Topical L-ascorbic acid: percutaneous absorption studies.

BACKGROUND: Reactive oxygen species generated by ultraviolet light result in photocarcinogenic and photoaging changes in the skin. Antioxidants protect skin from these insults. OBJECTIVE: This study defines formulation characteristics for delivering L-ascorbic acid into the skin to supplement the skin's natural antioxidant reservoir. METHODS: L-ascorbic acid or its derivatives were applied to pig skin. Skin levels of L-ascorbic acid were measured to determine percutaneous delivery. RESULTS: L-ascorbic acid must be formulated at pH levels less than 3.5 to enter the skin. Maximal concentration for optimal percutaneous absorption was 20%. Tissue levels were saturated after three daily applications; the half-life of tissue disappearance was about 4 days. Derivatives of ascorbic acid including magnesium ascorbyl phosphate, ascorbyl-6-palmitate, and dehydroascorbic acid did not increase skin levels of L-ascorbic acid. CONCLUSIONS: Delivery of topical L-ascorbic acid into the skin is critically dependent on formulation characteristics.

Modern approaches to photoprotection.

UV light reacts with skin to produce undesirable changes, including photoaging and skin cancer. Sunscreen strategies are useful for protection against UV-B and short-wave UV-A, but complete protection against long-wave UV-A has not been achieved. Because UV-A is especially efficient at generating reactive oxygen species, it is being recognized increasingly as an important cause of photoaging and skin cancer.

Microfine zinc oxide is a superior sunscreen ingredient to microfine titanium dioxide.

BACKGROUND: Microfine zinc oxide and microfine titanium dioxide are particulate sunscreen ingredients that absorb broad-spectrum ultraviolet (UV) irradiation. OBJECTIVE: We compare microfine zinc oxide and microfine titanium dioxide for their abilities to attenuate UVA radiation and their relative whiteness in cosmetic formulations. METHODS: UVA attenuation was measured by diffuse reflectance spectroscopy on normal human skin in vivo. Whiteness was determined by reflectance density of dried coatings on a black background of the two particulates at varying concentrations. RESULTS: Microfine zinc oxide demonstrates superior protection compared to microfine titanium dioxide in the UV spectrum between 340 and 380 nm. Microfine zinc oxide is less white than titanium dioxide at all concentrations. CONCLUSION: Microfine zinc oxide is superior to microfine titanium dioxide as a sunscreen ingredient. It is more protective against long-wave UVA and is less white at a given concentration.

Microfine zinc oxide (Z-cote) as a photostable UVA/UVB sunblock agent.

BACKGROUND: Microfine zinc oxide (Z-Cote) is used as a transparent broad-spectrum sunblock to attenuate UV radiation (UVR), including UVA I (340-400 nm). OBJECTIVE: Our purpose was to assess the suitability of microfine zinc oxide as a broad-spectrum photoprotective agent by examining those properties generally considered important in sunscreens: attenuation spectrum, sun protection factor (SPF) contribution, photostability, and photoreactivity. METHODS: Attenuation spectrum was assessed by means of standard spectrophotometric methods. SPF contribution was evaluated according to Food and Drug Administration standards. Photostability was measured in vitro by assessing SPF before and after various doses of UVR. Photoreactivity was evaluated by subjecting a microfine zinc oxide/organic sunscreen formulation to escalating doses of UVR and determining the percentage of organic sunscreen remaining. RESULTS: Microfine zinc oxide attenuates throughout the UVR spectrum, including UVA I. It is photostable and does not react with organic sunscreens under irradiation. CONCLUSION: Microfine zinc oxide is an effective and safe sunblock that provides broad-spectrum UV protection, including protection from long-wavelength UVA.

A common duplication in the lysyl hydroxylase gene of patients with Ehlers Danlos syndrome type VI results in preferential stimulation of lysyl hydroxylase activity and mRNA by hydralazine.

