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Institute of Food Research, Norwich Research Park, Colney; Norwich NR4 7UA, UK
3To whom correspondence should be addressed. E-mail: sian.astley{at}bbsrc.ac.uk.
KEY WORDS: • lycopene • carotenoids • DNA damage and repair • single cell gel electrophoresis • lymphocytes
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Lower incidences of cancer are associated with higher consumption of fruits and vegetables (3). These foods contain relatively high amounts of components with inherent antioxidant properties, including vitamins E and C, the carotenoids, and plant polyphenols. This has led to the suggestion that dietary compounds such as lycopene augment cellular defenses, helping to protect cellular components from oxidative damage. Some purportedly protective dietary components, however, have biological activities that are not directly attributable to antioxidant effects. It is becoming increasingly evident that the carotenoids, including lycopene, may also have bioactivities capable of modifying, for example, gene expression. Thus, an alternative mechanism for the action of lycopene might be enhancement of DNA repair.
How strong the evidence is that lycopene supplementation can modify biomarkers of oxidative DNA damage and repair in human lymphocytes is determined by the reliability of DNA oxidative biomarkers, the relative importance of DNA damage compared with repair, and the validity of lymphocytes as a surrogate tissue.
Biomarkers of oxidative DNA damage
8-Oxo-2'-deoxyguanosine (8-oxo-dGuo)4 is one of the most common oxidative lesions in DNA. Its presence can result in guanine-cytosine to thymine-adenine transversion, unless repaired prior to DNA replication (4), and may consequently induce mutations. This change, however, is not a unique marker for DNA oxidative damage. There are many different oxidative DNA lesions, although few of their biological consequences have been examined in detail. This paucity of information and the inconsistent application of different methods for measuring oxidative DNA damage (e.g., detecting oxidized bases and nucleosides and strand breaks using urine as well as target and surrogate tissues) means there is still a lack of consensus both in the measurement of oxidative DNA damage, as well as its repair, and in the interpretation of its significance for human health.
Although it is perhaps natural that different laboratories should have preferred ways of expressing their results, or even different protocols for analysis, this has led to mutual incomprehension when comparing data. Similarly, the group of methods (e.g., alkaline elution, alkaline unwinding, and the comet assay), which are dependent on converting lesions to DNA breaks and measuring the frequency of these breaks, although apparently simple to use, are subject to misinterpretation. The assays can be calibrated against X-rays, but the majority consider DNA damage semiquantitatively (e.g., % DNA present in the tail, comet assay). It is, however, incorrect to assume that single-strand breaks (SSBs) relate only to DNA damage. Repair, which occurs rapidly in most cells, can cause SSBs to accumulate as an indirect indicator of ongoing repair if cells are deficient in repair enzymes (5), are slow at removing the 3'-end blocking groups (6), have low stores of replacement nucleotides (7), or display reduced repair capacity because they are highly differentiated or nondividing (8). Lymphocytes are a case in point; they are highly differentiated and divide only with specific stimulation with, for example, a mitogen or CD3.
Surrogate and target tissues
Lymphocytes are often used as surrogate tissues in experiments following response to supplementation because these cells are easily accessible from healthy volunteers and their response may reflect that in other tissues (9,10). There are, however, no experimental data to support such a relation (11); factors responsible for oxidative DNA damage or repair vary in their magnitude and effect in different tissues, and differences in metabolic rate influence the rate of oxidative damage (12) as well as the availability of resources to respond to them. For example, activities of key antioxidant enzymes, including catalase, glutathione peroxidase, and superoxide dismutase, vary in different organs (13) and therefore, by implication, in different cells.
Biomarkers of oxidative DNA repair
Ultimately, provided DNA repair is timely and accurate, the extent or frequency of oxidative damage is perhaps less important than DNA repair. However, although there has been considerable interest in the types and frequency of DNA lesions, repair has been largely ignored in the context of nutrition. This may be because, like oxidative damage, DNA repair is not the result of a single process. There are numerous variations, including direct repair, mismatched-base repair, base excision repair (BER), nucleotide excision repair, double-strand break repair, and damage by-pass. Each repair mechanism is specific to the type of repair being undertaken, but many have common features that cannot be distinguished easily. For example, repair of SSBs, via BER, involves the identification of an incorrect or damaged base (e.g., uracil in DNA or 8-oxo-dGuo), its removal by a DNA glycosylase to create an abasic site (AP), nicking of the damaged DNA strand (by AP endonuclease) upstream of the AP and excision of the AP before the correct base(s) can be inserted by one of many DNA polymerases, and finally closure of the loose ends by DNA ligase. Bioactive compounds may affect any one of these process, directly or indirectly, but assays to determine anything more than the complete process (e.g., BER) are not available.
Epidemiology for lycopene and tomatoes
In Western diets, tomatoes are usually the major source of lycopene, although there are other minor sources, such as watermelon and pink grapefruit. Lycopene, a nonprovitamin A carotenoid, provides the familiar red coloration of tomatoes, but different varieties have a unique composition, and lycopene is not the only bioactive compound present; tomatoes contain small quantities of minerals, including iron, copper, and zinc, as well as vitamins C and E, folates, and various flavonoids and phenolic acids.
