Long-term cure of the photosensitivity of murine erythropoietic protoporphyria by preselective gene therapy.

Definitive cure of an animal model of a human disease by gene transfer into hematopoietic stem cells has not yet been accomplished in the absence of spontaneous in vivo selection for transduced cells. Erythropoietic protoporphyria is a genetic disease in which ferrochelatase is defective. Protoporphyrin accumulates in erythrocytes, leaks into the plasma and results in severe skin photosensitivity. Using a mouse model of erythropoietic protoporphyria, we demonstrate here that ex vivo preselection of hematopoietic stem cells transduced with a polycistronic retrovirus expressing both human ferrochelatase and green fluorescent protein results in complete and long-term correction of skin photosensitivity in all transplanted mice.

Preferential light absorption in atheromas in vitro. Implications for laser angioplasty.

Laser angioplasty, the in situ ablation of arterial obstructions with laser radiation, has been demonstrated in animal models and early clinical trials. A problem with this technique, however, is the possibility of thermal damage to adjacent or underlying normal tissues that also absorb the radiation. Using a spectrophotometer with an integrating sphere and a specially constructed tunable-dye laser-based spectrophotometer, we evaluated the transmittance and remittance of human cadaveric atheromas and adjacent normal aorta from 250 to 1,300 nm to identify wavebands where there is preferential light absorption by atheromas. Data were analyzed by both the Kubelka-Munk formalism and a Beer's law model. Both methods indicate that atheromas absorb more than normal aorta between 420 and 530 nm. At 470 nm the average Kubelka-Munk absorption coefficient of atheromas from 10 cadavers was 54 +/- 9 cm-1 compared with 26 +/- 6 cm-1 for normal aortic specimens from seven cadavers. Yellow chromophores responsible for the atheroma absorbance were extractable with xylenes. Thin-layer chromatography and absorption spectra identified the extracted chromophores as predominantly consisting of a mix of carotenoids, which are known constituents of atheromatous lesions. Preferential absorption of blue light by carotenoids in atheromas may permit selective ablation of atheromatous obstructions with appropriate pulses of laser radiation.

Amelioration of the metabolic defect in erythropoietic protoporphyria by expression of human ferrochelatase in cultured cells.

The cDNA for human ferrochelatase, the enzyme that is defective in the rare genetic disease erythropoietic protoporphyria (EPP), was tested for its ability to allow the expression of ferrochelatase in mammalian cells. The cDNA was ligated to the plasmid expression vectors pCD and pED6 and transfected into COS-1 and CHO-DUKX cells, respectively. In each case, ferrochelatase activity increased. The cDNA was also ligated into the retroviral vector pLXSN, and virus-packaging cells were produced. Supernatants from these cells were used to infect fibroblasts in vitro from a patient with EPP. We found that the infected cells containing the ferrochelatase cDNA had enzyme levels in the range of normal fibroblasts and that they did not accumulate protoporphyrin when grown in the presence of delta-aminolevulinic acid. We conclude that introducing the cDNA for normal ferrochelatase into fibroblasts from an EPP patient restores ferrochelatase enzyme activity to the normal range. These experiments suggest potential for genetic therapy in EPP.

The detection of carotenoid pigments in human skin.

Carotenoid pigments were extracted chemically from both epidermis and dermis obtained from non-carotenemic individuals at autopsy. Absorption maxima characteristic of beta-carotene were found in the extracts of specimens of epidermis following cantharidin application in volunteers made carotenemic by the ingestion of beta-carotene (180 mg/day for 10 weeks). These maxima were absent in the extracts of epidermis obtained from the volunteers before beta-carotene ingestion.

Plasma concentrations of carotenoids after large doses of beta-carotene.

