First draft prepared by
Dr G.J.A. Speijers
Section Public Health of the Centre for Substances & Risk Assessment, National Institute of Public Health and Environmental Protection, Bilthoven, Netherlands
and Professor P.A. van den Brandt
Department of Epidemiology, Maastricht University, Maastricht, Netherlands
Relationship between nitrite intake and oxidant stress in human erythrocytes
Nitrite occurs in the environment, in food and water, and is produced endogenous-ly. It is also used as a food additive, mainly as a preservative, antimicrobial agent and colour fixative. It is used in foods such as cheese and cheese products, raw and processed meats, edible casings, processed fish and fish products and roe.
Nitrite was reviewed by the Committee at its sixth, eighth, seventeenth, twentieth and forty-fourth meetings (Annex 1, references 6, 8, 32, 41 and 116). At its sixth meeting, the Committee allocated an ADI of 0–0.4 mg/kg bw to this substance, expressed as sodium nitrite. This ADI was based on a marginal reduction in body-weight gain at a dose of 100 mg/kg bw per day in a long-term study in rats. At its seventeenth meeting, the Committee lowered the ADI for sodium nitrite to 0–0.2 mg/kg bw and made it temporary At that time, the Committee used a safety factor higher than normal (500) because the ADI was based on a marginal effect and there is a possibility of the endogenous formation of N-nitroso compounds from nitrite and N-nitrosatable compounds present in food and the gastrointestinal tract. At its twentieth meeting, the Committee considered the reports of a WHO task group and of a working group of the International Agency for Research on Cancer on N-nitroso compounds but concluded that they did not provide sufficient evidence to revise the temporary status of the ADI.
After that meeting, numerous toxicological and epidemiological data became available, which the Committee considered at its forty-fourth meeting. Nitrite both with and without nitrosatable precursors was found to be genotoxic in vitro in several tests, but, with the exception of a test in Drosophila melanogaster, the results obtained in vivo were negative. The results of carcinogenicity studies with nitrite were negative, except those in which extremely high doses of both nitrite and nitrosatable precursors were administered simultaneously or premixed in the diet. In addition, the epidemiological data provided no evidence for an association between exposure of humans to nitrite and nitrate and the risk for cancer.
At its forty-fourth meeting, the Committee noted that several controlled laboratory studies had shown that N-nitroso compounds are formed endogenously when both nitrite and N-nitrosatable compounds are present together at high concentrations; it observed, however, that quantitative data were available only on those N-nitroso compounds that are readily formed endogenously, such as N-nitrosoproline, which is not carcinogenic. As there was no quantitative evidence of the endogenous formation of carcinogenic N-nitroso compounds at the levels of intake of nitrite and nitrosatable precursors achievable in the diet, a quantitative risk assessment of nitrite on the basis of endogenously formed N-nitroso compounds was considered to be inappropriate. The Committee at its forty-fourth meeting therefore based its safety evaluation on studies of toxicity with nitrite. The NOELs in these studies were 5.4 mg/kg bw per day (expressed as nitrite ion) in a 90-day study in rats, in which hypertrophy of the zona glomerulosa was observed, and 6.7 mg/kg bw per day (expressed as nitrite ion) in a 2-year study, in rats in which effects on the heart and lungs were observed. On this basis, the Committee allocated an ADI of 0–0.06 mg/kg bw, expressed as nitrite ion, by applying a safety factor of 100.
At its present meeting, the Committee reviewed the data that had become available on nitrite since its forty-fourth meeting, including data on the relevance and adverse nature of certain effects, with new information on the mode of action of effects on the adrenals and on toxicokinetics.
In an open, randomized, three-way cross-over study, seven women and two men (mean age, 23 ± 1.5 years; mean weight, 68 ± 3.3 kg) received a single oral dose of sodium nitrite at 0.06 or 0.12 mmol/mmol haemoglobin, corresponding to 140–190 mg or 290–380 mg, depending on the weight of the person, and 7 days later received one intravenous dose of sodium nitrite at 0.12 mmol/mmol haemoglobin, corresponding to 290–380 mg. All doses were administered after an overnight fast. Adverse effects, blood pressure, heart rate, haemoglobin concentra-tion, per cent methaemoglobin in blood and plasma nitrite and nitrate concentration were recorded frequently for 24 h after each administration of sodium nitrite.
Headache was the most frequent complaint during each treatment session, and lowered blood pressure accompanied by an increased heart rate were seen after each treatment. The maximum per cent methaemoglobin in blood was 8.4–12% after intravenous administration and 7.7–11% and 3.4–4.5% after the two oral doses of sodium nitrite. The maximum methaemoglobin level was reached about 1.2 h after intravenous or oral administration of sodium nitrite at 0.12 mmol/mmol haemoglobin and 0.7 h after administration of the low oral dose. Gastrointestinal absorption of sodium nitrite was rapid, with maximum plasma nitrite concentrations observed 15–30 min after dosing. Nitrite disappeared rapidly from plasma, with an average elimination half-life of 30 min. The bioavailability of sodium nitrite was 73–120% after the high oral dose and 70–110% after the low oral dose. The authors concluded that, under fasting conditions, 90–95% of orally administered sodium nitrite is absorbed from the gastrointestinal tract (Kortboyer et al., 1997a).
(a) In vitro
The methaemoglobin-inducing potential of nitrite was investigated in vitro in fresh, heparinized, human venous blood samples maintained at 37 OC. Ten doses of sodium nitrite with a molar ratio of nitrite to haemoglobin of 0.01–0.15 were tested in triplicate. The haemoglobin concentration, per cent methaemoglobin and the pH of the blood were determined at 2-h intervals after the start of the experiment. None of the blood samples showed visible haemolysis at the end of the experiment. Methaemoglobin contents up to 24% were induced. A linear relationship was established between the nitrite dose and the maximum methaemoglobin level induced in blood. Linear regression analyses showed an adjusted r2 of > 0.99 (p = 0.0001). The molar ratio of the nitrite dose to the maximum per cent methaemoglobin induced at Tmax was approximately 0.7 at all doses. A dose of sodium nitrite with a molar ratio to haemoglobin of 0.04 induced about 5% methaemoglobin ( Kortboyer et al., 1997b).
Contamination of food during manufacture and degradation of nitrate in vegetables appear to be important factors in the induction of methaemoglobinaemia. Stringent control of excessive use of nitrate and nitrites in food items such as refrigerated dim sum, stuffed pork and Chinese sausages, which are popular among some Asian populations, is required in order to prevent outbreaks of toxic methaemoglobinaemia. Persons with glucose-6-phosphatase deficiency, which is common in some Asian populations, may present with methaemoglobinaemia and intravascular haemolysis after exposure to oxidant drugs or chemicals. Methylene blue is inefficient and may exacerbate haemolysis in these patients; partial exchange transfusion may be required (Chan, 1996)
A case report described two incidents of illness due to intake of nitrite. In the first, 59% methaemoglobinaemia was found in 29 students and 20% methaemo-globinaemia in 14 of 49 students who ate leftover soup containing 460 mg/l nitrite. They also had blue lips and fingers, nausea, vomiting and headache. In the second incident, four of six office workers who drank leftover coffee containing 300 mg/l nitrite showed a 6–16% increase in methaemoglobin. No estimates of intake were made. In both cases, the nitrite originated from a contaminated hot-water tap (Centers for Disease Control, 1997).
In a case study, life-threatening methaemoglobinaemia associated with exposure to sodium nitrite was recorded in three previously healthy siblings: 4-year-old twin boys and their sister aged 2 years. Their father had used a solution of sodium nitrite as an insecticide. On the day of hospital admission, one of the children had mistaken a small bag of crystals for sugar, which was added to tea at concentrations of 5100 mg/l, 5000 mg/l and 4900 mg/l in the three cases, respectively (Finan et al., 1998).
