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Agricultural Sciences in China 2007, 6(10): 1246-1255

ScienceDirect

October 2007

Accumulation of Nitrate in Vegetables and Its Possible Implicationsto Human Health

DU Shao-ting, ZHANG Yong-song and LIN Xian-yong
Key Lab of Environmental Remediation and Ecosystem Health, Ministry of EducatiodCollege of Environmental and Resources Sciences, Zhejiang University, Hangzhou 310029, P.R.China

Abstract
In recent times, there are two kinds of completely opposite viewpoints about the impacts of nitrate on human health. To further objectively understand the effects of nitrate on human health, both of harmfulness and possible benefits of nitrate to human body, it is discussed in this review from the aspects of nitrate accumulation in vegetables, the source of nitrate ingested into human body, and the transformation of nitrate in human body, as well as the pathogenesis and physiological functions of nitrate metabolism. Key words: vegetable, nitrate, methemoglobin, carcinogenesis, human health, NO, bacteria

INTRODUCTION
Nitrate is a common chemical compound in the nature, and is widely found in soils, waters, and foods. Generally, nitrate in vegetables is considered to be the main source of dietary nitrate intake (Santamaria et al. 1998). Some previous researches suggested that the vegetables with high nitrate in the diet could put a human into the risk of gastrointestinal cancer and methemoglobinaemia (Bartsch et al. 1988; Slob et al. 1995). Therefore, there is great concern about the nitrate content in the daily diet, especially in vegetables. In recent decades, considerable researches have been done to minimize the nitrate accumulation in vegetables, mainly in plant nutrient management, horticultural technology, and breeding. However, some epidemiological studies also proposed that the nitrate may be advantageous to human health, such as protecting the intestine from harmful bacterial

infection (McKnight et al. 1999; Archer 2002; Dykhuizen et al. 1996). Besides, one of the reduced nitrate products, the NO, is known to be an important signal molecule in regulating many physiological functions in human body, and in addition, it has also served as an effective host defense against pathogens (McKnight et al. 1999; Archer 2002). Accordingly, whether the dietary nitrate is detrimental to human health and environment remains a disputed topic so far, especially after the publication of Nitrate andMan: Toxic, Harmless or Beneficial? by LHirondel J and LHirondel J-L in France in 1996 (English edition, 2001), which further activated the controversy. This review summarizes the accumulation of nitrate in vegetables, and the factors affecting it. Emphasis is also placed on the metabolism mechanism of the ingested nitrate and nitrite in human body and their deleterious and beneficial effects on human health, which can lead to a further understanding of it, and a just evaluation.

This article is translated from its Chinese version in Scieniia Agricultura Sinica. DU Shao-ting, Ph D candidate, E-mail: dushaoting@gmail.com; Correspondence ZHANG Yong-song, Professor,Tel: +86-571-86971151, Fax:+86-571-86971359, E-mail: yszhang@zju,edu.cn

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NITRATEACCUMULATION IN VEGETALBES
The AD1 (acceptable daily intake) of nitrate was allocated as 0-0.37 mg kg-' body weight, whereas, the exceeding of the AD1 limit has occurred frequently in daily human life (Boink and Speijers 2001). Slob et al. (1995) had shown that in adults, 15% of the daily intake exceeded the ADI, and might rise to 45% in young children. The daily nitrate intake occurs from three major sources: vegetables, drinking water, and meat products. Although great differencesexist in diary habits and water quality between different countries, vegetables are still the major source of nitrate intake (Table 1). They constitute 30-90% of the total nitrate intake. Assuming a daily consumption of 0.5 kg fresh vegetables per person, the maximum allowed nitrate levels in vegetables should be 432 mg kg-I. Generally, about 45% of the total nitrate in the vegetables can be reduced by pickling,