Patients with Ehlers Danlos Syndrome type VI (EDS VI) are biochemically characterized by a deficiency of lysyl hydroxylase (LH), an enzyme that hydroxylates lysine residues required in the formation of stable crosslinks in collagen biosynthesis. Recently, in 19% of 35 EDS VI families, a duplication of seven exons in the LH gene has been identified as a common cause of EDS VI. We have observed that in fibroblasts from patients with this duplication mutation, administration of hydralazine, an iron-chelating agent, and ascorbate, a cofactor for LH activity, stimulates LH activity and its mRNA significantly more than in other EDS VI patients who do not have this duplication. Administration of these reagents, either singly or in combination, to fibroblasts from five patients homozygous for the duplication stimulated the low basal level of LH activity (<20% of normal) by five- to sixfold (hydralazine +/- ascorbate) and by twofold (ascorbate alone) at 72 h. This paralleled the increase in the steady-state level of mRNA for LH measured in similarly treated fibroblasts from four of these patients. In contrast, the activity of LH in fibroblasts from six other EDS VI patients and the mRNA from four of these patients who did not have the duplication were increased only three- to fourfold by hydralazine +/- ascorbate. The mechanism for the preferential stimulation of LH activity and mRNA by hydralazine in the EDS VI cells carrying the duplication is unknown, but it could be attributed to the presence of, for example, an enhancer sequence within the duplicated region of the LH gene.

Vitamin C abrogates the deleterious effects of UVB radiation on cutaneous immunity by a mechanism that does not depend on TNF-alpha.

Acute low-dose treatment of murine skin with ultra violet B (UVB) light impairs induction of contact hypersensitivity (CH) to dinitrofluorobenzene (DNFB) in certain inbred strains of mice (termed UVB-susceptible), but not in others (termed UVB-resistant), and promotes tolerance. These deleterious effects of ultraviolet radiation (UVR) are mediated in part by TNF-alpha, which is released from UVR-exposed epidermal and dermal cells. Because UVR damage to skin has also been ascribed in part to the generation of reactive oxygen intermediates (ROIs) such as superoxide anion (O2-), hydrogen peroxide (H2O2), hydroxyl radical (OH-), and singlet oxygen ((1)O2), we investigated whether vitamin C (ascorbic acid), which can nullify ROIs, prevents the deleterious effects of UVR on the cutaneous immune system. We found that epicutaneous application of vitamin C (10% L-ascorbic acid solution) abrogated the deleterious effects of acute low-dose UVR on induction of CH and prevented the induction of tolerance. Vitamin C, however, did not reverse the effects of TNF-alpha on CH induction and tolerance. These results indicate that (i) ROIs generated intracutaneously by UVR contribute to the impaired ability of exposed skin to support the induction of CH and to promote the induction of tolerance and (ii) these effects are not dependent on TNF-alpha.

Ascorbic acid preferentially enhances type I and III collagen gene transcription in human skin fibroblasts.

Ascorbic acid is a potent stimulator for type I and III collagen expression in human skin fibroblasts; stimulation of type I and III collagen synthesis and their mRNA levels by ascorbic acid has been reported previously. Nuclear run-on experiments demonstrated that ascorbic acid enhanced the transcription of type I and III collagen genes 4- and 3.4-fold respectively, whereas transcription of type IV collagen was slightly stimulated (1.7-fold). The results suggest that ascorbic acid preferentially enhanced type I and III collagen transcription.

The expression of a functional, secreted human lysyl hydroxylase in a baculovirus system.

This study reports the expression of functional human lysyl hydroxylase (LH), a post-translational modifying enzyme that catalyzes the hydroxylation of the lysine residues essential for cross-linking in collagen biosynthesis. We have developed a novel baculovirus system for the expression of LH, a protein that exists normally within the lumen of the endoplasmic reticulum, using a powerful baculovirus signal sequence for secretion. The supernatant from Sf9 cells infected with the viral recombinant showed significant LH activity that increased linearly with supernatant concentration, whereas there was no detectable LH activity in the cell pellet. Silver staining of the fractions purified from the active supernatant by concanavalin A Sepharose chromatography and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated an 85-kDa protein (the expected size of the LH subunit) that was most prominent in those fractions with the highest LH activity. N-terminal amino acid sequencing verified that the N-terminal primary structure of this 85-kDa protein was identical to human LH. Moreover, the activity of the expressed protein was shown to be dependent on the presence of Fe++, ascorbate, and alpha-ketoglutarate, three essential cofactors for LH activity. We have therefore successfully developed a novel expression system that produces functional human LH and enables this normally nonsecretory enzyme to be secreted, facilitating its separation from the intracellular proteins of insect cells. Future applications should allow characterization of the LH active site by crystallographic studies and site-directed mutagenesis for structure-function comparison.

