Scientific Report 2005

Molecular and Experimental Medicine

Division Of Biochemistry

Preserving Vision in Glaucoma and Macular Degeneration: Neuroprotective Effects of the Flavonoids

A. Hanneken, J. Johnson, F.-F. Lin

Nerve cell damage is the primary cause of visual loss in patients with glaucoma, retinal detachments, and macular degeneration. Among people more than 75 years old, 30% have one of these conditions, and the incidence is expected to increase as the population ages. Restoring the function of injured nerve cells has been particularly difficult both in the CNS and in ocular diseases.

We formed an interdisciplinary research team with P. Maher and her colleagues at the Salk Institute, La Jolla, California, to explore the ability of flavonoids to prevent oxidative stress, the type of nerve injury thought to occur in the eye. This collaboration was started and the approach was validated after a large, 10-year clinical trial sponsored by the National Eye Institute indicated that antioxidants could reduce the progression of macular degeneration.

Flavonoids have 4 properties that make them promising candidates for the treatment of eye diseases. The compounds are potent antioxidants and free-radical scavengers, they induce neuronal recovery after injury, they are antiangiogenic (i.e., inhibit growth of blood vessels that occurs in macular degeneration and diabetic retinopathy), and they are well tolerated orally. Specific flavonoids can enhance the production of glutathione, block the production of reactive oxygen species, and prevent the late influx of calcium, all of which are activities that prevent specific events in the nerve cell death pathway. In addition, the flavonoids can activate the antioxidant response element, which induces the expression of genes that increase cells’ resistance to oxidative stress. The ability of flavonoids to restore the health of injured neuronal cells and induce the outgrowth of neurites gives these compounds a unique set of advantages compared with other antioxidants.

The goal of our research is to determine the specific classes and the chemical structures of dietary flavonoids that are the most effective at preventing oxidative stress–induced cell death in retinal ganglion nerve cells and retinal pigment epithelial cells. We are also investigating the mechanisms behind this protective activity. We screened multiple different flavonoids for their ability to protect these cell types from oxidative stress induced by hydrogen peroxide and tert-butyl hydroperoxide (Fig. 1). To date, we have identified several flavonoids that are both potent and effective in preventing cell death in each of these cell types (Table 1). We found other flavonoids that have limited or no effect.

Fig. 1. Chemical structures of the dietary flavonoids. EGCG = (–)-epigallocatechin gallate.

Table 1. The potency of various flavonoids in protecting primary human retinal pigment epithelium cells from oxidative stress–induced cell death*
Flavonoid	Free hydroxyl positions	Common name	EC50, μM		LD50, μM
			t-BOOH	H2O2
Flavone	5,6,7	Baicalein	8 ± 1	21 ± 1	>>100
	3´,4´,5,7	Luteolin	2 ± 1	3 ± 1	>50
Flavonol	3,6		7 ± 1	11 ± 1	>>50
	3,7		9 ± 1	8 ± 1	27
	3,5,7	Galangin	26 ± 1	61 ± 1	70
	3,3´,4´,7	Fisetin	3 ± 1	5 ± 1	>50
	3,4´,5,7	Kaempferol	~50	No	~50
	3,3´,4´,5,7	Quercetin	6 ± 1	11 ± 2	>50
	3,3´methoxy,4´,5,7	Isorhamnetin	>>50	No	>50
	3,3´,4´,5,5´,7	Myricetin	>50	No	>>50
Isoflavone	4´,5,7	Genistein	No	No	>50
Flavanone	4´,5,7	Naringenin	No	No	>>50
	3´,4´,5,7	Eriodictyol	7 ± 1	11 ± 1	>100
	3,3´,4´,5,7	Taxifolin	>50	No	>>50
Flavanol	3,3´,4´,5,7	Catechin	No	No	>>50
	3,3´,4´,5,7	Epicatechin	No	No	>>50
		Epigallocatechin-3-gallate	22 ± 1	30 ± 2	>100
Anthocyanidin	3,3´,4,4´,5,7	Cyanidin	No	No	>>50
* Half maximal effective concentrations (EC50) were determined by exposing retinal pigment epithelium cells to tert-butyl hydroperoxide (t-BOOH) or hydrogen peroxide (H2O2) in the presence of flavonoids. The doses of the compounds that caused 50% cell death (LD50) were measured by using exposure to flavonoids alone. Values represent the average of 2–3 independent experiments. Each study was conducted with 2–4 replicates. No = little or no efficacy at doses up to 50 μM.

As illustrated in Figure 2, luteolin, a flavonoid present in spinach, prevents oxidative stress–induced cell death in retinal pigment epithelial cells, the cells that die in patients with macular degeneration. Luteolin has no cellular toxic effects over a wide range of concentrations. Other flavonoids, including quercetin, fisetin, and eriodictyol, are also effective; genistein and myricetin are ineffective.

Fig. 2. Luteolin protects retinal pigment epithelial cells from oxidative stress–induced cell death. H2O2 = hydrogen peroxide; t-BOOH = tert-butyl hydroperoxide.

On the basis of these early findings, we compiled a list (Table 2) of the fruits and vegetables that contain the highest concentrations of the flavonoids that are the most effective at preventing oxidative stress–induced cell death in cells derived from the eye. Future studies will be directed toward validating and expanding these results.

Table 2. Dietary flavonoids that protect retinal cells from injury and death in macular degeneration
Flavonoid	Dietary source
Luteolin	Spinach, wild greens, hot peppers, celery, thyme, parsley, mint
Quercetin	Onions (especially yellow), cranberries, cocoa, wild greens, capers, fennel, spinach, chives, celery, cherries, blueberries, apples, kale, red wine
Eriodictyol	Peppermint, citrus juices (lemon, lime, sour orange)
Fisetin	Strawberries, tomatoes, onion, oranges, apples, peaches, grapes, kiwifruit, persimmons

This research is the result of a partnership formed between the Scripps Mericos Eye Institute and Scripps Research aimed at bringing together the promise of biomedical research and the practice of medicine.

Publications

Lin, F.-F., Maher, P., Hanneken, A. Flavonoids protect human retinal pigment epithelial cells from oxidative stress-induced death. Invest. Ophthalmol. Vis. Sci., in press.

Maher, P., Hanneken A. Flavonoids protect retinal ganglion cells from oxidative stress-induced death. Invest. Ophthalmol. Vis. Sci., in press.

Maher, P., Hanneken, A.The molecular basis of oxidative stress-induced cell death in an immortalized retinal ganglion cell line. Invest. Ophthalmol. Vis. Sci. 46:749, 2005.