Table 1 Estimation of daily nitrate intake and contribution of vegetables in various regions (Hambridge 2003)
Country Eastern Asia Africa Latin America Europe Daily nitrate intake from vegetables (mg d-I) 28 20 55 155 Contribution of vegetables (%) 45 30 65 90

and 75% reduced by cooking. Hence, the maximum allowable limit for nitrate can be increased to 785 mg kg-' for pickled vegetables and 1.440mg kg-' for cooked vegetables, respectively. In the case of a 60 kg body weight with the daily consumption of 100 g vegetables (2500 mg kg-' of nitrate content), there would be 13% excess of the ADI. Grouping of the nitrate content in edible parts, leafy and root vegetables show higher nitrate contents, whereas, the melon and nightshade show a much lower content (Table 2). The trends in Table 3 also show the similar results, which are classified as vegetable varieties. To date, no nitrate standards for vegetables have been introduced in the USA, whereas, a suggested maximum level of nitrate in vegetables of 3 100 mg kg-I has been established in China (Zhou et al. 2000). As shown in Tables, most vegetables can accumulate large amounts of nitrate. It is reported that the highest nitrate-accumulating vegetable is Diplotuxis, found in Italy, which can reach up to 9 300 mg kg-' (Santamaria et al. 1999). Moreover, there is always significant variance of nitrate content among different genotypes, and sometimes it can even reach several folds, such as spinach. The nitrate content in the full-wrinklegenotype, half-wrinkle genotype, and smooth genotype are 446.0, 315.9, and 194.0 mg kg-' FW, respectively, although under the same nitrate nutrition of 18 mmol L-' (Wang et al. 2000). Besides, the nitrate content also differs in

Table 2 Nitrate and nitrite content in edible part of vegetables (Wang et al. 2000)
Vegetable types Root vegetables Green vegetables Cabbage Vegetable varieties Carrot Datoucai leaf-mustard Lettuce Spinach Chinese cabbage Pakchoi Cabbage Cole Wax gourd Cucumber Eggplant NO; (mg kg-1FW) 0.02-0.23 0.12-0.64 0.08-2.15 0-0.73 0-0.65 0.09-2.42 0-0.41 3.64-5.35 0.01-0.06 0-0.11 0.07-0.49 NO; (mg kgl FW) 921-1 956 709-956 123-2 678 239-3 872 429-1 610 1023-3 098 259-1 250 766-1 365 358-680 12-143 250-424

Melon Nightshade

Table 3 Classification of vegetables according to nitrate content (mg kg-'FW) (Santamaria 2006)
~~ ~

Nitrate content (mg kg I Very low (< 200) Low (200-500) Middle (500-1000) High (1 000-2 500) Very high (> 2 500)

FW)

Vegetable varieties Artichoke, asparagus, broad bean, eggplant, garlic, onion, green bean, mushroom, pea, pepper, potato, summer squash, sweet potato, tomato, watermelon Broccoli, carrot, cauliflower, cucumber, pumpkin, 'puntarelle' chicory Cabbage, broccoli, dill, savoy cabbage, turnip Celeriac, Chinese cabbage, endive, fennel, kohlrabi, leek, parsley Celery, cress, chervil. lettuce, red beetroot, spinach, rocket

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various parts of the plant, such as the nitrate content in stem, petiole, and leaf in mustard is 376.0, 3 10.9, and 85.1 mg kg-' F W ,respectively. Generally, the vegetable organs can be listed in the decreasing order of the nitrate content as follows: petiole > leaf > stem > root > inflorescence > tuber >bulb > fruit > seed (Santamaria et al. 2001). However, when the media nitrate concentration is relatively low, the nitrate content will increase in the root, but decrease in the shoot. Contrarily, it will change on the high nitrate supply condition. The factors leading to the difference of nitrate contents in different vegetables are complex, and a number of studies on the mechanism of nitrate accumulation have been done, mainly on the nitrate uptake rate, nitrate reductase activity, and growth rate, which are closely related to carbon metabolism. The carbon metabolism in plant is determined by various growth conditions, including light, temperature, CO, level, and so on (Hageman and Flesher 1960; Toselli et al. 1999; Yu et al. 2006). Moreover, besides the genetic factor, the growth conditions also play a decisive role in affecting the nitrate accumulation in plant. Furthermore, the nitrogen absorbed by most vegetables from soil is nitrate which is sequentially reduced by nitrate reductase (NR), nitrite reductase (NiR), glutamine synthase (GS), and glutamate synthase (GOGAT). Although there are numerous researches on the mechanisms of nitrate accumulation in vegetables, it remains argumentative. However, most of the studies suggest that the imbalance between nitrate uptake and reduction should be the fundamental factor. In addition, once the increment of nitrate accumulation is stronger than its growth, growth lag would strengthen the nitrate accumulation through nutrient enrichment. The inconsistent distribution of nitrate and nitrate reductase in plant cells is another important factor resulting in the higher nitrate contents in some parts of vegetables. In recent years, many researches proposed the beneficial effects of nitrate on human health, and hence, the efforts done to alleviate nitrate accumulation in vegetables are doubted. Therefore, a clear understanding of nitrate metabolism in human body and its relationship with the hypothesized harmful and beneficial effects is the prerequisite for nitrate evaluation, which can also help us to set the allowable limit in food and water.