The mRNA and the activity of lysyl hydroxylase are up-regulated by the administration of ascorbate and hydralazine to human skin fibroblasts from a patient with Ehlers-Danlos syndrome type VI.

Lysyl hydroxylase (LH) catalyzes the formation of hydroxylysine required for the intermolecular cross-linking of collagen, which is an essential step in collagen biosynthesis. Dermal fibroblasts from patients with Ehlers-Danlos Syndrome type VI (EDS VI), an inherited connective tissue disorder, express decreased levels of LH activity. In the present study we have shown that both the mRNA and the enzyme activity of LH in skin fibroblasts from one EDS VI patient (AT750), a compound heterozygote for the LH gene, are increased by administration of ascorbate and hydralazine, either individually or in combination. Although the AT750 cells express only 24% of the LH activity found in normal cells, a similar fold increase in activities in both EDS VI and normal cells was observed following treatment with ascorbate (1.5- to 2-fold) and hydralazine (2- to 4-fold), which paralleled the increase in their steady state LH mRNAs. Ascorbate increased total collagen production by 2-fold from a similar basal level of collagen synthesis in each cell type. This was confirmed by protein gel analysis which showed increases in pro alpha 1(I), pro alpha 2(I), and pro alpha 1(III) collagen chains in both normal and EDS VI cells. This ascorbate-mediated increase of alpha 1(I) collagen resulted from increased mRNAs for alpha 1(I) collagen in both cell types. Hydralazine treatment, with or without ascorbate, severely decreased the alpha 1(I) collagen mRNAs in fibroblasts from both AT750 and the normal donor; total collagen synthesis was similarly reduced. This study shows that LH activity, which is severely deficient in fibroblasts from an EDS VI patient, can be upregulated by administration of ascorbate and hydralazine as a result of the increased mRNA for LH, suggesting that the mechanism for the regulation of the LH gene is functioning normally in this patient.

Comparative study of wound healing in porcine skin with CO2 laser and other surgical modalities: preliminary findings.

BACKGROUND: The CO2 laser is a common surgical modality in dermatology. To clarify conflicting reports on the histological healing properties of CO2 laser on incisional or ablative wounds, we have applied it in a miniature hairless porcine skin model at power settings similar to those used in clinical practice. METHODS: Histological parameters of wound healing in skin incisions using the CO2 laser were compared with those using scalpel, hot scalpel, and electrosection, and in dermal ablation using CO2 laser, fraize, wire brush, and electrofulguration alone or with curettage. RESULTS: In incisional wounds, tissue damage was most extensive in CO2 laser wounds, with delayed dermal healing and reepithelialization. In ablative wounds, CO2 laser caused a similar degree of tissue damage as did the electrosurgical modalities, and more damage than did fraize or wire brush. Reepithelialization was complete in CO2 laser, fraize, and wire brush wounds before electrosurgical wounds. Final histology of both incisional and ablative wounds at 6 weeks was similar with all surgical modalities. CONCLUSION: The CO2 laser and electrosurgery both produce greater focal tissue damage in incisional and ablative applications than the other modalities. Delayed epithelialization of the wound occurs with both modalities in incisional wounds but only with electrosurgery in ablative wounds. At 6 weeks, the appearance of the scar in all incisional and ablative modalities is similar grossly and histologically. Confirmation of these findings requires standardization of power density of the CO2 laser in incision and ablation.

Minoxidil stimulates elastin expression in aortic smooth muscle cells.

Minoxidil was found to inhibit the proliferation of smooth muscle cells in the proliferating phase, but not in the quiescent phase. Treatment of proliferating or quiescent cells with minoxidil resulted in a dose- and time-dependent stimulation of elastin synthesis specifically. Maximum stimulation (fourfold) occurred in cells treated with 1 mM minoxidil for 48 h. The stimulation of elastin synthesis was accompanied by a proportional increase in elastin mRNA level, and it was partially prevented by a K+ channel blocker (tetraethylammonium) and completely prevented by high K+ salt (0.1 M). Minoxidil had no significant effect on the extent of prolyl hydroxylation in newly synthesized elastin. These results indicate that minoxidil stimulates elastin synthesis at a pretranslational level by a mechanism unrelated to cell proliferation but one that may involve K+ efflux. As a pharmacological agent capable of stimulating elastin expression, minoxidil would be a useful drug for the treatment of abnormal elastin metabolism.