In blood, nitrite is rapidly oxidized by haemoglobin to yield methaemoglobin and nitrate; therefore, the oral bioavailability of nitrite cannot be calculated from the plasma nitrite concentration. A study in healthy volunteers was conducted to investigate the oral bioavailability of a substantial dose of nitrite in an aqueous solution. As the vasodilating and methaemoglobin-inducing potential of nitrite limit the dose that can be administered safely, the study was preceded by a range-finding study in three volunteers to establish the dose that induced methaemoglobin of not more 10–15%. The results showed that a dose of 0.12 mmo/ mmol haemoglobin (290–370 mg of sodium nitrite) induced formation of 11% methaemoglobin in the blood. This was considered the maximum safe dose for adult volunteers, and doses of 0.04, 0.08 and 0.12 mmol were used. At the maximum dose, all volunteers reported effects of mild intensity. In addition to lowered blood pressure, a compensatory increase in heart rate was seen at all doses, which remained within acceptable limits. The peak plasma nitrite concentrations were considered acceptable for a bioavailability study (Kortboyer et al., 1998).
Two forms of methaemoglobin—methaemoglobin and nitrite methaemoglobin—were found in native human erythrocytes in the presence of sodium nitrite in suspension. The interaction of intercellular oxyhaemoglobin with nitrite ions in normal erythrocytes resulted in the formation of methaemoglobin, whereas it led predominantly to the formation of nitrite methaemoglobin in metabolically exhausted erythrocytes. Nitrite methaemoglobin reacts with hydrogen peroxide to form reactive intermediates (e.g. peroxynitrous acid) and the products of haemoglobin destruction. During storage of erythrocyte suspensions containing methaemoglobin and modified nitrite methaemoglobin, the forms of erythrocytes and their degree of haemolysis changed. It was assumed that formation of methaemoglobin leads to destruction of erythrocytes (Starodubtseva et al.,1999)
A study was conducted in the USA to determine the dose of ascorbic acid required to inhibit N-nitrosoproline formation when 400 mg of nitrate were given 1 h before a standard 400-calorie meal with 500 mg of L-proline. Volunteers ate their normal diets but restricted their intakes of nitrate, proline, N-nitrosoproline and ascorbic acid. N-Nitrosoproline and N-nitrosarcosine were determined in urine after 18 h by methylation and then gas chromatography with thermal energy analysis. The mean yields of N-nitrosoproline were 11, 42, 33, 22 and 23 nmol in groups of 9–25 subjects taking proline alone, proline plus nitrate or proline plus nitrate plus 120, 240 or 480 mg of ascorbic acid, respectively. There was a significant trend to lower N-nitrosoproline yields as the dose of ascorbic acid increased. These results correspond to inhibition by ascorbic acid of 28%, 62% and 60% , respectively. Pairwise comparisons showed that each group taking ascorbic acid formed significantly less N-nitrosoproline than the group given only proline plus nitrate. The mean N-nitrosarcosine yields were 9 nmol when proline was taken alone and 17–24 nmol with proline plus nitrate plus ascorbic acid, with no trend to increasing individual values. It was concluded that 120 mg of ascorbic acid taken with a meal containing nitrate at 360 mg/day would significantly reduce nitrosamine formation in vivo ( Mirvish et al., 1998)
Groups of 10 male and 10 female B6C3F1 mice were given drinking-water containing sodium nitrite at a concentration of 0, 375, 750, 1500, 3000 or 5000 mg/l, equal to average daily doses of approximately 90, 190, 340, 750 and 990 mg/kg bw for males and 120, 240, 440, 840 and 1200 mg/kg bw for females, for 14 weeks. The body weights of males at the highest dose were significantly lower than those of controls at week 13. The relative weights of the spleen in males and the absolute and relative weights of the heart, kidney, liver and spleen of females at the two higher doses were greater than those of the control groups. Sperm motility was decreased in males at the highest doses, and the estrous cycles of females at the three higher doses were significantly longer than those of controls. Increased incidences of squamous-cell hyperplasia of the forestomach were seen in males and females at the two higher doses, of extramedullary haematopoiesis of the spleen in males at the two higher doses and in females at the three higher doses and of degenaration of the testis in males at the two higher doses. The NOEL was 190 mg/kg bw per day (National Toxicology Program, 2001).
As hypertrophy of the adrenal zona glomerulosa was observed at all concentrations of potassium nitrite tested, including the lowest (100 mg/l), a study was performed to establish a NOEL for nitrite. Groups of 10 male and 10 female 6-week-old Wistar (TNO) rats received drinking-water containing potassium nitrite at a concentration of 12, 25, 50, 100 or 3000 mg/l or sodium nitrite at a concentration of 81 or 2400 mg/l for 13 weeks, the nitrite content of the drinking-water in the last two groups being equal to that of the groups given the two higher concentrations of potassium nitrite. The potassium and sodium concentrations in the drinking-water of the corresponding control groups were equalized with KCl and NaCl, respectively.
General health, behaviour and survival were not affected by ingestion of nitrite. Body weight and food and water intake were slightly decreased in animals of each sex given the highest dose of either nitrite. The methaemoglobin concentration was significantly increased in rats at these doses in weeks 4 and 12, while a slight increase in erythrocyte variables was seen during week 12 in females given the potassium salt at 3000 mg/l. The relative weight of the kidney was increased at the highest dose of each nitrite. In week 4, plasma aldosterone and corticosterone concentrations were slightly decreased in males at the higher dose of sodium nitrite, and plasma corticosterone was slightly decreased in females at the highest dose of potassium salt; these effects were not seen in week 13. Systolic blood pressure was not affected. Microscopic examination revealed slight hypertrophy of the adrenal zona glomerulosa in rats given potassium nitrite at 100 or 3000 mg/l and at the lower dose of the sodium salt, and plasma aldosterone and corticosterone concentrations were slightly decreased in males at the highest dose of sodium nitrite, the incidence and degree being dose-related. The results seen with potassium nitrite at 100 and 3000 mg/l in drinking-water were comparable to those found at the same concentrations in the previous 90-day study. The effects of sodium nitrite were similar to those of potassium nitrite. The NOEL for potassium nitrite was 50mg/l in drinking-water, equivalent to 5 mg/kg bw per day (Til et al., 1997).
In a study of the effect of age on the toxicity of nitrite, groups of 10 young adult and four aged male Wistar rats were allocated to four subgroups. Three subgroups received potassium nitrite in the drinking water at a concentration of 6 mmol/l (supplemented with 30 mmol KCl/l), 18 mmol/l (supplemented with 18 mmol KCL/l) or 36 mmol/l, equivalent to 83, 250 and 500 mg/kg bw, and a control group received 36 mmol/l. Blood samples were collected for measurement of methaemoglobin every 2 weeks, starting before the start of nitrite administration. In week 4, the rats were placed in metabolism cages, and 24-h urine samples were collected for qualitative (dipstick) and quantitative (volume, creatinine concentration, osmolality, N-acetylglutamate activity, nitrate and total protein concentrations) analysis. At the end of the urine collection period, blood was collected for clinical chemistry. In week 5, a test for phenolsulfthalein was conducted. In week 6, the rats were autopsied and examined macroscopically, and blood was collected for measurement of the concentrations of insulin, thyroxine and free thyroxine. Heart, liver, adrenals, brain, pituitary, spleen and thyroid gland, were weighed and fixed for histopathological examination. In addition, mesenteric lymph nodes and all organs showing macroscopic lesions were prepared for examination.
