Vitamin C stimulates sphingolipid production and markers of barrier formation in submerged human keratinocyte cultures.

Human keratinocytes differentiate in vitro in response to a variety of stimuli, but neither the levels nor the spectrum of ceramides approach those seen in vivo. Ceramide production increases when human keratinocytes are grown at an air-liquid interface, and alterations in ceramide content occur when vitamin C is added to air-exposed, organotypic culture systems (Ponec et al. J Invest Dermatol 109:348, 1997). Here, we assessed whether vitamin C stimulates sphingolipid production in human keratinocytes independent of differentiation and air exposure. When submerged, human keratinocytes were grown in 1.2 mM calcium and serum-containing medium with vitamin C (50 microg per ml) for 9 d, total lipid content remained unchanged, but both glucosylceramide and ceramide content increased. Moreover, selected ceramide and glucosylceramide species: i.e., nonhydroxy ceramide 2 and both alpha- and omega-hydroxylated sphingolipids, increased preferentially [ceramide 4 (6-hydroxy-acylceramide), ceramide 5 (alpha-hydroxyceramide), ceramide 6 (4-hydroxy-alpha-hydroxyceramide), and ceramide 7 (6-hydroxy-alpha-hydroxyceramide); and acylglucosylceramide, glucosylceramide-B, and glucosylceramide-D], whereas ceramide 1, ceramide 3, glucosylceramide-C, and sphingomyelin remained unchanged. Synthesis of the corresponding ceramide and glucosylceramide fractions was enhanced by vitamin C, attributable, in part, to increased ceramide synthase activity (over 2-fold, p = 0.01); both serine palmitoyltransferase and glucosylceramide synthase activities remained unaltered. Finally, increased vitamin C-stimulated sphingolipid production correlated with the presence of lamellar bodies with mature internal contents, an increase in covalently bound omega-hydroxyceramide, and the appearance of prominent, corneocyte-bound lipid envelopes, whereas cornified envelope formation was unchanged. Thus, in submerged human keratinocytes, vitamin C induces both increased sphingolipid production and enhancement of permeability barrier structural markers.

Lovastatin enhances the photocytotoxicity of UVA radiation towards cultured N.C.T.C. 2544 human keratinocytes: prevention by cholesterol supplementation and by a cathepsin inhibitor.

The effect of the hydroxymethylglutaryl-CoA (HMG-CoA) inhibitor lovastatin on the UVA-induced photocytotoxicity has been investigated in cultured human N.C.T.C. 2544 keratinocytes. In the absence of irradiation, 5 x 10(-7) M lovastatin did not exhibit any significant cytotoxic effect towards this cell line. Although the drug cannot act as a photosensitizer, because it does not absorb in the UVA range, it markedly increased the UVA-induced cellular damage (about 70% reduction in cell viability at 5 x 10(-7) M). This effect was not accompanied by an increase in the lipid peroxidation product content of cells as compared with treatment with UVA alone. Medium supplementation with 0.01 mg/ml free cholesterol totally prevented the enhancement of UVA photocytotoxicity induced by lovastatin. A protective effect was also observed when cells were supplemented with an amount of low-density lipoprotein giving the same cholesterol concentration in the culture medium. Finally, E64 [L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane], a lysosomal cathepsin inhibitor, also prevents the cell death induced by UVA in cells treated with lovastatin. These results suggest that HMG-CoA reductase inhibitors could increase the sensitivity of skin cells to UVA radiation, and that this phenomenon is related to lysosomal enzyme release.

Polyunsaturated fatty acid enrichment increases ultraviolet A-induced lipid peroxidation in NCTC 2544 human keratinocytes.

The influence of cell enrichment with fatty acids with increasing degree of unsaturation on the ultraviolet A-induced formation of lipid-peroxidation products (thiobarbituric acid reactive substances [TBARS]) has been investigated in NCTC 2544 human keratinocytes. A 48-h preculture of cells in controlled medium supplemented with unsaturated fatty acids resulted in a marked increase in TBARS appearance under ultraviolet A exposure. This effect was dependent upon the degree of unsaturation of the fatty acids, with the following order of efficiency: arachidonic > linolenic > linoleic > oleic acid. For arachidonic acid (AA), the potentiating effect on ultraviolet A-induced lipid peroxidation was dependent upon the fatty acid concentration, with about a 2.5-fold increase in TBARS formation in cells pre-cultured with 5 x 10(-5) M AA, then exposed to a UVA dose of 13 J/cm2. The increase in TBARS formation by AA was almost totally prevented by supplementation of cells with 5 x 10(-5) M vitamin E, whereas buthionine sulfoximine, a chemical which depletes cell glutathione, potentiated lipid peroxidation. These results suggest that the nature of the fatty acids of cellular lipids could influence the response of keratinocytes to ultraviolet A, and especially the ultraviolet A-induced lipid peroxidation.

Teratogenic effect of the cholesterol synthesis inhibitor AY 9944 on rat embryos in vitro.

AY 9944 [trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride] is an amphiphilic cationic molecule. This chemical is an established inhibitor of cholesterol synthesis and is teratogenic in rats. The mechanisms of this teratogenicity remain to be clarified. This study used cultured rat whole embryos to ascertain whether AY 9944 had a direct effect on embryos, or whether its action was indirect, via the maternal cholesterol metabolism. Four experimental conditions were investigated: (A) controls; (B) 10 day untreated embryos were cultured in serum of treated rats; (C) 10 day untreated embryos were cultured in serum containing added AY 9944 (0-1,000 micrograms/ml); and (D) 10 day embryos from females treated on day 4 of gestation were cultured in normal serum. In group B there was no growth retardation; some slight nonspecific abnormalities were not significant. In group C, direct addition of AY 9944 to culture medium retarded growth and differentiation in a dose-dependent manner. No malformation was observed, but histological examinations showed numerous areas of cell necrosis, especially in the CNS. In group D, not only was growth retardation observed, but also characteristic malformations of AY 9944 teratogenesis, including pituitary agenesis. These results show that AY 9944 teratogenicity is initiated prior to day 10.