Tranilast, a selective inhibitor of collagen synthesis in human skin fibroblasts.

Effects of tranilast, N-(3,4-dimethoxycinnamoyl)anthranilic acid, on collagen synthesis in cultured human skin fibroblasts were studied. Tranilast was found to inhibit collagen synthesis in a dose-dependent manner to a maximum of 55% at 300 microM during 48 h of treatment; the synthesis of type I and type III collagens was equally affected. Administered simultaneously or subsequently, tranilast reduced the stimulatory effect of transforming growth factor beta 1 (2.5 ng/ml) on collagen synthesis without affecting the accompanying stimulation of noncollagen protein synthesis. It did not affect prolyl or lysyl hydroxylase activity in vitro and in cells. The content of pro alpha 1(I) collagen mRNA was decreased 60% by tranilast. Tranilast prevented the TGF beta 1-mediated increase in pro alpha 1(I) collagen mRNA. These results indicate that tranilast specifically inhibits collagen production at a pretranslational level by interfering with TGF beta 1 effects. Tranilast also inhibited collagen synthesis in scleroderma fibroblasts to the same extent and in keloid fibroblasts to a greater extent than in normal fibroblasts, attesting to its therapeutic potential as an antifibrotic drug.

Effects of ascorbic acid on proliferation and collagen synthesis in relation to the donor age of human dermal fibroblasts.

Several events are associated with cellular aging: alterations in the extracellular matrix, loss of the cell's proliferative capacity, and decreased responsiveness to growth factors. In skin, a major component of the extracellular matrix is collagen; an important regulator of collagen synthesis is ascorbic acid, which may also have growth factor-like properties. To investigate the relationship of the extracellular matrix and proliferative capacity to aging, we examined the effects of ascorbic acid on cell proliferation and collagen expression in dermal fibroblasts from donors of two age classes, newborn (3-8 d old) and elderly (78-93 years old). In the absence of ascorbic acid (control) proliferative capacities were inversely related to age; newborn cell lines proliferated faster and reached greater densities than elderly cell lines. However, in the presence of ascorbic acid both newborn and elderly cells proliferated at a faster rate and reached higher densities than controls. To determine whether there are age-related differences in extracellular matrix production and ascorbic acid responsiveness we examined and found that collagen biosynthesis (collagenase-digestible protein) was inversely related to age, but the stimulation by ascorbic acid appeared age independent. The increase in collagen synthesis was reflected by coordinate increases in steady-state pro alpha 1(I) and pro alpha 1(III) collagen mRNAs, suggesting a pretranslational mechanism. Ascorbic acid appears capable of overcoming the reduced proliferative capacity of elderly dermal fibroblasts, as well as increasing collagen synthesis in elderly cells by similar degrees as in newborn cells even though basal levels of collagen synthesis are age dependent.

Inhibition of collagen hydroxylation by lithospermic acid magnesium salt, a novel compound isolated from Salviae miltiorrhizae Radix.

We have screened several chinese medicinal herbs for the presence of antifibrotic agents. An aqueous extract of Salviae miltorrhizae Radix was found to inhibit collagen secretion by human skin fibroblasts without affecting DNA or noncollagen protein synthesis. We have subsequently purified the material exhibiting the inhibitory activity and identified it as magnesium lithospermate. From its chemical structure this compound was predicted to be an inhibitor of the post-translational modifying enzymes prolyl and lysyl hydroxylases in collagen biosynthesis. Accordingly, it decreased the extent of prolyl and lysyl hydroxylations in collagen by approx. 50%. Added to cell extracts it inhibited both prolyl and lysyl hydroxylase activities, but only lysyl hydroxylase activity when added to intact cells. Oral administration of this compound to mice led to a significant reduction of prolyl hydroxylation in newly-synthesized skin collagen. This naturally-occurring compound thus offers a potential means for treating fibrotic diseases, such as systemic scleroderma and keloid.

