Karyotype modifications in human malignant melanoma cell cultures after treatment with azelaic acid.

Azelaic acid (AzAc) is a C9 dicarboxylic acid which has recently been shown to have some therapeutic applications in skin diseases of different aetiologies. In order to study the in vitro activity of AzAc five human malignant melanoma primary cell cultures were treated for up to 60 days with 10 mM C9 2Na; the growth characteristics were defined by growth curve and the cytogenetics by Giemsa standard technique and GTG banding technique. Our data demonstrated an inhibition in replication of all five melanomas and the disappearance of the clones with chromosomal markers in four out of five melanomas after AzAc treatment.

Saturated dicarboxylic acids as products of unsaturated fatty acid oxidation.

Upon chemical, radiation-induced or enzymatic oxidation, cis-polyunsaturated fatty acids, i.e., C18:2(n-6), C18:3(n-3), C20:2(n-6), C20:3(n-6), C20:3(n-3), C20:4(n-6), C20:5(n-3), C22:2(n-3), C22:4(n-6), C22:6(n-3), were found to generate saturated short and medium-chain length dicarboxylic acids, which can be regarded as a distinctive feature of the particular double bonds positions in the polyunsaturated fatty acid molecule. Two different dicarboxylic acids, which were unambiguously quantified by GC-MS, were produced from a single fatty acid: one deriving from the oxidative splitting at the level of the first double bond in the molecule, the other being two-carbon-atoms lower homologous. Formation of dicarboxylic acids occurred also from triacylglycerols and phospholipids containing cis-polyunsaturated fatty acids. In this case, following oxidation, the diacids remained covalently bound to the starting molecule and transesterification was necessary for identification. Being extremely stable and easily detectable compounds, dicarboxylic acids may be considered potential markers of oxidative attack to both free and esterified unsaturated fatty acids.

Squalene peroxides may contribute to ultraviolet light-induced immunological effects.

Ultraviolet (UV) irradiation is capable of producing a dose-dependent decomposition of skin surface lipids and particularly of squalene, with the concomitant generation of active lipoperoxides. The biological effects of UV-peroxidated squalene were tested, compared with those produced by synthetic lipoperoxides (cumene hydroperoxide), on some immunological parameters in vivo modified by UVB irradiation. Application of UV-peroxidated squalene as well as cumene hydroperoxide significantly inhibited the induction of contact hypersensitivity to dinitrofluorobenzene in mice, which was associated with a decrease in the number of ATPase positive cells. The effect was dose-dependent (over 40 micrograms for peroxidated squalene and over 20 micrograms for cumene) and relevant after 2 d of treatment. Down-regulation towards the applied hapten was demonstrated. The results indicate that UV-induced lipoperoxides of squalene are capable of inhibiting the induction of contact hypersensitivity in mice and suggest that, among the other photoproducts generated in humans, squalene peroxides may play a role as biochemical messengers of the biological effects of UV irradiation of the skin.

Ultrastructure of melanocytes in chronically sun-exposed skin of elderly subjects.

Chronically sun-exposed facial skin of three females aged 68, 71, and 78 years, and of a male aged 78, was examined by electron microscopy in order to study the condition of the epidermal melanocytes. Considerable heterogeneity of morphological and functional characteristics of the cells was observed. The majority of melanocytes were large, active, with occasionally lobulated or double nuclei, an appearance indicative of hyperstimulation. Some cells exhibited an appearance of having reached the end of an active life cycle and were labelled "aged." Others, in the upper end of the outer root sheath of hair follicles and adjacent interfollicular epidermis, presented a typically inactive appearance, indistinguishable from that of fetal melanocytes, or of those in unexposed skin of younger subjects. A cell with indented nucleus, fully melanised melanosomes, and hypertrophic Golgi apparatus was sporadically seen. Minute foci of dissociation of keratocytes were present, and melanocytes included in these were frequently disrupted. Swelling of mitochondria and cytoplasmic lipid droplets occurred sporadically within all the above variants of melanocytes. It proved difficult to establish criteria of specific sun damage of melanocytes. It is suggested that either the melanocytes exhibiting stimulation or the relatively inactive ones could be the precursors of the proliferating cells of lentigo maligna.