The known effects of nitrite, methaemoglobinaemia and hypertrophy of the zona glomerulosa of the adrenals, were observed in both young and aged rats. Hypertrophy of the zona glomerulosa was seen at the two higher doses, the NOEL thus being 6 mmol/l (equivalent to 84 mg/kg bw per day). Aged rats drank less water than controls (31% less) and the young adults (14% less at the highest dose). As nitrite is rapidly converted to nitrate, the plasma concentration of nitrate can be interpreted as a reflection of internal nitrite dose. The plasma nitrate concentrations of the aged rats were approximately three times higher than those of young adult rats. Because aged rats have more fat than young adults, the distribution volume is not proportional to body weight, leading to an increase in plasma nitrate. More efficient renal retention of water (and consequently of nitrite/nitrate) may also have contributed to the higher nitrate concentrations in the plasma of aged rats. Therefore, the internal nitrite dose of the aged rats was adequate and at least comparable to that of the young adults. No further treatment-related differences in effect were found between the groups. The NOEL in this strain of Wistar rats was 50 mg/kg bw per day, which is considerable higher than the NOEL in the strain used by Til et al. (1997), even though both NOELs were for hypertrophy of the renal zona glomerulosa. The authors concluded that the aged rats and young adult rats were equally susceptible to the toxic effects of nitrite (Boink et al., 1996, 1997).
Groups of 10 males and 10 females rats were given drinking-water containing sodium nitrite at a concentration of 0, 380, 750, 1500, 3000 or 5000 mg/l, equal to average daily doses of 30, 55, 120, 200 and 310 mg/kg bw per day for males and 40, 80, 130, 220 and 340 mg/kg bw per day for females, for 14 weeks. Further groups of 15 males and 15 females were given water containing the same concentrations for 70 and 71 days. One female at 220 mg/kg bw per day died before the end of the study.
The body weights of males at 310 mg/kg bw per day were significantly lower than those of controls. Males at 310 mg/kg bw per day and females at the two higher doses drank less water than controls in weeks 2 and 14. The clinical findings related to treatment included brown discolouration of the eyes and cyanosis of the mouth, tongue, ears and feet of males at 200 and 310 mg/kg bw per day and of females at the three higher doses. Reticulocyte counts were increased in both males and females at the two higher doses. The erythron was decreased on day 19 but increased by week 14 in males and females at the highest dose. Methaemoglobin concentrations were elevated in almost all treated groups throughout the 14-week study: the per cent of total haemoglobin found as methaemoglobin was 0.002% in control males, 4.4% in males at 200 mg/kg bw per day and 17% in males at 310 mg/kg bw per day, and 0.37% in control females, 5.8% in females at 220 mg/kg bw per day and 11% in females at 340 mg/kg bw per day. The authors concluded that a NOEL could not be identified for this effect. The relative weights of the kidney and spleen of males and females at the two higher doses were increased, and sperm motility was significantly decreased in males at 120 and 300 mg/kg bw per day. Increased erythropoietic activity in the bone marrow of males and females at the two higher doses was observed as an increased number of reticulocytes. The incidence of squamous-cell hyperplasia of the forestomach was significantly increased in males and females at the highest dose. As methaemoglobin formation of less than 3% is considered not to be adverse, the NOEL was 55 mg/kg bw per day on the basis of reduced sperm motility in rats at 120 mg/kg bw per day (National Toxicology Program, 2001).
Groups of 50 male and 50 female B6C3F1 mice were given drinking-water containing sodium nitrite at a concentration of 0, 750, 1500 or 3000 mg/l, equal to average daily doses of 60, 120 and 220 mg/kg bw for males and 45, 90 and 160 mg/kg bw for females, for 2 years. The survival rate of the treated groups was similar to that of the controls. The mean body weights of females at the highest dose were lower than those of controls throughout the study, and the treated groups generally consumed less water than the control groups. The incidences of squamous-cell papilloma and carcinoma (combined) in the forestomach of female mice showed a positive trend. The incidence of hyperplasia of the glandular epithelium was significantly greater in males at the highest dose than in controls. There was thus equivocal evidence of carcinogenic activity on the basis of the positive trend in the incidence of squamous-cell papilloma and carcinoma (combined) of the forestomach (National Toxicology Program, 2001).
The effects of long-term administration of powdered fish meal with sodium nitrite were studied in six groups of 50 Fischer 344 rats of each sex. The first three groups were fed diets supplemented with 8% (basal diet), 32% or 64% fish meal and given drinking-water containing 0.12% sodium nitrite. The other three groups were given the same concentrations of fish meal and tap water. At week 104, all surviving animals were killed and examined histologically.
Treatment with fish meal increased the incidence and multiplicity of atypical tubules, adenomas and renal-cell carcinomas in male rats in a dose-dependent manner. Females were less susceptible than males to renal tumour induction. The incidence and multiplicity of atypical tubules were also significantly increased in males given 64% fish meal as compared with those given 8% fish meal. Nephropathy occurred in a clearly dose-dependent manner in groups given fish meal, irrespective of the sodium nitrite concentration, and was more frequent in males than in females. N-Nitrosodimethylamine (NDMA) was found in the stomach contents after 4 weeks of treatment with 64% fish meal plus 0.12% sodium nitrite, at a level twice that in the group given 8% fish meal plus 0.12% sodium nitrite. Thus, concurrent administration of fish meal and sodium nitrite induced renal epithelial tumours. The high intake of protein should be noted (Furukawa et al., 2000).
Groups of 50 male and 50 female Fischer 344/N rats were given drinking-water containing sodium nitrite at a concentration of 0, 750, 1500 or 3000 mg/ l, equal to average daily doses of 35, 70 and 130 mg/kg bw for males and 40, 80 and 150 mg/kg bw for females, for 2 years. In studies of the toxicokinetics of plasma nitrite and blood haemoglobin, 10 male and 10 females were given the same concentrations for 12 months.
The survival of treated groups was similar to that of controls. The mean body weights of males and females at the highest dose were lower than those of controls throughout the study. Males and females at this dose drank less water than controls throughout the study, and the water consumption of the other treated group was generally lower only after week 14. The incidences of hyperplasia of the forestomach epithelium in males and females at the highest dose were significantly higher than in controls. The incidence of fibroadenoma of the mammary gland was significantly increased in females at the intermediate dose, and the incidences of multiple fibroadenoma were increased in females at the two lower doses; however, these neoplasms occur at a high background incidence, and no increase was seen at the highest dose. The incidences of mononuclear-cell leukaemia were significantly decreased in males and females at 80 or 150 mg/kg bw per day. Under the conditions of this study, there was no evidence of carcinogenicity (National Toxicology Program, 2001).
Sodium nitrite was tested for its effects on reproduction and fertility in Swiss CD-1 mice. Data on food and water intake, body weight and clinical signs in a dose range-finding study were used to select concentrations of 0.06%, 0.12% and 0.24% (w/v) in drinking-water for the continuous cohabitation phase. Water consumption was reduced by 10–17% in the group given the highest concentration of sodium nitrite, although the body weights of the F0 generation during the cohabitation phase were not reduced. The data on body weight and water consumption allowed calculation of daily nitrite consumption, which was 120, 260 and 420 mg/kg at the low, intermediate and high doses, respectively. Nine animals died during this phase, comprising three at 0.06%, four at 0.12%, none at 0.24% and one control. There was no-treatment-related reduction in the mean number of litters per pair, the number of pups per litter or the viability or weight of the pups. The days to delivery of each litter were not affected. Each dam was allowed to nurse the last litter from the cohabitation phase until weaning. While mortality during nursing was not increased by treatment with nitrite, pup body-weight gain was reduced in the group at the highest dose on postnatal days 7–21 by 12–17%. This effect may have been due to reduced maternal water intake, with consequent lower milk production.