A patient with Ehlers-Danlos syndrome type VI is a compound heterozygote for mutations in the lysyl hydroxylase gene.

In the present study, we have isolated and sequenced the complementary DNAs of two mutant alleles for lysyl hydroxylase (LH) in fibroblasts from one patient (AT750) with Ehlers-Danlos syndrome type VI (EDS VI). We have identified a putative mutation in each allele which may be responsible for the patient's decreased LH (normalized to prolyl hydroxylase) activity (24% of normal). Intermediate levels of LH activity were measured in the patient's parents, who are clinically normal (father 52%; mother 86%). After the cloning of cDNAs and amplification by PCR, sequence analysis revealed two equally distributed populations of cDNAs for LH in the AT750 cell line. Each allele revealed different but significant changes from the normal sequence. In one allele (allele 1), the most striking change was a triple base deletion that would result in the loss of residue Glu532. The most significant difference in the other allele (allele 2) was a G-->A change which would produce a Gly678-->Arg codon change in a highly conserved region of the enzyme. Restriction analysis identified that allele 1 was inherited from the proband's mother and allele 2 from the father. This study represents the first example of compound heterozygosity for the LH gene in an EDS VI patient, and it appears that there is an additive effect of each mutant allele on clinical expression in this patient.

Sequence analysis of a cDNA for lysyl hydroxylase isolated from human skin fibroblasts from a normal donor: differences from human placental lysyl hydroxylase cDNA.

Using polymerase chain reaction, we have isolated and sequenced a 3-kb cDNA for lysyl hydroxylase (LH) from human skin fibroblasts from an normal donor. Apart from two polymorphic sites, no differences were observed between the 2184 nt coding regions of LH cDNA from fibroblasts and placenta. However, four differences were observed in the 3' non-coding regions of the two cDNAs; three were single base changes and the fourth a deletion of a single base. The absence of the single nucleotide in the LH cDNA from fibroblasts resulted in the loss of an HpaII site that is present in the placental LH cDNA; this was confirmed in HpaII digests of fibroblast and placental LH cDNAs from the same donor. Northern blots showed that the LH gene was strongly expressed in fibroblasts and placenta and, to a lesser extent, in aorta, lung, vein, cartilage, and artery.

Regulation of lysyl oxidase mRNA in dermal fibroblasts from normal donors and patients with inherited connective tissue disorders.

Lysyl oxidase (LO) is an extracellular copper-dependent enzyme that catalyzes the initial reaction in the formation of lysine or hydroxylysine-derived crosslinks during collagen biosynthesis. We have isolated a cDNA for human LO from skin fibroblast poly(A+)RNA by PCR using primers based on the recently published sequence of human LO. This cDNA probe detects a major mRNA of 4.2 kb on Northern blots of RNA from normal fibroblasts. The level of LO mRNA was not significantly affected by cell density or by ascorbate treatment. Treatment of skin fibroblasts with hydralazine (50 microM), which increases the mRNAs for both the alpha and the beta subunits of prolyl hydroxylase (PH) and the mRNAs for lysyl hydroxylase, also increased LO mRNA by fourfold over a 72-h time course. In contrast, hydralazine dramatically decreased the mRNAs for alpha 1(I) collagen. Administration of minoxidil (500 microM), which specifically decreases LH activity without affecting PH activity or collagen biosynthesis in skin fibroblasts, stimulated the level of LO mRNA. Neither the administration of penicillamine (100 microM), which interferes with collagen cross-linking, nor the administration of beta-aminopropionitrile, which is a strong irreversible inhibitor of LO, to fibroblasts significantly changed the levels of LO mRNA over a 72-h time course. However, bleomycin (0.6 microgram/ml) significantly decreased the 4.2-kb LO mRNA in contrast to the levels of the alpha 1(I) collagen mRNAs, which were unchanged. No significant change was observed in the steady-state levels of LO mRNAs in fibroblasts isolated from patients with certain connective tissue disorders, including Marfan syndrome, Menkes disease, cutis laxa, and pseudoxanthoma elasticum.