MECHANISM OF NITRATE IN HUMAN BODY

Nitrate widely exists in the drinking water and food, and as a result, it inevitably enters the human food chain. Therefore, understanding the metabolism mechanism of the ingested nitrate is a prerequisite for proper evaluation of the hypothesized harmful and beneficial effects on human health. Thus, the mechanism of the nitrate in human body will be demonstrated initially.

Translation of nitrate in oral cavity

As the vegetables first enter into the body by swallowing, the conversion of nitrate in the mouth is particularly important. The dorsal surface of the tongue symbiotically harbors a specialized flora of anaerobic nitrate reducing bacteria, which can rapidly reduce nitrate to nitrite (Duncan et a l . 1995), hence, the contents of nitrite in saliva are generally higher, and h can reach 200 p 4 (Benjamin 2000). Some antibiotic inhibition studies showed that amoxicillin (a broad spectrum antibiotic that can inhibit bacteria growth) significantly reduces the nitrite concentrationin the saliva (Dougall et al. 1995),suggestinga critical role of bacteria in the conversion of nitrate to nitrite. However, the nitrite will not be further reduced in the mouth because of the lack of nitrite reductase. Nevertheless, it will be absorbed in the salivary glands, therefore, the nitrite concentration in saliva is actively concentrated by a factor of ten from the plasma, even 50-100 folds of the stomach (Leifert et al. 1999).

Conversion of nitrite in stomach

Because of the absence of nitrite reductase in the oral cavity, nitrite will convert to varieties of nitrogen compounds in the stomach. The main products are NO and N-nitroso compounds. Converting to NO NO generation in human body can be divided into the endogenous and exogenous pathways. Nitrite formed in the mouth can be acidified in two ways: direct acidifying in the acid environment around the teeth caused by microorganisriis (such as Lactobacillus or Streptococcus mutans); or acidifying after being swallowed, in the acidic stomach, which is thought to be more important. However, acidification

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of nitrite produces nitrous acid, which is unstable, spontaneously decomposing to manifold nitrogen compounds. The most important product is NO, and the whole process is as follows: NO; + H HNO,; 3HN0, H,O + 2N0 + NO, + H+;2HN0, H,O + N,O,; N,O, NO + NO, Because the NO generates from the nitrate in food, this process can be defined as an exogenous pathway. It is reported that the NO concentration formed in the stomach can be increased when nitrate intake is increased, but on the contrary, it can also be reduced when stomach acidification is impaired (Lundberg et al. 1994). Recent researches indicate there is a significant correlation between NO generation and nitrite content in plasma (Kleinbongard et al. 2003), and thereby the nitrite is suggested to be the storage of NO (Dejam et al. 2004). Moreover, it is likely that reducing substances secreted in the stomach, such as ascorbic acid and reduced thiols, are responsible for the enhanced NO production (DeGroote and Fang 1995; de Belder et al. 1994). The chemistry of this process is complex, and will generate different products under varying conditions of initial concentrations, pH, and oxidoreductive potential. Hereby, it still awaits further study. Furthermore, a high concentration of NO could be generated in the acidic stomach. But it was also suggested that the total nitrite in saliva would generate about only one tenth of the NO that is actually measured in the stomach (McKnight et al. 1999), indicating that other NO generation pathways should exist. It was considered that the endogenous NO was synthesized from NO synthetase acting on L-arginine (Moncada and Higgs 1993), and this NO generation pathway was defined as the endogenous pathway. Converting to N-nitroso compounds N-nitroso compounds (NOCs) include nitrosamine and nitroamide. The nitosoproline formation can be detected in urine after proline or nitrate-rich beet juice is intaked, suggesting that endogenous NOCs can be synthesized in human body (Ohshima and Bartsch 1981). It demonstrates that the formation of NOCs is because of the acidification of salivary nitrite on entering the stomach, where it undergoes nitrosation reactions. In the acid environment, nitrite can be converted to active nitrosating agents, such as, nitrous anhydride (N,O,), nitrosyl isothiocyanate (GN-NCS), nitrosyl