As no effects on reproduction were noted during this phase, only the controls and mice at the highest dose were retained after weaning and examined for potential reproductive toxicity. Water consumption was measured several times during this part of the study and was found to be reduced by 8%. The body weights of F1 mice at the beginning of the week of mating were similar in all groups. The fertility and reproductive success of F1 mice during the last phase were not affected by treatment, and there was no change in the ability of mice to mate, become pregnant or deliver live young. The number, weight and viability of the young were not reduced by treatment. After delivery and analysis of the F2 pups, the F1 mice were killed and necropsied. The terminal body weights of treated and exposed mice did not differ, and there were no treatment-related changes in the weight of any organ. The length and pattern of the estrous cycle were unchanged during the 12 days of evaluation, and the motility, concentration and morphology of epididymal sperm were also unchanged. Histologically, the liver and kidneys of the mice at 0.24% nitrite and the control mice were similar. Thus, sodium nitrite had no adverse effect on reproduction or reproductive performance at necropsy in mice treated at doses up to 420 mg/kg bw, which was the NOEL for reproductive toxicity (Chapin et al., 1997).
Sodium nitrite was mutagenic in Salmonella typhimurium strain TA100, with and without Aroclor 1254-induced hamster and rat liver enzymes; no mutagenicity was observed in strain TA98. Intraperitoneal injection of sodium nitrite to male mice and rats did not induce micronucleus formation in bone marrow, and a test for micronuclei in peripheral blood from mice in the 14-week study described above also gave negative results (National Toxicology Program, 2001).
(a) Effects on the immune system
A pronounced decline in the percentage of CD4+ T lymphocytes was found in mesenteric lymph nodes from groups of four B6C3F1 mice infected with Trichinella spiralis and given nitrite for 10 or 25 days. Less pronounced differences were observed in the percentage of CD8+ T lymphocytes in both mesenteric lymph nodes and Peyer patches. Infection with T. spiralis alone induced a pronounced decline in CD4+ T lymphocytes in mesenteric lymph nodes, whereas the percentage of CD8+ T lymphocytes remained at the level of controls. Treatment with sodium nitrite alone caused a clear increase in the percentage of CD8+ cells as compared with untreated, uninfected controls. The latter effect may result in a depressed immune response, causing more severe T. spiralis infections in sodium nitrite-treated mice (Bany et al., 1995).
(b) Effects on the vascular system and adrenals
Studies of blood pressure and the role of the adrenals in the mode of action of nitrite in hypertrophy of the adrenal zona glomerulosa have been reviewed. Nitrite and organic nitrates have vasodilating properties (Nickerson, 1975), and dilatation and thinning of the intramuscular coronary blood vessels were seen in rats given drinking-water containing sodium nitrite at concentrations of 100–300 mg/l for 2 years (Shuval & Gruener, 1972). A fatal fall in blood pressure after nitrate poisoning was reported in cows given nitrate (Dinkla, 1976); as nitrate is efficiently converted to nitrite in ruminants, the fall in blood pressure was probably due to nitrite rather than to the ingested nitrate.
The effects of physiological amounts of nitrite on generation of nitric oxide and relaxation of rat aorta were studied in vitro with the pH reduced to that found in tissues during hypoxia or ischaemia. The relaxing effect of nitrite was greater in an acidic buffer solution (pH 6.6) than at neutral pH; the EC50 for nitrite was reduced from 200 to 40 µmol/l. Nitrite-evoked relaxation was effectively prevented by co-administration of an inhibitor of soluble guanylyl cyclase and was further potentiated by addition of ascorbic acid. Nitric oxide was generated from nitrite for 11 days in a pH-dependent manner, with even larger amounts seen after addition of ascorbic acid, and was correlated to the degree of relaxation of rat aorta (Modin et al., 2001)
The effect of sodium nitrite on blood pressure was investigated to test the hypothesis that nitrite induces hypertrophy of adrenal zona glomerulosa by reducing the blood pressure and stimulating the renin–angiotensin axis. Blood pressure was evaluated with a radiotelemetry system in conscious, freely moving animals. Two male Wistar rats (weighing 350 and 375 g) were maintained on a 12-h light–dark cycle for 11 days, and systolic and diastolic blood pressure and heart rate were measured every 5 min; mean arterial pressure was calculated. Potassium nitrite decreased the mean arterial pressure and increased the heart rate. Potassium chloride had no effect. A previous study in anaesthetized rats showed rapid conversion of plasma nitrite into nitrate (Vleeming et al., 1995a,b). As intravenouis administration of sodium nitrate at a concentration of 100 µmol/kg bw over 5 min did not decrease the mean arterial pressure, it was concluded that nitrite is responsible for hypotension and therefore for activation of the renin–angiotensin axis (Vleeming et al., 1996).
In order to test the hypothesis that the relatively low blood pressure of vegetarians is due partly to a high dietary load of nitrate, which is reduced to nitrite and finally to nitric oxide, spontaneously hypertensive rats received drinking-water containing nitrite at a concentration of 0, 25, 50 or 100 mmol/l for 56 days. Food was offered ad libitum or was restricted by 20% (pair-feeding) to simulate the lower energy consumption of vegetarians. Blood pressure, which was monitored at regular intervals, was lowered in a dose-dependent manner by nitrite. The effect was reversible and was not enhanced by energy restriction. In volunteers, the plasma nitrate concentration increased by a factor of 8–32 after ingestion of a nitrate-rich meal, and the mean methaemoglobin concentration increased from 1.2% to 2.4%, indicating endogenous formation of nitrite under these conditions (Beier et al., 1995).
To investigate whether long-term administration of nitrite lowers the blood pressure of spontaneously hypertensive rats and prevents secondary lesions induced in organs by hypertension, 96 of these rats received drinking-water containing sodium nitrite at concentrations of 50–75 mmol/l or equimolar amounts of sodium bicarbonate for 4, 8 or 12 months. At each time, arterial blood pressure (determined by the tail-cuff method) was significantly lower in the group receiving sodium nitrite than in the controls, indicating that no significant tolerance towards nitrite had developed. There was also a tendency to reduced cardiac hypertrophy and renal atrophy in this group, which was not statistically significant. Drinking-water containing sodium nitrite at 75 mmol/l was not well tolerated by young rats (Haas et al., 1999)
Intravenous administration of nitrite to anaesthetized rats induced an immediate, dose-dependent decrease in blood pressure, which preceded an increase in methaemoglobin concentration, suggesting that hypotension is the primary effect of nitrite. A single dose of 30 µmol/kg bw caused a 10–20% decrease in blood pressure. Rats drink about 50 ml/kg bw of water each day. If the drinking-water contains nitrite at 36 mmol/l, the total daily dose will therefore be 1800 µmol/kg bw. Thus, assuming 100% bioavailability, 1/60 of the total daily consumption was able to decrease the blood pressure by 10–20%. As the blood pressure had returned to normal within 30 min, continuous monitoring is necessary to detect the effect of nitrite on blood pressure. Recording of blood pressure and heart rate by means of a telemetry device in a freely moving rat given drinking-water containing potassium nitrite at a concentration of 36 mmol/l (equivalent to 500 mg/kg bw) revealed transient hypotension (Vleeming et al., 1997).
Morphometry and histology of the adrenals of 10 Wistar rats of each sex given drinking-water containing potassium nitrite at a concentration of 3.6, 12 or 36 mmol/l showed slight hypertrophy of the zona glomerulosa after 28 days, which persisted unchanged throughout the 90-day treatment but disappeared slowly after cessation of treatment. Slight focal and slight diffuse hypertrophy were still present after a 30-day recovery period but had disappeared by 60 days. Enlargement of the zona glomerulosa was not seen by morphometry in rats at the two lower doses, but histological examination showed that the adrenals of eight rats at 12 mmol/l and three at 3.6 mmol/l were minimally hypertrophic. This effect was dose-related; no NOEL could be identified. Nitrite is readily converted to nitrate, the plasma half-life of nitrite in various species being estimated as < 30 min, while the half-life of nitrate ranges from 4 h (sheep and horses) to 44 h (dogs), suggesting that nitrate plays a role in the causation of hypertrophy of the adrenal zona glomerulosa. However, no hypertrophy was observed by means of morphometry in rats given drinking-water containing a concentration of 36 mmol/l for 90 days, and histological examination revealed minimal hypertrophy in only two of 10 rats. Thus, the hypertrophy must be attributed to nitrite ion alone.
