Pro-Carcinogenic Action of Beta-Carotene Homer S. Black Department of Dermatology Baylor College of Medicine Houston, Texas, USA Good afternoon ladies and gentlemen. I want to thank Professor Lesley Rhodes for inviting me to participate in this symposium on mechanisms related to systemic photoprotection, and specifically I want to talk to you about the Anti- and Pro-carcinogenic action of two potential systemic photoprotectants. In this talk I want to define photoprotection as protection to the ultimate end-point of UV exposure, i.e. skin cancer

Dietary Recommendations* (to reduce cancer risk) Caloric reduction, i.e., lowering the level of free-radical reactions arising in the course of normal metabolism Minimize dietary components, e.g., polyunsaturated fats, that tend to increase the level of free-radical reactions Supplement the diet with one or more free-radical reaction inhibitors, i.e., antioxidants It is this recommendation and mechanism of photoprotection that requires closer scrutiny and is an area that I have had some experience with two potential systemic photoprotectants that I would like to share. *Harman D. The free radical theory of aging. In: Pryor WA, ed. Free Radicals in Biology V. New York: Academic Press, 1982.

Butylated Hydroxytoluene (BHT) The first of these examples is BHT, a synthetic phenolic compound used as a food antioxidant since 1954. BHT is known to provide protection against UV-induced carcinogenesis, both with respect to tumor latency and multiplicity and, as shown here, severity of the tumors that do occur. It is assumed that the mechanism by which BHT exerts its anti-carcinogenic activity involves the quenching of reactive oxygen species and lipid soluble radicals. Control BHT

Carotenoids Lycopene Zeaxanthin Canthaxanthin -Carotene The antioxidant I wished to speak about today is a carotenoid, β-carotene, a tetraterpenoid. It has no effect on epidermal absorption . Canthaxanthin -Carotene

“Human cancer risks are inversely correlated with (a) blood retinol and (b) dietary ß-carotene” Peto, R, Doll, R, Buckley, JD. And Sporn, MB. Can dietary beta-carotene materially reduce human cancer rates” Nature 290: 201-208, 1981. From a epidemiologic study in 1981 in which individuals that consumed greater levels of green, leafy vegetables seemed to exhibit lower risk for cancer. Because these foods are rich in beta-carotene and, based upon the carotenoids specific capacity to quench singlet oxygen, scavenge oxy-radicals, and terminate free radical reactions, it was suggested that this caroetnoid might be responsible for this anti-carcinogenic potential.

β-Carotene Photoprotection Mathews-Roth MM. Antitumor activity of β-carotene, canthaxanthin, and phytoene. Oncology, 1982; 38:33-37. Mathews-Roth MM, Krinsky NI. Carotenoid dose level and protection against UV-B-induced skin tumors. Photochem Photobiol 1985; 42: 33-38. Indeed. Earlier studies had demonstrated that it could inhibit UVR-carcinogenesis.

Influence of β-Carotene on Tumor Incidence However, we found that it actually exacerbates UV-carcinogenesis under certain dietary conditions. Shown here when semi-defined diets are supplemented with the carotenoid, tumor latent period is significantly shortened.

And tumor multiplicity, i. e. , number of tumors per animal And tumor multiplicity, i.e., number of tumors per animal., is increased.

Influence of Diet on β-Carotene Modulated UVR-Carcinogenic Expression Med.Tumor Time, wk Tumors/Animal Closed Formula Control 20.6 0.52 0.07% β-car 20.0 0.60 Semi-Defined 19.5 17.2 (p<0.002) 1.63 (p<0.03) In an effort to discern what could be responsible for the discortant observations of photoprotection and exacerbation, comparisons were made with closed-formula rations and semi-defined diets….

-Electron Transfer Scheme- Proposed Mechanism by which -Carotene Interacts with Vitamins E and C to Quench Radical Reactions -Electron Transfer Scheme- George Truscott and colleagues offered a mechanism by which beta-carotene might act as a pro-oxidant at high oxygen tensions or oxidative stress. Based upon the relative electron transfer rate constants for interaction between β-carotene, α-tocopherol, and ascorbic acid, a mechanism was proposed by which -carotene interacts with both vitamins E and C to quench radical reactions. Lack of adequate vitamin C, in the case of smokers, or excess vitamin C under oxidative stress, e.g., UV exposure, could lead to an increased level of un- repaired -carotene radical cation by stoichiometrically blocking the redox pathway and result in a pro-oxidant/pro-carcinogenic state. Edge R, Land EJ, McGarvey D, Mulroy L, Truscott TG, Relative One Electron Reduction Potentials of Carotenoid Radical Cations…..J Amer Chem Soc 120, 4987, 1998.