--

halide (NOX), and protonic nitrite (H,NO,). These compounds can easily nitrosate with secondary amines to produce NOCs, and the reactions are as follows (Bai and Sun 2002): 2HNO,-+N,O, + H,O NO; + HCl = HNO, + C1R,N + H, R,NH (secondary amines) + H R,N-NO + HNO, R,NH + N,O, Actually, nirosation is the nucleophilic substitution reaction between secondary amines and nitrosating agents, converting to nitroamide probably by protonic nitrite: 2HN0,-H2N0,+ + NO; or HNO, + H+-H,NO,

--

NH + H,NO,+ RCO Amide

R\

\ N -NO + H,O RCO Nitroamide

+ H+

The in vitru kinetics of exogenous nitrosation researches indicated that the nitrosation reaction was related to pH and the reactants concentration. The pH in the stomach of healthy humans is 1-3. In such an acidic condition, the growth of nitrate reducing bacteria will be inhibited and almost no exogenous nitrate can be converted to nitrite. Once achlorhydria takes place, the gastric pH will rise and then nitrate reducing bacteria will be increased and activated, along with a large amount of nitrate being reduced to nitrite. Thereby, there will be more nitrite converted in the chronic atrophic gastritis patients, easily leading to the formation of NOCs. Besides, gastric pH is generally increased with age, and hence the NOCs synthesize more easily in older people. It is reported that more than 20% of people over 50 years of age were achlorhydric because of the high gastric pH (Hill 1999). Nitrate recirculation between saliva and gastrointestinaltract After entering into human body, approximately 25% of dietary nitrate derived from food and water is recirculated between the saliva and the gastrointestinal tract as follows: food or water oral cavity esophagus stomach intestine plasma saliva oral cavity (Duncan et al. 1995). It was first reported in 1961, that humans excrete more nitrate than they consume, which was further confirmed in the 1970s and 1980s (Benjamin 2000), suggesting that there also exists a self-secrete pathway, except for exogenous nitrate intake through food and water. It is speculated that both these methods can provide 70-75

--

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mg nitrate per day (Hibbs et al. 1992). Generally, the self-secrete pathway is known to exist because of the formation of nitric oxide by mammalian cells from the amino acid L-arginine (Fig.) (Moncada and Higgs 1993). Although it is still unclear whether all endogenous nitrate synthesis are derived from this route, the I5N-labelled arginine resulted that the enrichment of urinary nitrate with this heavy isotope is only about one half of the total lSNarginine enrichment (Li et al. 1997), indicating that other nitrate synthesis pathways should exist.

BENEFICIAL AND HARMFUL EFFECTS OF NITRATE

Generally, it is considered that high nitrate intake increased the risk of cancer and methemoglobinaemia, even hyperparathyroid, children polyuria, hypertension, and so on (McKnight et al. 1999). However, in recent years, many researches found that the nitrate has lots of beneficial effects on human health. Therefore, these two contrary conclusions cause a controversy.

Nosogenetic effects of nitrate and nitrite

The safety of nitrate on human health was first doubted in 1945, when the infantile methemoglobinaemia was associated with dietary water. However, L'Hirondel J and L'Hirondel J-L (2001) indicated that it should be caused by the insanitary condition and not by the nitrate

in the dietary water. Further studies also suggested that it was not the nitrate itself, but its metabolite, the nitrite, which could be the compound leading to infantile methemoglobinaemia. It is reported that the toxicity doses f o r methemoglobin formation, as a criterion, ranged from 33 to 350 mg nitrate ion kg-' body weight, especially in an infant, and it could easily lead to death (Speijers 1996). Generally, in the early weeks of life, the infant's gut is susceptible, colonized by many bacteria (e.g., Campylobacter, Salmonella, and E. coli), and hence, nitrate in the feed is readily reduced to nitrite in the stomach and small intestine by bacterial nitrate reductase, and consequently the methemoglobin can easily accumulate through the reaction between nitrite and hemoglobin [4NO, + 4oxyHb (Fe2+)+ 4H' 4metHBb (Fe3+) 4NOi + 0, + 2H,O], which greatly + reduces the 0,-binding capacity and therefore the infant tissues are starved of 0, and result in cyanosis. It is generally accepted that 60% of the hemoglobin in the form of methemoglobinis a lethal level (Hill 1999). Later in life the infant develops a gastric acid barrier and so the bacteria colonized on the upper gut decrease to very low levels, minimizing nitrite formation. Besides, an erythrocyte enzyme is formed, which destroys the nitrite and protects the hemoglobin from nitrite toxicity, reducing the methemoglobin to hemoglobin [metHBb (Fe3+)+ oxyHb (Fez+)]. Consequently, children and adults are far less susceptible to methemoglobinaemia than young infants, unless the accidental and