Plasma concentrations of beta-carotene were determined in healthy men ingesting 180 mg beta-carotene/d during studies on the effects of beta-carotene on sunburn prevention. This dose is also used in the treatment of light-sensitive skin diseases. beta-carotene concentrations were found to reach a plateau in 1.5 to 4 wk, although there was much individual variation in the actual serum concentrations achieved. Carotenodermia was present in most subjects. No evidence of toxicity was found, confirming the findings of previous photosensitivity-prevention studies, which also reported no significant toxicity attributable to beta-carotene.

Beta-carotene therapy for erythropoietic protoporphyria and other photosensitivity diseases.

This paper describes the development of the use of carotenoid pigments in the treatment of light-sensitive skin diseases. It also discusses the animal and human studies involved in determining whether carotenoids have any anti-cancer activity. The possible mechanisms of carotenoid photoprotective and anti-cancer actions are briefly discussed.

Erythropoietic protoporphyria. 10 years experience.

The clinical and laboratory findings in 32 patients with erythropoietic protoporphyria as well as a review of the pertinent literature on this relatively recently described form of porphyria are presented. The disease is thought to be transmitted in an autosomal dominant fashion with variable penetrance and was characterized in these 32 patients by the onset in childhood of burning (97 per cent) and itching (88 per cent) of the skin on exposure to sunlight. This was accompanied by edema (49 per cent) and erythema (69 per cent) of the exposed areas. Vesicles, petechiae and residual scarring occurred less frequently. Associated abnormalities included cholelithiasis (12 per cent), anemia (27 per cent) and abnormal liver function studies (4 per cent). Reports of associated liver disease including nine cases of fatal hepatic failure, are reviewed. Current methods of diagnosis as well as theories of pathophysiology of the disease are presented. Nineteen of 23 of these patients recently treated with beta-carotene responded with significant increase in their tolerance to sun exposure.

Carotenoids in erythropoietic protoporphyria and other photosensitivity diseases.

Studies in bacteria, animals and humans have demonstrated that carotenoid pigments can prevent or lessen photosensitivity by endogenous photosensitizers such as chlorophyll or porphyrins, as well as by exogenous photosensitizers such as dyes (e.g., toluidine blue) or porphyrin derivatives. The carotenoids beta-carotene and canthaxanthin have been found to be effective in the treatment of the photosensitivity associated with EPP and certain other photosensitivity diseases. No serious toxicity has been reported from their use, although the use of canthaxanthin is not recommended because of its propensity to form retinal granules. The pigments perform their protective function by quenching excited species formed by the interaction of porphyrins or dyes, light and air, thereby preventing the cellular damage which leads to the symptoms of photosensitivity.

Beta carotene therapy for erythropoietic protoporphyria and other photosensitivity diseases.

We treated with high doses of oral beta carotene (Solatene) (15 to 180 mg/day) 133 patients suffering from erythropoietic protoporphyria (EPP), 27 patients with polymorphous light eruption, six patients with solar urticaria, three patients with hydroa aestivale, one patient with porphyria cutanea tarda, and two patients with actinic reticuloid to relieve the photosensitivity associated with these diseases. Eighty-four percent of the patients with erythropoietic protoporphyria increased by a factor of 3 or more their ability to tolerate sunlight. On the other hand, only nine of the patients with polymorphous light eruption, and one fifth of the patients with all of the other forms of photosensitivity treated showed similar improvement. We conclude that beta carotene is an effective treatment for EPP, but that other forms of photosensitivity will need empirical therapeutic study with beta carotene to determine the range of effectiveness, if any, of this compound in conditions other than EPP.

Dietary beta-carotene and 13-cis-retinoic acid are not effective in preventing some features of UVB-induced dermal damage in hairless mice.

