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.

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.

A double-blind study of cysteine photoprotection in erythropoietic protoporphyria.

In an open trial in 9 patients, we found that the ingestion of 500 mg of cysteine twice a day could prevent or lessen the photosensitivity associated with erythropoietic protoporphyria (EPP). We report here the results of a double-blind cross-over design trial in 16 patients. Patients with EPP were randomized into two groups, one that received 500 mg twice daily of cysteine during the first period and then placebo during the second period, and the second that received placebo first and then cysteine. Each drug ingestion period lasted 8 weeks, and there was a 1 week wash-out period between them. Patients were seen before the first period, at the end of the first period and at the end of the second period for phototesting, an objective method of assessing treatment efficacy, and for blood tests to monitor cysteine safety. The subjects also were asked to keep light-exposure diaries, a subjective method of assessing efficacy. We found that cysteine significantly prolonged the time it took to develop erythema by phototest, objectively demonstrating efficacy. In addition, analysis of patients' diaries showed that cysteine ingestion significantly increased the length of sun exposure it took to develop symptoms of photosensitivity. We conclude that cysteine ingestion may be an effective therapy in the prevention of photosensitivity associated with EPP.

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.

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.

Effects of carotenoid administration on bladder cancer prevention.

Bladder cancer was induced in male B6D2F1 strain mice by the administration of N-butyl-N-(4-hydroxybutyl)nitrosamine. Mice supplemented with beta-carotene for 5 weeks before receiving the carcinogen and maintained on beta-carotene for an additional 26 weeks developed significantly fewer tumors than did unsupplemented mice. Mice receiving canthaxanthin for the same time period showed no protection against the development of bladder cancer.

Distribution of [14C]canthaxanthin and [14C]lycopene in rats and monkeys.

The absorption and distribution of [14C]-canthaxanthin and [14C]lycopene were studied in rats and in rhesus monkeys following the oral administration of [14C]canthaxanthin or [14C]lycopene in olive oil supplemented with 1 mg alpha-tocopherol/mL. For canthaxanthin and lycopene, peak accumulation of radioactivity in plasma occurred between 4 and 8 h in rats and between 8 and 48 h in monkeys. In rats, the liver contained the largest amount of both kinds of radioactive pigments. In monkeys, with the exception of one stomach sample, liver was also the major depot organ for both canthaxanthin and lycopene. The other organs tested accumulated various amounts of pigment. No labeled metabolic products of either canthaxanthin or lycopene were found.

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.

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.

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.

The metabolism of [14C]beta-carotene and the presence of other carotenoids in rats and monkeys.

The metabolism of beta-carotene has been studied in both rats and Rhesus monkeys, following the oral administration of [14C]beta-carotene in olive oil supplemented with 1 mg/mL alpha-tocopherol. In the rats, peak serum accumulation of [14C]retinol occurred 4 h after a single oral dose, but we were not able to detect [14C]beta-carotene in rat sera at any time up to 72 h after dosing. Small amounts of [14C]beta-carotene were found in the livers, although 88-94% of the recovered radioactivity was localized in the retinol fraction after saponification. Although radioactivity was also found in fractions other than beta-carotene and retinol, the amounts were too small to allow characterization. In the monkeys, peak accumulation of [14C]retinol in serum occurred between 8 and 24 h after supplementation. Some [14C]beta-carotene was also present. Most of the absorbed radioactivity was stored in the liver as [14C]retinol, although 2-8% was present as [14C]beta-carotene. Other organs also contained [14C]beta-carotene, confirming the ability of the monkey to absorb intact beta-carotene. In addition, monkey livers and other organs were found to contain lutein, zeaxanthin, alpha-cryptoxanthin, beta-cryptoxanthin and beta-carotene, presumably arising from dietary sources.

Enhancing the carotenoid content of atherosclerotic plaque: implications for laser therapy.

Selective laser ablation of human atherosclerotic plaque is possible because endogenous carotenoid pigments found in atherosclerotic plaque confer a twofold preferential absorption of laser radiation at 450 to 500 nm. In this study, patients with carotid endarterectomy were pretreated with oral beta carotene to determine if the carotenoid content and therefore laser selectivity of plaque could be increased in vivo. Beta carotene-treated patients had a significant, nearly twofold increase in their plaque carotenoid concentration, which increased from 0.22 to 0.40 microgram beta carotene/mg cholesterol. These results suggest that selective ablation of atherosclerotic plaque may be enhanced by pretreating patients with doses of oral beta carotene for short periods of time.

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.