IEndogenouI
Cell ___)

Amino acid

Nitric oxide synthetase ~ ~ ~ i ~ Nitric oxide i ~

+ -

1
I

II
Exogenous Nitrate in food

N-nitroso compounds

Hypochlorhydria

Microorganisms MouthSaliva Nitrite

Lr
I
L--

Nitrate

,I

Sweat Tear

Nitrate-1
I ,

I
Strong acidic condition Nitric oxide Reducers such as Vc

I Urine

Stomach-

Saliva gland 4 Recycle with blood plasma

I' /

Kidney

Fig. Mechanisms of translocation and transformation of nitrate in the human body.

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occasionally fatal cases. The main possibilities why young infants are more susceptible are concluded by McKnight et al. (1999): First, young infants have a higher ) gastric pH (pH >4 ;second, there are low concentrations of reducing agents, such as, ascorbic secreted; third, the methemoglobin reductase system is not mature. However, the capacity of the reaction rate of reducing methemoglobin to hemoglobin is 250 times greater than the rate at which hemoglobin is normally oxidized. Therefore, methemoglobin, because of the nitrite, will occur only if the dose is higher than the methemoglobinreductase capacity (Boink and Speijers 2001). Of late, there are still a few cases of methemoglobinaemia, which are considered to be caused by bacterial contamination.

Nosogenetic effects of N-nitroso compounds

The carcinogenic feature of NOCs has been generally established. Some epidemiological studies have been conducted to show that the correlation coefficient between gastric cancer mortality and nitrate intake is 0.88. Hitherto, the incidence of gastric cancer in Japan is 6-8 times higher than in USA, along with three folds of nitrate intake than in America (Mirvish 1985). According to the results of Gulis et al. (2002), on cancer, there was a positive association between nitrate level and incidence of non-hodgkin lymphoma and colon cancer, with an excess risk among women. In contrast, many epidemiological researches indicated that nitrate intake did not link to cancer. They had, however, produced contradictory effects (Archer 2002). Furthermore, the investigation by the British Ministry of Agriculture (1992) evidenced that no association existed between high nitrate content vegetables and gastric cancer. Longterm consumption of water at the concentration of 5.5 mg L-' also would not increase the incidence of bladder cancer (Ward et al. 2003). Hill (1999) considered that it had a remarkably low toxicity in adults, whereas, become toxic once the nitrite formed by bacteria. Vegetarianshad a nitrate intake that was three times higher than nonvegetarians, 185-194mg person-' day-' compared to 61 mg person-' day-', however, a 20-40% reduction in death from cancer was shown for the vegetarian groups. Researches from 1986 to 1989 also showed that only those who ate meat or low ascorbic acid food had colon cancer easily, when they took high nitrate

concentration water (de Roos et al. 2003). Consequently, the carcinogenesiseffect of nitrate was clearly correlated to other special factors, such as hygiene levels and food preparation methods. In conclusion, nitrate itself has no carcinogenic effect. Of late, more and more evidence suggested the carcinogenesis of NOCs, the metabolites from nitrate. S i n c e the f i r s t d i s c o v e r y of c a r c i n o g e n i c nitrosodimethylamineby Magee and Barnes (1956), lots of researches have been conducted on the relationship of NOCs and carcinogenesis. To date, more than 75% of the 300 species of NOCs were found to have carcinogenesis, with no case of any animal in remission. Animal and epidemiological investigations suggest the harmful effects of carcinogenic NOCs, especially on the incidence of liver cancer, nasopharyngeal cancer, esophageal cancer, and gastric cancer (Bartsch et al. 1988; Slob et al. 1995). Although both nitrosamine and nitroamide are two species of NOCs, the mechanism are not the same. Nitrosamine is more stable, and no direct teratogenesis effect has been found on the organ, tissue, or cell to date. However, it can still cause acarbon hydroxylation on the alkyl side by microsomes hydroxylase, NADPH and molecular oxygen, generating formaldehyde, and methylnitrosamine, which will be further converted to diazo dydroxide, and will finally decompose into alkyl cations, whereas, the alkyl cations always occur on the seventh guanine to form the 7alkyl guanine. Nucleic acid alkylation, the prelude of sabotageand carcinogenesis,will change the cell genetics and disrupt the protein synthesis. However, nitroamide also has the carcinogenic effect by decomposition to produce the same intermediatesas nitrosamine activation as it is unstable under the physiological pH conditions (Bai and Sun 2002). Besides carcinogenesis,the nitrosating substances also have teratogenic effects because they can cause DNA alkylation. Chronic high nitrate ingestion by a mother may affect the fetus either by germ-cell mutation or by transplacental transmission of tumourigenic nitroso compounds, finally leading to malformation.