Albino hairless mice were fed diets containing 10 g/kg feed of beta-carotene and 200 mg/kg feed of 13-cis retinoic acid to assess the ability of these molecules to prevent UVB-induced dermal damage. Diets were administered for 12 weeks prior to UVB exposure and were continued throughout the 20 week irradiation period. The UVB source was a bank of FS-20 sunlamps (280-400 nm: peak 313 nm). Exposures were thrice weekly at 0.1 J/cm2 per exposure for the first 10 weeks and 0.2 J/cm2 per exposure for the second 10 weeks. Histologic evaluation of skin biopsies revealed no difference, between animals fed active or placebo diets, in UVB-induced elastosis, collagen changes or amounts of glycosaminoglycans and proteoglycans of the ground substance.

Protective role of butylated hydroxytoluene and certain carotenoids in photocarcinogenesis.

Butylated hydroxytoluene (BHT) and certain carotenoid pigments have been found to inhibit photocarcinogenesis in animal models. In addition, BHT protects against UV-B-induced erythema and UV-B induction of ornithine decarboxylase. Studies on the photoprotective mechanism(s) of BHT suggested that changes in the physico-chemical properties of the keratin of the stratum corneum layer of skin occurred, leading to increases in UV absorption of that tissue. These changes might be exerted via the anti-radical action of BHT that retards oxidation and prevents cross-linking of the keratin chains, resulting in a diminution of UV-B radiation reaching potential target sites. The carotenoids beta-carotene, canthaxanthin and phytoene also inhibit UV-B carcinogenesis. beta-Carotene and canthaxanthin are excellent quenchers of singlet oxygen, and all three pigments can quench free radicals. beta-Carotene and canthaxanthin have been shown to quench singlet oxygen/free radical reactions in the skin of porphyric mice, and these two pigments as well as phytoene have been found to quench excited species formed on irradiation of mouse skin by UV-B.

Lack of genotoxicity with beta-carotene.

A literature review was conducted on adverse effects of carotenoids on human and animal development. The data suggest that beta-carotene administration prevented genetic damage caused by mutagens both in bacterial and cell culture systems, and that large doses of pure beta-carotene do not cause embryotoxicity in rodents. In addition, studies of individuals with congenitally high levels of plasma carotenoids and babies born carotenemic because of their mothers' intake of large amounts of carotenoid-containing foods during pregnancy reveal no abnormalities attributable to the carotenoid molecule.

Carotenoid functions in photoprotection and cancer prevention.

Carotenoid pigments in in vitro and in vivo systems are able to quench excited species, such as singlet oxygen and free radicals. The U.S. Food and Drug Administration has approved beta-carotene for use in humans for prevention of the photosensitivity associated with the orphan disease, erythropoietic protoporphyria. Although the usual adult dose used is 180 mg/day, intake up to 300 mg/day is allowed. No serious toxicity to beta-carotene has been reported. Carotenoids have demonstrated some anticancer activity in certain animals. Clinical trials in populations at high risk for developing certain types of cancer are presently underway, using doses of beta-carotene ranging from 15 to 50 mg/day, or much lower doses than used for photosensitivity prevention. There is no way of predicting which dose will be effective in preventing cancer: The lower doses were chosen to avoid development of marked carotenodermia; they are sufficient to cause an increase in the serum carotenoid level but may, occasionally, in individuals, cause the development of some degree of carotenodermia. This article reviews development of the use of beta-carotene for preventing photosensitivity in humans, studies investigating the anticancer properties of the carotenoids, and studies aimed at understanding how the carotenoids exert their protective functions.

Systemic photoprotection.

At present, three classes of compounds are used as systemic photoprotective agents, but only for specific indications, not for general use in healthy individuals. Beta-carotene prevents or lessens photosensitivity in most patients with erythropoietic protoporphyria and in some patients with other photosensitivity diseases. The antimalarial drugs can clear up skin lesions in patients with polymorphous light eruption and solar urticaria who cannot obtain relief with topical sunscreens and in some patients with porphyria cutanea tarda. Oral psoralens and controlled exposure to sunlight or artificial sources of UVA radiation can increase tolerance to sunlight in fair-skinned individuals and in certain patients with vitiligo or polymorphous light eruption.