The adrenals regulate blood pressure via the renin–angiotensin–aldosterone axis. When blood pressure decreases, baroreceptors in the juxtaglomerular apparatus in the kidneys stimulate release of renin. Renin catalyses the conversion of angiotensinogen to angiotensin I, which in turn is converted to angiotensin II by a converting enzyme. Angiotensin II effectively contributes to restoration of normal blood pressure by its vasoconstrictive effect on blood vessels and by stimulating the release of aldosterone from the zona glomerulosa of the adrenals. Aldosterone enhances the reabsorption of sodium in the renal tubules, leading to expansion of the extracellular volume. This rise in blood volume also causes an increase in blood pressure. As described above, administration of nitrite to rats in the drinking-water caused repeated falls in blood pressure, thus repeatedly activating the renin–angiotensin–aldosterone-axis, which may have caused hypertrophy of the adrenal zona glomerulosa. To investigate this suggestion further, a limited number of rats were given ramipril, an inhibitor of angiotensin-converting enzyme, in their diet concurrently with nitrite. No hypertrophy of the zona glomerulosa occurred, strongly suggesting that slight hypertrophy of the adrenal zona glomerulosa represents a physiological adaptation to repeated episodes of hypotension caused by nitrite (Boink et al., 1998).
(c) Effects on tumour induction
A number of studies have addressed the effect of administration of 0.2–0.3% sodium nitrite on the incidence of forestomach tumours induced in rats by well-known carcinogens (N-methyl-N’-nitro-N-nitrosoguanidine, phenolic compounds, catechol, 3-methoxycatechol and butylated hydroxyanisole). These concentrations of nitrite, equivalent to 200–300 mg/kg bw, enhanced the incidence of forestomach tumours induced by the carcinogens. Lower doses were not examined. As forestomach tumours are of limited relevance for humans, however, these findings have no impact on the safety assessment of nitrite in food (Hirose et al., 1993; Kawabe et al., 1994; Yoshida et al., 1994; Miyauchi et al., 2002). In a recent study of the effect of 0.2% sodium nitrite (equivalent to 200 mg/kg bw) on the incidence and volume of mammary tumours in rats induced by 2-amino-1-methyl-6-phenylimidazol[4,5-b]pyridine, the nitrite did not affect the incidence of mammary tumours but appeared to reduce their volume. This finding at a relatively high dose has no implications for the safety assessment of nitrite in food (Hirose et al., 2002).
To determine the relative contributions of endothelial nitric oxide and intravascular nitrogen oxide species in the regulation of blood flow, simultaneous measurements were made of forearm blood flow and arterial and venous concentrations of plasma nitrite, low-molecular-mass S-nitrosothiols, high-molecular-mass S-nitrosothiols and erythrocyte S-nitrosohaemoglobin. Measurements were made at rest and during regional inhibition of nitric oxide synthesis, followed by forearm exercise. Unexpectedly, significant gradients in circulating arterial–venous plasma nitrite, representing a novel delivery source for intravascular nitric oxide, were observed. Further support for the notion that circulating nitrite is bioactive was provided by the finding that use of nitrite increased significantly with exercise during inhibition of regional endothelial synthesis of nitric oxide. It was concluded that circulating nitrite is bioactive and provides a gradient for delivery of intravascular nitric oxide (Gladwin et al., 2000).
Epidemiological studies can be ranked in increasing order, from ecological (or correlation) studies to cross-sectional studies, case–control studies, cohort studies and intervention trials. This classification of epidemiological study designs with respect to their potential for bias and, consequently, the strength of evidence they provide and the costs involved has been described in detail (e.g. van den Brandt et al., 2002). Intervention trials provide the strongest evidence for a causal relationship with risk and (because of the possibility to control for confounding and bias) have the least possible bias; however, they are usually the least feasible and the most expensive. Less expensive cohort studies allow assessment of exposure and selection of study participants before the health outcome of interest occurs and thus provide relatively strong evidence. Although the less expensive case–control studies generally involve assessment of exposure retrospectively in subjects with and without the health outcome, the resulting evidence is more debatable. This is particularly so in the case of dietary intake, because of the possibility of selection bias, recall bias and/or bias due to the presence of disease. Cross-sectional studies suffer from the additional problem that exposure and disease are measured at the same time, making it impossible to draw conclusions about cause and effect. Correlation studies cost the least, but they provide weak evidence and are much more susceptible to bias. In addition to the problem of extrapolating the data to the individual level (as the units of measurement are population groups), most such studies include limited data on exposure, rely on mortality (rather than incidence) rates and often do not include consideration of the induction period. Some investigators have stated that observational studies cannot, by definition, establish the causality of a relationship on the basis of a statistical association. However, when several studies of high quality, such as those in which the biases are shown to be minimal, are available and these consistently show a dose–response relationship, observational studies may well contribute to conclusions about causality. The power of observational epidemiological studies was established 50 years ago, when such studies revealed that smoking caused lung cancer.
(a) Cross-sectional studies
The concentration of nitrite in gastric juice and of several N-nitroso compounds and other analytes in urine were measured in nearly 600 residents of an area of Shading, China, where precancerous gastric lesions are common and the rates of stomach cancer are among the world’s highest. The concentration of nitrite in gastric juice was considerably higher in people with pH values for gastric juice > 2.4 than in those with values < 2.4, and nitrite was detected more often and at higher concentrations in people with late-stage gastric lesions, especially when the gastric pH was high. Of patients with intestinal metaplasia, 18% had detectable gastric nitrite, whereas only 7.2% of people with less advanced lesions had this analyte. The odds ratio (OR) for intestinal metaplasia in people with detectable nitrite relative to those with undetectable nitrite increased from 1.5 (95% confidence interval [CI], 0.6–4.1) to 4.1 (95% CI, 1.8–9.3) for people with low and high nitrite concentrations, respectively. Urinary acetaldehyde and formaldehyde concentrations also tended to be higher among patients with more advanced disease, particularly dysplasia. However, the urinary excretion of total N-nitroso compounds and several nitroamino acids was similar among patients with chronic atrophic gastritis and intestinal metaplasia and dysplasia, as was reported by the authors to have been found in recent studies in Colombia, France and the United Kingdom. The results for this high-risk population suggested that elevated concentrations of gastric nitrite, especially in a high pH milieu, were associated with advanced precancerous gastric lesions, although specific N-nitroso compounds were not implicated (You et al., 1996). This cross-sectional study is of limited value, as cause and effect are confusingly mixed.
The possible role of N-nitroso compounds in the induction of upper aerodigestive tract tumours was considered in a case–control study conducted in the Valle d’Aosta, an Italian region with a high incidence of these neoplasms. Nitrate, nitrite and labile and stable N-nitroso compounds were analysed in the saliva of 36 patients with these cancers and 23 healthy individuals. After allowing for tobacco use, the presence of nitrate, nitrite and N-nitroso compounds in saliva was not associated with an increased risk for upper aerodigestive tract cancers. The OR for continuous units of total N-nitroso compounds was 0.99 (95% CI, 0.9–1.1). Thus, salivary nitrate, nitrite and N-nitroso compounds might not be suitable markers for assessing the risk for cancers at these sites, although a role for N-nitroso compounds could not be excluded (Airoldi et al., 1997).