Repair of the Carotene Cation Radical by Ascorbic Acid CAR   + AscH CAR + Asc + H+ Β-carotene can act as a pro-oxidant at high oxygen concentrations and under oxidative stress conditions, exhibits either limited antioxidant properties or even as a pro-oxidant effect. It has been shown that many strongly oxidizing species, especially peroxyl radicals, convert the carotenoid to the one-electron oxidized form resulting in the radical cation. This radical cation exhibits a reduction potential of about 1000 mv and represents a strong oxidizing agent, itself. Furthermore, depending upon the microenvironment, has a rather long life-time, suggesting if un-repaired, the radical cation could inflict considerable damage to biological membranes, and thus, explain the pro-oxidative/carcinogenic influence of β-carotene. It has been demonstrated that -carotene reacts with the tocopherol radical cation to produce the carotenoid radical cation which, in turn, could be repaired by ascorbic acid.

Tumor Incidence: -carotene,  vit C However, when vitamin C was either removed or increased (6-fold) in -carotene supplemented semi-synthetic diets, no influence on the carotenoids exacerbative effect was observed, as shown here. These findings weaken the argument for such a repair mechanism, but can explain the pro-oxidative properites of β-carotene . It should be noted that, unlike humans, mice have no dietary requirement for vitamin C and it is still possible that vitamin C supplementation could protect against the exacerbation of lung cancer in smokers.

What Repairs the β-Carotene Radical Cation? CAR●+ + ? CAR + H● + ? How the Carotene radical cation might be repaired in the closed formula diets is unknown. It may be isomers of the carotenoid, other carotenoids, other phytochemicals or a combination of a broad array of these ingredients.

Tumor Incidence: Beta-carotene, low Vit E; No Vit C Nor did 10-fold increased levels of vitamin E influence -carotene mediated UV-carcinogenic expression. However, reduction in the level of dietary vitamin E augmented -carotene –mediated exacerbation, confirming a vitamin E and -carotene interaction.

Influence of Varying Levels of Vitamins E and C on β-Carotene Modulated Tumor Multiplicity Control (-βC) + βC + βC – Vit C + βC – Vit C, low E 1.05 3.2 3.45 5.9 Control – semi-defined diet containing 110 mg vitamin E and 990 mg vitamin C/kg diet. +BC -0.07% BC (0.79 g/kg – as you see, increasing the tumor burden 3-fold. BC – Vit C – you can see there is no significant effect of removing vitamin C from the diet +BC-Vit C, low Vit E - reducing vit E to 49 mg/kg – there is a 6-fold increase compared to control and nearly 2-fold increase compared to BC.

“Until further insight is gained, ß-carotene should not be recommended for use in cancer prevention in the general population and it should not be assumed that ß-carotene is responsible for the cancer protecting effects of diets rich in carotenoid containing fruits and vegetables” IARC Working Group on the Evaluation of Cancer Preventive Agents. IARC Handbooks of Cancer Prevention, “Carotenoids”, Vol. 2, Lyon, 1998. “A general recommendation to consume supplements for cancer prevention might have unexpected adverse effects” and thus “Dietary supplements are not recommended for cancer prevention” World Cancer Research Fund/American Institute for Cancer Research, Food, Nutrition, Physical Activity, and the Prevention of Cancer: A Global Perspective. Washington, D.C: AICR, 2007. In conclusion, -carotene suffers even greaterlimitations as a photoprotectant. It has been used at 0.07% w/w of the diet – but that is 160-fold higher that the 50 mg daily doses used in most clinical trials. And there is the potential for it to exacerbate UV-carcinogenesis, depending upon dietary parameters. Indeed, the IARC and World Cancer Fund have made a sea change in regard to antioxidant suppoementation.

Recommendation The best recommendation for maintaining a strong defensive antioxidant system may be the consumption of a balanced diet containing adequate green leafy and yellow vegetables that are known to be rich in a broad range of antioxidants and from which the original observations of reduced cancer risks were based. Recommendations and this should guide us in a search ofo a systemic photoprotectant.