Role of skin surface lipids in UV-induced epidermal cell changes.

Ultraviolet irradiation is capable of affecting skin surface lipids, especially squalene and cholesterol, both in vitro and in vivo, with generation of active lipoperoxides. The photodecomposition of the skin lipid component was carefully evaluated by capillary gas-chromatography. The effects of UV-induced lipoperoxides on human keratinocytes in culture and on guinea pig ear slices were compared with those of synthetic lipoperoxides, i.e. cumene hydroperoxide and 13-hydroperoxylinoleate. A time- and dose-dependent effect on protein synthesis and mitotic activity was observed. In cell culture low concentrations (0.05-5 micrograms/ml) of peroxidated squalene and synthetic lipoperoxides stimulated the incorporation of radiolabelled thymidine and phenylalanine, while higher concentrations (greater than 10 micrograms/ml), or longer periods of treatment, induced cellular damage. In guinea pig ear slices, the lipoperoxides (5-50 micrograms/ml) increased aminoacid incorporation and the number of epidermal pigment cells; higher concentrations (greater than 100 micrograms/ml) caused a derangement of epidermal structure. The results suggest that UV irradiation of skin generates lipoperoxides from the surface lipids which, in vitro, are capable of producing a number of changes in epidermal cells.

Scavenging activity of azelaic acid on hydroxyl radicals "in vitro".

Azelaic acid is an aliphatic dicarboxylic acid (HOOC-(CH2)7-COOH) which has recently been shown to have some practical therapeutic applications in skin diseases of different etiologies. It possesses diverse biological activities and its mechanisms of action are still under investigation. Azelaic acid, as disodium salt (C(9)2Na), at concentrations from 0.05 mM to 1.0 mM is capable of inhibiting significantly the hydroxylation of 1-tyrosine to 1-DOPA due to hydroxylradicals (HO.) produced by Fenton reaction. Similarly C(9)2Na significantly inhibits the heterogeneous photocatalytic oxidation of toluene to cresols, and the peroxidation of arachidonic acid (C20:4,n6), due to HO. formed by dissolved oxygen in the presence of UV-irradiated semiconductor TiO2 (photo-Fenton type reaction). C(9)2Na decomposition and its by-products formation are quantifiable only at high HO. concentrations. On the contrary, C(9)2Na is not a scavenger of O2-. generated by xanthine-xanthine oxidase system. Under the same experimental conditions, mannitol behaves like C(9)2Na. These data indicate that HO. scavenging capacity of C(9)2Na in vitro, and represent a useful tool for further investigations on the mechanisms of action of azelaic acid in biological systems.

The oxyradical-scavenging activity of azelaic acid in biological systems.

We have previously shown that azelaic acid, a C9 dicarboxylic acid, as disodium salt (C(9)2Na) is capable of inhibiting significantly the hydroxylation of aromatic compounds and the peroxidation of arachidonic acid due to reactive hydroxyl radicals (HO.). In this paper we have investigated the ability of C(9)2Na to inhibit the oxyradical induced toxicity towards two tumoral cell lines (Raji and IRE1) and normal human fibroblasts (HF). Oxyradicals were generated either by the addition of polyphenols to the medium, or by direct irradiation of phosphate buffered-saline in which cells were incubated from 15 min prior to incubation in normal medium. The effects of C(9)2Na were compared with those obtained by mannitol (MAN), superoxide dismutase (SOD) and catalase (CAT). C(9)2Na, MAN, SOD and CAT significantly decreased the polyphenol toxicity towards cell lines cultured up to 24 h. After 48 h of incubation the above compounds lost the capability of protecting cells from polyphenol toxicity. This suggests that the toxic role of oxyradicals (O2-., H2O2, HO.) persists for about 24 h and, subsequently other toxic mechanisms must be involved, which are not affected by oxyradical scavengers. SOD and CAT did not show any protective effect on UV induced cytotoxicity, while both C(9)2Na and MAN were capable of reducing significantly the UV damage towards cell lines, even after 48 h incubation. This can be explained by the fact that UV cytotoxicity depends mainly on the generation of HO., that can be "scavenged" by C(9)2Na or MAN, but not by SOD or CAT. C(9)2Na and MAN were not significantly degraded in the period during which they afford protection against HO..