The excretion of nitrate, nitrite, apparent total N-nitroso compounds and volatile nitrosamines was measured in 24-h urine from 61 Egyptians, divided into four groups: controls, Schistosoma haemotobium-infected patients and bladder cancer patients with and without a history of schistosomal infection. The urinary concentration of nitrate in S. haemotobium-infected patients was significantly higher than in the other three groups. The concentration of nitrite was below the limit of detection of the method (< 0.015 µg/mg creatinine) in all but one of the control samples. S. haemotobium infection significantly increased urinary nitrite to 0.9 ± 1.16 µg/mg creatinine (mean ± SD, p = 0.001), and the concentration of nitrite in both groups of patients with bladder cancer was about 20 times that in S. haemotobium-infected patients without bladder cancer. Excretion of apparent total N-nitroso compounds paralleled that of nitrite, and, overall, a good correlation was observed between these two variables (r = 0.71, p = 0.0001). NDMA was present in all the samples analysed, and S. haemotobium infection significantly increased the urinary concentration over that of controls (4.0 ± 1.6 and 2.0 ± 3.0 ng/mg creatinine, respectively; p = 0.01). Among the cancer patients, the concentration of NDMA was higher than that in controls only in those with schistomal infection. The presence of N-nitroso compounds, including NDMA, in the urine of S haemotobium-infected patients both before and after the development of cancer and the observation that these compounds also occur in bladder cancer patients with no history of schistosomal infection suggest that these compounds might have a role not only in initiation of the carcinogenic process but also in its progression (Mohsen et al., 1999). As endogenous nitric oxide production can also result from schistosomiosis, cause and effect can be reversed.
A study was conducted in Egypt, an area with high environmental levels of nitrosating agents, to investigate whether salivary nitrate and nitrite and the activity of nitrate reductase in saliva affect the risk for oral cancer. The concentrations of salivary nitrite (8.3 ± 1.0 µg/ml) and nitrate (44 ± 3.7 µg/ml) and the activity of nitrate reductase (74 ± 10 nmol/ml per min) were significantly (p < 0.05) higher in 42 oral cancer patients than in 40 healthy individuals (nitrite, 5.3 ± 0.3 µg/ml; nitrate, 27 ± 1.2 µg/ml; nitrate reductase activity, 46 ± 4 nmol/ml per min). The adjusted ORs and the 95% confidence intervals for oral cancer, categorized by the values for salivary nitrate and nitrite and nitrate reductase activity, showed a risk associated with a nitrite concentration > 7.5 µg/ml (OR, 3.0; 95% CI, 1.0–9.3), a nitrate concentration > 40 µg/ml (OR, 4.3; 95% CI, 1.4–13.3) and nitrate reductase activity > 50 nmol/ml per min (OR, 2.9; 95% CI, 1.1–7.4). The findings suggest that increased dietary intake of nitrate and nitrite is associated with elevated levels of salivary nitrite. Together with the increased activity of salivary nitrate reductase, these observations may explain, at least in part, the role of nitrate and nitrite in the development of oral cancer in individuals in areas with a high burden of N-nitroso precursors (Badawi et al., 1998).
(b) Case–control studies
A case–control study on gastric cancer and diet conducted in Marseille (France) included 92 patients with histologically confirmed adenocarcinoma and 128 controls undergoing functional reduction for injuries or trauma. The participants were interviewed by a trained dietician who administered a questionnaire about usual diet during the year preceding the first symptoms for cases or preceding the interview for controls. The intakes of nitrite, nitrite and pre-formed NDMA from food were estimated from a food composition table compiled ad hoc. Odds ratios were calculated after adjustment for age, sex, occupation and calorie intake. A high intake of preformed NDMA was associated with an increased risk for gastric cancer, the ORs for the second and third tertile of NDMA intake being: OR2 = 4.1 (95% CI, 0.93–18) and OR3 = 7.0 (95% Cl, 1.8–26). Intake of nitrate and nitrite was not associated with an increased risk for stomach cancer. Consumption of vegetables was protective in general, independently of their estimated nitrate content (Pobel et al., 1995).
A study was conducted to investigate whether consumption of foods and beverages containing nitrosamines, nitrite and nitrates affects the risks for laryngeal, oesophageal and oral cancer. In a population-based case–control study in western Washington State, USA, dietary consumption of these substances was measured for 645 cases (169 laryngeal, 125 oesophageal and 351 oral cancer ) and 458 controls. After adjustment for tobacco and alcohol use and other known risk factors, the risk for upper aerodigestive tract cancer was found to be 52% lower in persons who consumed larger amounts of nitrate (upper tertile) as compared with the lowest tertile (p < 0.001 for trend). Nitrate intake was associated with a reduction in the risk for cancers at all three sites. The reduction in risk for oesophageal cancer with increasing nitrate consumption was more evident in frequent tea drinkers than in other persons. The ORs for oesophageal cancer per category of nitrite intake were: 1.0 (reference) for < 1.1 mg/day, 1.2 for intakes between 1.1 and 1.6 mg/day and 1.6 (95% CI, 0.73–3.4) for intakes > 1.6 mg/day (p for trend = 0.20). There was no significant association between nitrite consumption and the risk for laryngeal or oral cancer; however, for individuals with a history of canker sores (an indicator of possible endogenous nitrosation), the risk for oesophageal cancer was seven times greater in those with a high nitrite intake than in those with a low intake. Consumption of foods with high concentrations of NDMA was associated with a 79% increase in the risk for upper aerodigestive tract cancer (p = 0.037 for trend). Daily consumption of beer and of nitrite-containing meats was associated with increased risks for oesophageal cancer (OR, 2.5 and 1.8, respectively). The OR for cancer of the oral cavity was also increased for people who drank beer daily (OR, 1.8). Persons who consumed large amounts of ascorbic acid from foods and from supplements were less likely to develop upper areodigestive tract cancer than were individuals with lower ascorbic acid intake (p for trend = 0.003) (Rogers et al., 1995).
Many cases of lower urinary tract cancer cannot be attributed to known risk factors, such as cigarette smoking and certain occupational exposures to chemicals. Data from a case–control study conducted on Oahu, Hawaii, between 1979 and 1986 were used to examine the role of other factors, such as total fluid intake and dietary intake of nitrites, nitrosamines and selected foods. A total of 195 male and 66 female patients of white and Japanese ancestry with lower urinary tract cancer were matched to two population-based controls on age, sex and race. Total fluid intake, and that of tap water in particular, showed an inverse dose–response relationship with cancer risk among women (OR for highest relative to lowest quartile of total fluid intake = 0.3; p for trend < 0.01). This association was stronger among smokers than nonsmokers. Although fluid intake showed no overall association among men, the findings for smokers suggested an effect similar to that found in women. Current intake of dietary nitrites and nitrosamines was positively associated with risk in Japanese men (for nitrites, OR for highest relative to lowest tertile = 2.0; p for trend = 0.05; for nitrosamines, OR for highest tertile relative to lowest tertile = 3.0; p for trend = 0.01). Consumption of processed meats, in particular bacon, sausage and ham, was also significantly associated with increased risk only in Japanese men, although associations with consumption of sausage in Japanese women and of bacon in white women were suggested. Unfortunately, it was not possible to determine whether these elevated risks were due to the fat, nitrite or sodium content of the processed meats or to their having been fried (Wilkens et al., 1996).