Beneficial effects of NO
Nitrate is recycled in the blood, concentrated by the salivary gland, and repeatedly recirculated through the

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stomach rather than being secreted immediately, suggesting the beneficial physiological function which is attributed to the metabolite of nitrate -NO. Signal transduction NO can be generated by the nitrate both from exogenous nitrate intake and endogenous synthesis. From the 1980s, many studies have already demonstrated the signal transduction function of NO in signal transduction, which is an effective vasodilator agent and platelet aggregation inhibitor (McKnight et al. 1999). Therefore, NO deficiency will cause many diseases, such as arteriosclerosis, diabetes, low blood pressure, and coagulation diseases (Coss et al. 2004). In addition, Stuehr and Marletta (1985) reported that macrophage could produce nitrate when it was suffering from the toricity of bacterial compound, and their further analysis showed that once macrophages were attacked by toxic elements, they released NO to increase the phagocytosis before the NO was converted to nitrite and nitrate. Later, DeGroote and Fang (1995) confirmed the results above, and also found that the phagocytosis would be impaired if NO synthesis was inhibited, demonstrating the important antibacterial role of NO during stress induction. Cardiovascular protection Many theories have been proffered over the last few decades in an effort to explain the relation between cardiovascular diseases and diets with a high content of vegetables and fruits. Undoubtedly, the antioxidant vitamins and folic acid have an important effect on the health of the cardiovascular system, but the mechanism is still unclear (Goldin and Gorbach 1996). McKnight et al. (1997) found that large quantities of diffusible NO gas are generated in the stomach, which would readily lead to S-nitrosated formation with a diet that contains thiols in the acidic environment. The platelet adhesion and aggregation are critically related to the vascular occlusive disease, while NO, S-nitrosothiol,and S-nitrosoglutathione are known to be platelet aggregation inhibitors (de Belder et al. 1994). Therefore, nitrate intake can strengthen platelet function, preventing thrombotic episodes (McKnight et al. 1999). Abrams (2002) also considered that the symptoms of congestive heart failure can be reduced by an appropriate amount of nitrate intake, such as clinical nitroglycerin, but the patients who have used nitrates for over a period of years can also suffer from weak health. Other functions McKnight et al. (1999) found that

bacterial survival was shorter after the 2 mM nitrate drink. Also, Benjamin and McKnight (1999) reported that a lower incidence of diarrhea happened when the volunteer took nitrate tablets. Therefore, nitrate will have an important physiological role in killing ingested pathogens and reducing the normal time required. However, nitrate itself has no bactericidaleffects because of its chemical characteristics. Recently, it was stressed that the nitrate metabolites, including nitrite, nitrite acid, and NO, should have the antimicrobial activity for intestinal pathogen prevention (Duncan et al. 1995; Lundberg et al. 1994). In other studies, addition of nitrite (same concentration found in saliva) to hydrochloric acid solution with pH 2-4 increased its antimicrobial activity up to a 100-fold, further demonstrating the significant correlation between the antimicrobialactivity and acidity. Here, also the harmful effects of nitrite on the microorganisms that are beneficial to human health cannot be ignored. NO can easily diffuse across membranes from the vascular endothelium to the skin surface. Diffusion of NO from human skin can readily be detected using a simple apparatus (Weller et al. 1996). NO release can be increased by enhanced nitrate intake or strengthened humoral acidity (Lundberg et al. 2004). The healthy human sweat usually contains about 5 pM of nitrite, and this concentrationis precisely in line with the amount of NO release from human skin (Benjamin 2000). This observation maybe a host defence mechanism to protect against pathogenic (especially for fungi) skin infection. However, the source of these nitrites is not clear, generally from the bacterial reduction of sweat nitrate by skin organisms.