[Mechanism of azelaic acid action in acne]. / Meccanismo di azione dell'acido azelaico nell'acne.

The physiopathologic mechanism of acne seems to be dependent on four main factors: a) sebum production and excretion; b) type of keratinization of the follicular channel; c) microbial colonization of the pilosebaceous unit and d) inflammatory reaction of the perifollicular area. Azelaic acid is effective in the treatment of acne because it possesses an activity against all of these factors. Azelaic acid is a competitive inhibitor of mitochondrial oxidoreductases and of 5 alpha-reductase, inhibiting the conversion of testosterone to 5-dehydrotestosterone. It also possesses bacteriostatic activity to both aerobic and anaerobic bacteria including Propionibacterium acnes. Azelaic acid is an anti-keratinizing agent, displaying antiproliferative cytostatic effects on keratinocytes and modulating the early and terminal phases of epidermal differentiation.

Effect of dicarboxylic (C6 and C9) acids on a human squamous carcinoma cell line in culture.

In tissue culture, azelaic acid (C9) has been shown to have an anti-proliferative and cytotoxic effect on human and murine malignant melanocytes, with inhibition of mitochondrial oxido-reductase enzymes and DNA synthesis, and damage to mitochondria. Recent reports of effects on differentiation of normal keratocytes have led to the present study of its effects on a squamous carcinoma cell line. Cells were exposed to single doses of disodium salts of azelaic (C9(2)Na) and adipic (C6(2)Na) acids at concentrations of 10(-2)M and 5 x 10(-2)M for 48 hrs. Only C9(2)Na at 5 x 10(-2) M for 4 hrs., and longer, significantly affected proliferation, and the cells exhibited massive swelling of mitochondria with loss of cristae. The results further confirm the probable value of azelaic acid as a general anti-tumoral agent rather than a specifically melanocytotoxic one. They could justify clinical studies on the effect of topical azelaic acid therapy on squamous cell carcinoma in vivo.

[Azelaic acid in the treatment of acne]. / L'acido azelaico nella terapia dell'acne.

This review is an update of the literature accumulated over the past 6 years following the original observation that topically applied azelaic acid, a non-toxic C9 dicarboxylic acid, has a beneficial therapeutic effect on acne vulgaris. These studies have shown that azelaic acid has a modulating influence on the process of keratinization, and that it acts as a keratolytic and anti-comedogenic agent. There is evidence that it inhibits mitochondrial and microsomal oxido-reductases, including 5-alpha-reductase, and that it may interfere with the process of sebogenesis. It has a spectrum of antimicrobial activity, both in vitro and in vivo, against aerobic microorganisms and is effective against the anaerobic Propionibacterium acnes. Extensive multi-centre clinical trials have established that topical azelaic acid (a 20% cream) is an effective treatment for all types of acne. It compares well with other agents, such as topical tretinoin or benzoyl-peroxide, or oral tetracycline. It is non-irritant, and does not give rise to allergic or photo-toxic reactions. Its use is not associated with teratogenicity, possible endocrine unbalance, or the disadvantages of antibiotic treatment. It can be applied for long periods, in recurrences, and as maintenance "spot" therapy against individual lesions.

Effect of dicarboxylic acids (C6 and C9) on human choroidal melanoma in cell culture.