Careful examination of the epidemiology demonstrates that daily tomato consumption is associated with reduced risk of cancer of the respiratory and digestive tracts, stomach, and lung; there are insufficient data to support a role in prevention elsewhere (3). There is also no difference in the protection offered by cooked tomato products compared with raw tomatoes, with the exception of prostate cancer, where consumption of cooked tomato sauces is substantially better (14). However, there is little agreement between the effects of tomatoes and lycopene. For example, in the EURAMIC study, Spanish volunteers with the lowest risk of heart attack also had the lowest plasma concentration of lycopene (15). Similarly, despite health statistics suggesting high rates of cardiovascular disease and cancer in Northern Ireland, compared with southern European countries, the EU-funded Five Countries Study reported that plasma lycopene was lowest in Spain and highest in Northern Ireland (16). These data appear to make nonsense of the overwhelming evidence in favor of consumption of fruits and vegetables if taken at face value.
Modern analytical techniques and instrumentation make it possible to describe the complex chemical nature of foods with ever-increasing degrees of accuracy and sensitivity. However, the types and absolute quantities of dietary compounds, such as lycopene, in foods may have very little bearing on their contribution to human health because only a portion is absorbed and ultimately utilized. Only when examined more closely does the real situation become apparent; the epidemiology is not well founded. Already biased by researcher preference, epidemiological analyses should be based on good food-composition data. But, until recently, it was difficult to measure lycopene quantitatively, and there is still a lack of information available describing lycopene bioavailability or, more specifically, its absorption, digestion, metabolism, and excretion.
Studies in vitro and in vivo with lycopene supplementation
Because reports on the effects of carotenoids are conflicting, the authors examined the similarities and differences from contiguous studies in vitro and in vivo (17). Single-cell gel electrophoresis was employed to measure the frequency of SSBs in the lymphocyte cell line MOLT-17 (as a model system) and human peripheral blood lymphocytes (PBLs) with and without oxidative challenge in the form of hydrogen peroxide (100 µmol/L, 5 min, 4°C). In this way, background levels of DNA SSBs and resistance of the DNA to oxidative challenge were assessed simultaneously, before and after supplementation, in an in vitro model system and ex vivo.
The MOLT-17 human lymphocyte cell line, derived from a 5-y-old female patient with acute lymphocytic leukemia (18), was supplemented with ß-carotene, lutein, or lycopene at concentrations from 0 to 8 µmol/L. Similarly, lutein, lycopene, and ß-carotene, in the form of natural isolate capsules (15 mg/d for 4 wk), were used to supplement the diets of the same apparently healthy male volunteers, at the same time of year, over 3 y in 3 separate randomized double-blind studies.
Lutein, lycopene, and ß-carotene were selected because they are 3 of the most commonly consumed carotenoids in the UK: lutein from green and yellow vegetables, in particular green peas; lycopene from tomatoes and tomato products; and ß-carotene from carrots and green vegetables. The range of concentrations used for supplementing the cells in vitro was selected on the basis that 1) it included typical plasma concentrations found in northern and southern Europe; 2) the upper concentration (8 µmol/L), which is >4 times the usual plasma concentration found in a UK population (19,20), is at the upper limit of plasma concentrations that might reasonably be achieved by dietary manipulation (i.e., without use of isolate supplements); and 3) 8 µmol/L is twice the concentration at which antioxidant protection had previously been shown to be reduced, suggesting a shift toward pro-oxidant behavior (21).
Although supplements were used in these studies, the doses (15 mg/d) are comparable with the addition of 
150 g/d of an equivalent food source (e.g., 150 g/d of carrots, tomatoes, or green peas). Foods were not used because of confounding factors, including the presence of other compounds with bioactive potential, which could not be recreated in vitro, and personal taste preferences.
The findings of these studies are described and discussed elsewhere in detail (22). Briefly, carotenoid uptake in vitro was shown to be dose dependent. Media ß-carotene concentration had no effect on SSBs in control cells, but incubation with lycopene or lutein above 2 µmol/L increased the number of SSBs in control cells. MOLT-17 DNA was less susceptible to oxidative damage following incubation with carotenoids between 0.5 and 1 µmol/L; above 1 µmol/L, the effects were ambiguous.
Supplementation of the diet of apparently healthy male volunteers with lutein, ß-carotene, and lycopene raised plasma concentrations to different extents. As a consequence, although lycopene and lutein had no effect on SSBs in control PBLs or following oxidative challenge, increased plasma ß-carotene was associated with more SSBs in control cells whereas PBL DNA resistance to oxidative damage ex vivo was unaffected.
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FOOTNOTES |
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2 Supported by the Ministry of Agriculture, Fisheries and Food (UK), the Food Standards Agency (UK), The Food and Drink Federation (UK), and the Biotechnology and Biological Sciences Research Council (BBSRC), and performed at the Institute of Food Research, Colney, Norwich, UK.
4 Abbreviations used: 8-oxo-dGuo; 8-oxo-2'-deoxyguanosine; AP, abasic site; BER, base excision repair; PBL, peripheral blood lymphocyte; SSB, single-strand break.
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LITERATURE CITED |
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