crystal structure determined. Two dodecamers form a B-form duplex. Electron density maps clearly show that one of the two mo(4)C residues forms a pair with a guanine residue of the opposite strand, the geometry being the canonical Watson-Crick type, and that the other mo(4)C residue forms a wobble pair with the opposite guanine residue. These two pairings are ascribed to the tautomerization of the methoxylated cytosine moieties between the amino and imino forms.

Distribution of hydrolytic activity catalyzes the biotransformation of prednisolone 21-acetate in human skin.

We investigated the distribution of hydrolytic enzymes which metabolize prednisolone 21-acetate (PNA) to prednisolone (PN) in human skin. Km (Michaelis-Menten constant) and Vmax (maximum rate) of hydrolytic enzyme in human skin was 25.1 microM and 0.46 nmol/min/mg protein, respectively. Specific activities of hydrolysis in dermis and epidermis were similar and, in consideration to their thickness, hydrolytic activity in epidermis was 12.1 times higher than in dermis. Moreover, the highest amount of metabolite (PN) was found at 80-120 microm from the skin surface by skin slicing. Therefore, hydrolytic activity which metabolized PNA was distributed in epidermis, especially in the basement membrane area; epidermis borders dermis in this area and the papillary plexus is reached just beneath the dermal papillae. These results suggest that the distribution of hydrolytic activity in human skin may prevent certain substances from entering the systemic circulation in their unhydrolyzed form.

Metabolism of prednisolone 21-acetate in hairless mouse skin.

We investigated the hydrolytic activity of prednisolone 21-acetate (PNA) to prednisolone (PN) in an enzyme solution composed of esterase and skin homogenates from hairless mice. The values of the Michaelis-Menten constant obtained from hairless mouse skin and esterase solution were 14.2 and 10.2 microM, respectively; conversely, the value of the maximum rate from hairless mouse and esterase solution were 0.67 and 1,886 nmol/min/mg protein, respectively. To examine the effect of enzymatic inhibitors on hydrolytic activity, five enzymatic inhibitors, 3,4-dichloroisocoumarine (DCIC), N-tosyl-L-phenylalanine chloromethyl ketone, iodoacetamide, p-hydroxymercuribenzoic acid (HMBA) and sodium dodecylsulfate, were added to the enzyme solution. Sixty-eight percent of hydrolytic activity in skin homogenates were not deactivated by DCIC which completely inhibited the enzymatic activity in esterase solution. We also studied the localization of hydrolytic enzyme with a subcellular faction: 66 and 11% of specific activity existed in microsome (Ms) and cytosol (Cp) fractions, indicating that the hydrolytic activity of PNA was included mainly in the Ms fraction. Hydrolytic activity in Ms and Cp fractions was different from sensitivity to enzymatic inhibitor; DCIC inhibited activity in the Ms fraction and, on the other hand, HMBA inhibited it in the Cp fraction. Therefore, Ms and Cp fractions in skin homogenates include a different esterase isoform and the metabolism of PNA to PN in hairless mouse skin is mediated by these isoforms.

X-ray analyses of two DNA dodecamers containing N4-methoxy-cytosine paired with adenine or guanine.

To investigate mismatch of base-pairings in relation to mutagenesis by oxyamines, crystal structures of two DNA dodecamers with the sequence d(CGCZAA TTmo4CGCG) (Z = A or G), containing N4-methoxy-cytosine (mo4C), have been determined by X-ray analysis. These dodecamers essentially form right-handed B-form duplexes, respectively. In the dodecamer with Z = A, the two mo4C residues are adapted in imino form with the anti methoxyl group to form pairs with A on the opposite strand in a manner of Watson-Crick fashion. While in the dodecamer with Z = G, one mo4C in amino form with the anti methoxyl group forms a normal Watson-Crick pair with G, but the other one in imino form with syn methoxyl conformation wobbles with G. Based on these results, possible mutation mechanism has been proposed.

Crystallographic studies on damaged DNAs. II. N(6)-methoxyadenine can present two alternate faces for Watson-Crick base-pairing, leading to pyrimidine transition mutagenesis.