In cell culture, azelaic acid (C9) has been shown to have an antiproliferative and cytotoxic effect on human and murine malignant cutaneous melanocytes. Normal melanocytes are unaffected, as are normal choroidal melanocytes. Here, effects on cell kinetics and ultrastructure of cells of a human choroidal melanoma line have been studied. Cells were exposed to single doses of disodium salts of azelaic (C(9)2Na) and adipic (C(6)2Na) acids at concentrations of 10(-2) M and 5 X 10(-2) M for 48 hr. C(9)2Na at 5 X 10(-2) M had a significant effect on proliferation at 24 and 48 hr and this was not reversible on removal of diacid. At 5 X 10(-2) M for 24 hr, C(6)2Na had no effect and at 5 X 10(-2) M for 48 hr had an effect which was marginally significant, but reversible. Swelling and disruption of mitochondria was seen in cells exposed to C(9)2Na at 5 X 10(-2) M for 1 hr and longer, but even at 10(-1) M, cells exposed to C(6)2Na were minimally affected. The results could encourage further investigations of the feasibility of azelaic acid therapy for uveal and ocular adnexal melanoma.

Azelaic acid--biochemistry and metabolism.

Medium chain length dicarboxylic acids (DA) from C8 to C13 are competitive inhibitors of tyrosinase in vitro. The introduction of electron acceptor groups or electron donor groups into the 2 and/or the 8 position of the molecule enhances or reduces respectively the inhibitory effects of DA. In addition to tyrosinase, DA can reversibly inhibit thioredoxin reductase, NADPH cytochrome P450 reductase, NADH dehydrogenase, succinic dehydrogenase and H2CoQ-Cytochrome C oxidoreductase. Among DA, azelaic acid (AA, C9 dicarboxylic acid) is extensively used because: 1) it is much cheaper than other DA; 2) it has no apparent toxic or teratogenic or mutagenic effect; 3) when administered perorally to humans, at the same concentrations as the other DA, it reaches much higher serum and urinary concentrations. Serum concentrations and urinary excretion obtained with intravenous or intra-arterial infusions of AA are significantly higher than those achievable by oral administration. Together with AA, variable amounts of its catabolites, mainly pimelic acid, are found in serum and urine, indicating an involvement of mitochondrial beta-oxidative enzymes. Short-lived serum levels of AA follow a single 1 h intravenous infusion, but prolonging the period of infusion with successive doses of similar concentration produces sustained higher levels during the period of administration. These levels are consistent with the concentrations of AA capable of producing a cytotoxic effect on tumoral cells in vitro. AA is capable of crossing the blood-brain barrier: its concentration in the cerebrospinal fluid is normally in the range of 2-5% of the values in the serum.

Hyperpigmentary disorders--mechanisms of action. Effect of azelaic acid on melanoma and other tumoral cells in culture.

Azelaic acid has been shown to have a dose- and time-dependent inhibitory effect on both proliferation and cell viability of murine and human melanoma cells at a concentration of 10(-3) M and higher. It also has an inhibitory effect on DNA synthesis and plasminogen activator activity, and causes swelling and vacuolation of mitochondria. These effects have also been observed with other tumoral cells in culture-lymphoma and leukaemia derived cell lines, and human squamous cell carcinoma. Normal cells in culture are not generally affected by exposure to azelaic acid. Tissue culture experiments have confirmed the clinical activity and efficacy of azelaic acid, and biochemical conclusions as to its mode of action.

Ten years' experience of treating lentigo maligna with topical azelaic acid.

Topically applied azelaic acid led to complete clinical and histological resolution of lentigo maligna in more than 50 patients. The therapeutic results are highly durable, in fact 27 out of the 50 are still disease-free, 5-10 years after treatment. There was a recurrence in 11 cases, but all resolved on renewing treatment. The effect of azelaic acid is illustrated in a patient with lentigo maligna monitored clinically, histologically and ultrastructurally over the past 5 years.

Transmission and scanning electron microscopy and freeze-fracture replication of normal human melanocytes and human melanoma cells in tissue culture.