A case–control study conducted in northern Italy on 746 cases of histologically confirmed gastric cancer and 2053 controls who had been admitted to hospital for acute non-neoplastic, non-digestive-tract disorders addressed the interaction between gastric cancer and the intake of methionine, salt and nitrite. In relation to people with low methionine (< 1500 mg/day) and low salt intake (not defined), the ORs were 2.4 (95% CI, 1.5–4.0) for patients with low salt and high methionine intake (> 1900 mg/day), 1.5 (95% CI, 1.0–2.2) for patients with high salt and low methionine intake and 2.8 (95% CI, 1.9–4.2) for patients with high salt and high methionine intake. Likewise, in relation to people with low methionine and low nitrite intake (< 2.7 mg/day), the ORs were 1.9 (95% CI, 1.3–2.6) for high methionine and low nitrite, 1.5 (95% CI, 2.1) for low methionine and high nitrite (> 2.7 mg/day) and 2.5 (95% CI, 1.9–3.2) for high methionine and high nitrite intake. All these estimates were statistically significant. Thus, the findings from this large-scale case–control study conducted in a relatively high-incidence area for gastric cancer suggest that a diet rich in methionine, salt and nitrite is associated with an increased risk for gastric cancer (La Vecchia et al., 1997).
Studies of nasopharyngeal carcinoma have associated elevated risks with higher consumption of salted fish and preserved foods, particularly during childhood. Although these foods can contain high concentrations of nitrosamines, exposure to these compounds was not measured directly in most studies. In a case–control study conducted in Taiwan (China) to evaluate dietary intake and risk for nasopharyngeal carcinoma, interviews were conducted with a food-frequency questionnaire administered to 375 patients (99% response rate) and 327 controls (88% response rate) about their diet at age 10 and as an adult. The mothers of 96 cases and 120 controls were interviewed about their own diet during breastfeeding and the diets of their children during weaning, at the age of 3 and up to the age of 10. Concentrations of nitrosamines and nitrite were assigned to 66 foods on the basis of published values. Intake of nitrosamines and nitrite as an adult was not associated with an increased risk for nasopharyngeal carcinoma, but high intakes of nitrosamines and nitrite from foods other than soya bean products during childhood and weaning were associated with increased risks. The adjusted ORs for the highest quartile were 2.2 (95% CI, 0.8–5.6) for children aged 10, 2.6 (95% CI, 1.0–7.0) for children aged 3 and 3.9 (95% CI, 1.4–10) for the weaning diet. The intakes of nitrite and nitrosamines from soya bean products during childhood and weaning were inversely associated with risk, perhaps because soya beans contain known inhibitors of nitrosation. The results suggest that intake of nitrosamines and nitrite during childhood plays a role in the development of nasopharyngal carcinoma (Ward et al., 2000). The validity of the recall of childhood diets is uncertain, however.
In view of the rapidly rising incidence rates of adenocarcinoma of the oesophagus and gastric cardia in the USA, nutrient intake was studied as a risk factor for these cancers in a population-based case–control study in Connecticut, New Jersey and western Washington State. Interviews were conducted with 282 patients with histologically confirmed oesophageal adenocarcinoma, 255 with adenocarcinoma of the gastric cardia, 206 with oesophageal squamous-cell carcinoma, 352 with gastric adenocarcinoma other than in the cardia and 687 population controls. Associations between nutrient intake and risk for cancer were estimated as adjusted ORs, with a comparison of the 75th and 25th percentiles of intake. The nutrients that were significantly inversely associated with the risks for all four tumour types were fibre, beta-carotene, folate and vitamins C and B6. In contrast, dietary cholesterol, animal protein and vitamin B12 were significantly positively associated with risk for all four tumour types. Dietary fat was significantly associated with the risk for oesophageal adenocarcinoma only (OR, 2.2; 95% CI, 1.3–3.8), and dietary nitrite was associated only with non-cardia gastric cancer (OR, 1.6; 95% CI, 1.3–2.2). Use of vitamin C supplements was associated with a significantly lower risk for non-cardia gastric cancer (OR, 0.60; 95% CI, 0.41–0.88). Higher intake of nutrients found primarily in plant-based foods was associated with reduced risks for adenocarcinomas of the oesophagus and gastric cardia, whereas higher intake of nutrients found primarily in foods of animal origin was associated with increased risks (Mayne et al., 2001).
A study of risk factors for childhood brain tumours was conducted to investigate the association between source of residential drinking-water during pregnancy and the occurrence of brain tumours among offspring. Dipstick measurements were made of nitrates and nitrites in tap water in the houses of a subset of women who were living in the same house in which they had lived during their pregnancy. A total of 540 patients with childhood brain tumours and 801 controls were identified in Los Angeles County and the San Francisco Bay Area of California and western Washington State, USA. Overall, no increased risk for childhood brain tumours was found in offspring of women for whom wells were the source of water; however, an increased risk (OR, 2.6; 95% CI, 1.3–5.2] was observed among offspring of women in Washington State and a decreased risk (OR, 0.2; 95% CI, 0.1–0.8) among those in Los Angeles County who relied exclusively on well water. Among the small subset of participants for whom dipstick measurements of tap water were available, the risk for childhood brain tumours associated with the presence of measurable nitrite and/or nitrate was 1.1 (95% CI, 0.7–2.0). Given the crude measurement method used and the fact that measures were often obtained years after the pregnancy, the relevance of the dipstick findings is unclear. The lack of consistency in the findings for residential water source does not support the hypothesis of an increased risk with consumption of well water; however, regional differences in the well water content of nitrite may exist, and the increased risk observed in western Washington State deserves further evaluation (Mueller et al., 2001).
Although brain tumours are the main cause of death from childhood cancers, the causes of most of these tumours remain obscure. Few chemicals cause brain tumours experimentally after systemic administration of low doses; a notable exception is one group of N-nitroso compounds, the nitrosamides (in particular the nitrosoureas). Thus, feeding pregnant animals nitrosamide precursors (e.g. sodium nitrite and an alkylamide such as ethylurea) causes a high incidence of nervous system tumours in the offspring. A population-based epidemiological study was therefore designed to test the hypothesis that maternal consumption during pregnancy of meats cured with sodium nitrite increases the risk for brain tumours in their offspring. Intake of vitamins C and E inhibits endogenous formation of N-nitroso compounds and was expected to be protective. The mothers of 540 children under the age of 20 during 1984–91 in whom a primary brain tumour was diagnosed at that time and 801 control children in the same 19 counties on the west coast of the USA were interviewed. The risk increased with increasing frequency of eating processed meats (OR, 2.1 for consumption at least twice a day compared with no consumption; 95% CI, 1.3–3.2; p = 0.003). The risk also increased with increasing average daily amount of cured meats or of nitrite from cured meats (p for each < 0.005) but not with nitrate from vegetables. Daily use of vitamins throughout pregnancy decreased the risk (OR, 0.54; 95% CI, 0.39–0.75). The risk of mothers who consumed nitrite from cured meat at above the median was higher when vitamins were not taken (OR, 2.4; 95% CI, 1.4–3.6) than when they were (OR, 1.3). These effects were seen for each of the three major histological types of brain tumour and across social classes, age groups and geographical areas. This is the largest study to date of maternal diet and childhood brain tumours and suggests that exposure to endogenously formed N-nitroso compounds during gestation is associated with tumour occurrence (Preston-Martin et al., 1996).
In a study to evaluate the roles of maternal nutrition during gestation and subsequently in the etiology of childhood brain tumours, all 300 incident cases of nervous system tumours diagnosed in children under 18 in Israel between 1984 and 1993 were identified. Two matched population controls per case were selected (n = 574). Personal interviews were conducted with a semi-quantified three-step food frequency questionnaire. Univariate analysis showed that increased consumption of vegetable fat (p for trend = 0.01; 95% CI, 1.1–3.2), carbohydrates (p for trend = 0.05; 95% CI, 1.0–5.9) and vitamin E (p for trend = 0.05; 95% CI, 1.0–3.3) during childhood was significantly associated with risk for brain tumour. No associations were found with nitrate, nitrite or vitamin C. A significant positive association with potassium consumption during gestation (p for trend = 0.01; 95% CI, 1.1–3.7) was noted. In a multivariate analysis, the only persistent associations were with vegetable fat (OR, 1.4; 95% CI, 1.1–1.7) in the diet during childhood and potassium intake during gestation (OR, 1.4; 95% CI, 1.0–2.0) (Lubin et al., 2000).