In a previous paper, 2'-deoxy-N(6)-methoxyadenosine (mo(6)A) was shown to form a mismatch base-pair with 2'-deoxycytidine with a Watson-Crick-type geometry. To fully understand the structural basis of genetic mutations with damaged DNA, it is necessary to examine whether the methoxylated adenine residue still has the ability to form the regular Watson-Crick pairing with a thymine residue. Therefore, a DNA dodecamer with the sequence d(CGCGmo(6)AATTCGCG) has been synthesized and its crystal structure determined. The methoxylation has no significant effect on the overall DNA conformation, which is that of a standard B-form duplex. The methoxylated adenine moieties adopt the amino tautomer with an anti conformation around the C(6)-N(6) bond to the N(1) atom, and they form a Watson-Crick base-pair with thymine residues on the opposite strand, similar to an unmodified adenine residue. It is concluded that methoxylated adenine can present two alternate faces for base-pairing, thanks to the amino<-->imino tautomerism allowed by methoxylation. Based on this property, two gene transition routes are proposed.

X-ray analysis of a DNA dodecamer containing 2'-deoxy-N4-methoxycytidine.

The crystal structure of DNA dodecamer with the sequence of d(CGCAAATTXGCG), where X is 2'-deoxy-N4-methoxycytidine, has been determined by X-ray analysis. The dodecamers form a double helix with B-form conformation. The electron density indicates that the two modified cytosine bases respectively make a pair with the adenine bases on the opposite strand in a manner of Watson-Crick geometry and that the methoxy groups are in anti conformation to the N3 atom.

Effect of ultrasound application on skin metabolism of prednisolone 21-acetate.

PURPOSE: The effect of ultrasound on skin penetration and metabolism of prednisolone (PN) and prednisolone 21-acetate (PNA) was investigated in the hairless mouse skin in vitro. METHODS: The abdominal skin excised freshly was pretreated under different ultrasound intensities (4.32, 2.88, and 1.50 W/cm2) for 10, 30, and 60 min. The penetration/metabolism rate of PNA and its metabolite (PN) was then measured using a side-by-side diffusion cell. RESULTS: The skin penetration of PN was enhanced by the ultrasound pretreatment. This enhancement was attributed to the decrease in the stratum corneum barrier capacity by ultrasound energy. The steady-state appearance rate of PN following the skin bioconversion of PNA decreased appreciably with increasing the product of the duration of pretreatment (Dp, min) and the intensity of ultrasound applied (Iu, W/cm2). When the product value was less than 40 W/cm2 . min, the steady-state appearance rate of the PN hardly increased in spite of the penetration enhancement of PNA. CONCLUSIONS: These findings indicated a possible deactivation of the skin enzymes by ultrasound energy.

Binding of prednisolone and its ester prodrugs in the skin.

PURPOSE: Skin binding of prednisolone and its esters was investigated in the hairless mouse skin in vitro. METHODS: The distribution of the amount of drugs bound in the skin was determined by a skin slicing technique. The model drugs used were prednisolone (PN, M.W. 360) and its esters, senesyonate (PN-C5, M.W. 442), geranate (PN-C10, M.W. 510), farnesylate (PN-C15, M.W. 578), and geranylgeranate (PN-C20, M.W. 646). RESULTS: The distribution of bound drug was nonhomogeneous in the skin; the concentration of PN-C10 and PN-C15 in the skin increased gradually with the distance from the skin surface. The parent drug, PN, however, was hardly bound in the viable skin. CONCLUSIONS: These findings suggest that the prodrugs of prednisolone may prolong the dermal retention of the parent drug and minimize to delivery into the systemic circulation of the prodrug and metabolite.

Diffusion and metabolism of prednisolone farnesylate in viable skin of the hairless mouse.

The diffusion and metabolism of prednisolone 21-farnesylate were investigated in viable skin of the hairless mouse in vitro. The prodrug ester was extensively metabolized in viable skin, while it was stable in the donor and receptor solutions. The rate of appearance of the prodrug and its metabolite prednisolone was markedly influenced by the direction of the skin placed between the in vitro diffusion half-cells. The rate of bioconversion of the prodrug was determined as a function of the distance from the surface of the skin. The prodrug was increasingly metabolized with the distance from the surface of the skin, indicating that the responsible enzymes are enriched in the lower layers of the viable skin. A model with linearly increasing enzyme activity in the viable skin accounts for the in vitro profiles of the diffusion/metabolism of the prodrug in the viable skin of hairless mouse.