DISCUSSION AND PROSPECT

Although nitrate itself has no toxicity, its metabolites, such as nitrite and NOCs, have a harmful effect on human health, especially the NOCs which are related to many cancers. Therefore, the hazards of nitrate should be mainly caused by unhygienic husbandry conditions or improper eating habits (LHirondel J and LHirondel J-L 2001). Contrarily, the NO from nitrate metabolism is beneficial to human health. However, the beneficial effects of nitrate cannot be negated just because of its chemical relationship with NOCs, and both beneficial

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and harmful effects should be justly evaluated. In conclusion, the beneficial effects of nitrate on human health may occur when its dose is in a certain concentration, but its harmful effects may happen when its ingested amount is excessive. However, nitrate is the precursor substance of NOCs, the inducement factors in its generation are also unclear. Moreover, human body can secrete endogenous nitrate, accounting for 50%, which means that reducing the nitrate intake could be an acceptable way of cancer prevention (Lundberg et al. 2004). Fresh vegetables have low nitrite accumulation and plenty of protective antioxidants, which can decrease the incidence of caner. But part of the nitrate in fresh vegetables will be converted to nitrite when it is improperly stored after harvest. Similarly, cured meat products also contain high nitrite. Once the AD1 of nitrite intake is exceeded (0-0.06 mg kg-' body weight), the amine reaction easily occurs in human body, forming strong carcinogenic nitrosamines (Slob et al. 1995). In conclusion, the diets with more fresh vegetables, few pickled and smoked foods, low nitrate and nitrite content meat products and drinking water should be an important way to effectively prevent cancer, methemoglobinaemia, and other diseases. Besides, the legislation on human nitrate exposure to food and water is also necessary (DeGroote and Fang 1995). The AD1 of nitrate was allocated as 0-0.37 mg kg-' as a nitrite ion by the WHO, but it is always broken because of daily habits (Boink and Speijers 2001). However, whether consumption exceeding the limits would be hazardous to human health is still unknown to date. It is believed that as long as such unclear risks exist, it is very reasonable to limit the nitrate content in vegetables and drinking water by legislation. Furthermore, it is important to investigate clinical medicine and gain a comprehensive understanding on the mechanism of nitrate in human body and its affect factors, including the relationshipbetween different levels of health status and age structure and various metabolites from nitrates; different dietary habits, and various metabolites from nitrates, such as how to effectively decrease the harmful metabolites in the body, how to orient the conversion of accumulated nitrate to a beneficial direction, for example, converting it to a beneficial level of NO. Only with mastering these problems will it be possible to develop an effective

treatment or diet program for different patients, avoid or minimize the harm to human health, and make it a useful orientation for humans. In short, the mechanism of nitrate and its metabolites in human body is still critically important for future research, especially the generation and conversion of endogenous nitrate and the condition of NO synthesis, which has an important guiding role in related researches. Meanwhile, there should also be a continuous concern about the measures for reducing nitrate content in vegetables and water bodies, and formulation of relevant standards, to ensure public health. Besides diet habits and legislative attention, agricultural researches should also be conducted to reduce the accumulation of nitrate in vegetables. Generally, the nitrate content between different vegetable species is greatly different, even in the same variety (Wang et al. 2000). Accordingly, the selection of low nitrate accumulation varieties should be a feasible way and the agronomic measures for coordinating the plant metabolism between carbon and nitrogen, which is related to plant growth, snould be further investigated. In the previous research, it was found that carbon dioxide fertilization in a greenhouse using agricultural bio-fermentation could effectively reduce the nitrate content in vegetables by promoting carbon metabolism (Yu et al. 2006). Moreover, some antioxidants in vegetables can also help us prevent cancers, therefore, it is valuable to investigate the agricultural methods to improve the carotenoids, vitamins C and E, dietary fiber, and plant sterols in the vegetables.

Acknowledgements
This study was financially supported by the Program for Changjiang Schlors and Innovative Research Team in University, China (IRT0536), the National Basic Research Program of China ( 9 7 3 Program, 2002CB4 10806), the National Natural Science Foundation of China (NSFC, 30571083),and the Natural Science Foundation of Zhejiang Province, China (2303465).

References
Abrams J. 2002. How to use nitrates. Cardiovascular Drug and Therapy, 16, 5 11-514. Archer D L. 2002. Evidence that ingested nitrate and nitrite are

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