Basic LM, TEM, SEM, and FFR appearances of a pure line of normal human melanocytes derived from foreskin, and a human melanoma line, in cell culture are described. Normal melanocyte cultures exhibit side by side, cells of widely different melanogenic activities--possible clones--and melanosomes of bizarre shape and internal structure are frequent. Aggregates of melanosomes, with or without associated amorphous material, and with no discernible limiting membrane are present within many cells, and occasional simple specialised contacts occur between apposed cells. On replicas of plasma membrane of normal melanocytes, particle densities and diameters on P and E fracture faces were within the ranges for cells in general, and equivalent data for the melanoma cells were not significantly different. Similarly, there was no difference in density of distribution or diameter of nuclear pores between the normal and the tumoural cells.

Azelaic acid.

This review is an update on the literature accumulated over the past 10 years following the original observation that azelaic acid, a naturally occurring and nontoxic C9 dicarboxylic acid, possesses significant biologic properties and a potential as a therapeutic agent. These studies have shown that azelaic acid is a reversible inhibitor of tyrosinase and other oxidoreductases in vitro and that it inhibits mitochondrial respiration. It can also inhibit anaerobic glycolysis. Both in vitro and in vivo it has an antimicrobial effect on both aerobic and anaerobic (Propionibacterium acnes) microorganisms. In tissue culture it exerts a dose- and time-dependent cytotoxic effect on malignant melanocytes, associated with mitochondrial damage and inhibition of deoxyribonucleic acid (DNA) synthesis. Tumoral cell lines not containing tyrosinase are equally affected. Normal cells in culture exposed to the same concentrations of the diacid that are toxic for tumoral cells are in general not damaged. Radioactive azelaic acid has been shown to penetrate tumoral cells at a higher level than normal cells of the corresponding line. Topically applied (a 20% cream), it has been shown to be of therapeutic value in skin disorders of different etiologies. Its beneficial effect on various forms of acne (comedogenic, papulopustular, nodulocystic) has been clearly demonstrated. Particularly important is its action on abnormal melanocytes, which has led to the possibility of obtaining good results on melasma and highly durable therapeutic responses on lentigo maligna. It is also capable of causing regression of cutaneous malignant melanoma, but its role in melanoma therapy remains to be investigated.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative cytotoxicity of phenols in vitro.

Two melanotic human melanoma cell lines, IRE 1 and IRE 2, and the lymphoma- and leukaemia-derived cell lines Raji and K 562, were exposed to different concentrations (from 5 X 10(-3) M to 10(-5) M) of phenols, both substrates (s) and non-substrates (ns) of tyrosinase, in the presence or absence of the oxygen-radical-scavenger enzymes superoxide dismutase, catalase and peroxidase. Monophenols were tyrosine (s), 4-hydroxyanisole (s) and butylated hydroxyanisole (ns); diphenols were L-3,4-dihydroxyphenylalanine (s), dopamine (3,4-dihydroxyphenethylamine) (s), terbutylcatechol (s), hydroquinone (s) and resorcinol (ns); triphenols were 6-hydroxydopa (3,4,6-trihydroxyphenylalanine) (s) and methyl gallate (s). Triphenols and o- and p-diphenols underwent complete oxidation in culture medium within 24 h of incubation and were significantly more toxic than monophenols and the m-diphenol resorcinol, which, under the same cultural conditions, were much more stable. No significant differences in percentage survival were found among the different cell lines for each drug tested. The major component of toxicity up to 24 h of di- and tri-phenols is due to toxic oxygen species acting outside the cells and not to cellular uptake of these phenols as such. In fact the addition of oxygen-radical-scavenger enzymes significantly (P less than 0.01) decreased the adverse effect of these drugs on all cell lines. The lower toxicity of monophenols and resorcinol as compared with that of di- and tri-phenols is due, in our opinion, to the fact that they are less oxidized under the conditions existing in the culture medium, and therefore do not produce sufficient levels of oxygen radicals. For these compounds, a primary intracellular action has to be taken into account to explain their cytotoxicity.