(c) Cohort studies
The association between intake of nitrate or nitrite and gastric cancer risk was investigated in a prospective cohort study begun in 1986 in the Netherlands, of 120 852 men and women aged 55–69 years. At baseline, data on dietary intake, smoking habits and other covariates were collected by means of a self-administered questionnaire. For data analysis, a case–cohort approach was used, in which the person–years at risk were estimated for a randomly selected subcohort of 1688 men and 1812 women. After 6.3 years of follow-up, 282 microscopically confirmed incident cases of stomach cancer were detected, with 219 in men and 63 in women. The rate ratios for gastric cancer with increasing quintiles of mean nitrite intake were: 1.0 (intake, 0.01 mg/day), 1.2 (0.04 mg/day), 1.2 (0.09 mg/day), 0.88 (0.16 mg/day) and 1.4 (0.35 mg/day). The 95% confidence interval for the rate ratio derived by contrasting the highest versus the lowest intake quintile was 0.95–2.2. A test for trend in rate ratios across quintiles gave a p value of 0.24. The authors concluded that the study did not support a strong positive association between nitrite intake and gastric cancer risk (van Loon et al., 1998).
A cohort study was conducted to investigate the relationship between intake of nitrates, nitrites and NDMA and the risk for cancers of the gastrointestinal tract in 9985 adult Finnish men and women who were initially free of cancer. During a follow-up period of up to 24 years, 189 gastrointestinal cancer cases were diagnosed in the cohort. The intakes of nitrate, nitrite and NDMA were estimated from data on food consumption obtained during an interview about the total diet of the participants over the previous year. The mean daily intake of nitrates was 77 mg, and that of nitrites was 5.3 mg. Nitrates were provided mainly by vegetables (92%), whereas nitrites were derived mainly from cured meats and sausages (94%); dietary NDMA was provided by smoked and salted fish (52%) and cured meats and sausages (48%). The mean daily intake of NDMA from the diet was 0.052 µg, and that from beer was 0.071 µg. A significant positive association was observed between intake of NDMA and subsequent occurrence of colorectal cancer, with a relative risk between the highest and lowest quartiles of intake of 2.1 (95% CI, 1.0–4.3). Of the various sources of N-nitroso compounds, smoked and salted fish were found to be significantly associated with the risk for colorectal cancer (relative risk, 2.6; 95% CI, 1.2–5.5), and intake of cured meat was nonsignificantly associated (relative risk, 1.8; 95 % CI, 0.98–3.5). No such association was observed with intake of other fish or other meat. No significant association were observed between NDMA intake and cancers of the head and neck or of the stomach or between nitrate or nitrite intake and the risk for cancers of the gastrointestinal tract (Dich et al., 1996; Knekt et al., 1999).
(d) Intervention trial
The concentration of salivary nitrite in patients with cancer is twice that in healthy individuals, indicating that endogenous nitrite is correlated with cancer occurrence but not necessarily causally. In a culture medium modified with vitamin B1 or potassium chlorate, many bacteria found in the human body (especially the bowels) nitrified ammonium ion to nitrite ion. Nitrite ions can combine with a secondary amine to form nitrosamines, which are carcinogenic. A method for preventing cancer was tested in an experiment involving 7392 workers in a Chinese flax textile mill. Persons with a salivary concentration of nitrite ion > 10 mg/l for 3 consecutive days and with symptoms were targets for preventive treatment with antibiotics and ‘nitrosamine destroyers’. The average cancer incidence rate in the control group was 55% higher than in the experimental group over 9 years. The difference between the two groups was statistically significant (Huang et al., 1996). Unfortunately, this study is difficult to interpret, because there were problems with follow-up, no standardization for age seems to have been performed, the rates in the control group changed dramatically over the study period and the incidences of cancers potentially related to nitrite intake, such as stomach cancer, were proportionally increased in the experimental group. In fact, the experiment addressed not nitrite but antibiotics, vitamin C and other ‘nitrosamine destroyers’.
Erythrocytes in vitro took up 0.8 mmol/l nitrite with a half-time of 11 min. Nitrite uptake was sensitive to temperature and to the pH and ionic solution of the medium but was not inhibited by a specific anion-exchange inhibitor. About 25% of nitrite uptake occurred on the sodium-dependent phosphatase transporter and the rest as diffusion of nitrous acid or other species across the plasma membrane. Methaemoglobin formation increased in proportion to the intracellular nitrite concentration. Nitrite reacted with erythrocyte ascorbate, but ascorbate loading of cells decreased nitrite-induced methaemoglobin formation only at high nitrite concentrations. It was concluded that nitrite rapidly enters erythrocytes and reacts with oxyhaemoglobin but does not exert a strong oxidant stress on these cells (May et al., 2000).
The Committee considered a new study of toxicokinetics in human volunteers given sodium nitrite in their drinking-water. The maximum plasma nitrite concentrations were observed 15–30 min after dosing, and nitrite disappeared rapidly from plasma, with a half-life of approximately 30 min. The bioavailability of sodium nitrite was similar and virtually total after low and high oral doses. An intravenous dose of sodium nitrite of 290–380 mg per person induced methaemoglobinaemia, with maximum percentages of 8.4–12%. After oral administration, the maximum concentration of methaemoglobin was reached 0.70 h after dosing.
Several new studies were available on the acute and short-term toxicity of nitrite in humans, in which the severity of methaemoglobinaemia was reported after accidental, high intake of nitrite. Most were case reports of poisoning incidents. None of these studies added data that could be used for the safety evaluation.
The Committee considered studies recently completed within the National Toxicology Program in the USA. In 14-week studies of the toxicity of sodium nitrite administered in drinking-water to mice at doses equal to 0, 90, 190, 340, 750 or 990 mg/kg bw per day and to rats at doses equal to 0, 30, 55, 120, 200 or 310 mg/kg bw per day, elevated levels of methaemoglobinaemia were observed in rats in all treatment groups, but not in mice. The Committee considered a level of methaemoglobin formation of up to 5% not to be adverse. The lowest NOEL value was observed in a study in male rats and was equal to 55 mg/kg bw per day (37 mg/kg bw per day expressed as nitrite ion) on the basis of reduced sperm motility at higher doses. Reduced sperm motility was also seen in mice at higher doses. The results of the 2-year carcinogenicity studies with sodium nitrite added to drinking-water of mice and rats at a concentration of 750, 1500 or 3000 mg/l (equal to 45, 90 and 160 mg/kg bw per day in mice and to 35, 70 and 130 mg/kg bw per day in rats) led to the conclusion that nitrite is not carcinogenic in rats or male mice. The authors noted that there was equivocal evidence of carcinogenic activity of sodium nitrite in female mice, in view of the positive trend in the incidence of squamous-cell papilloma and carcinoma (combined) in the forestomach. As no evidence of genotoxicity in vivo or of cytotoxicity in the forestomach was found, the mode of action of sodium nitrite in inducing forestomach neoplasia is unclear.
A study of reproductive toxicity in mice given drinking-water containing sodium nitrite at concentrations up to 0.24% w/w (equivalent to 240 mg/kg bw per day) did not reveal adverse effects on reproductive performance or on end-points examined at necropsy.
New studies on genotoxicity were available. Sodium nitrite was mutagenic in a test for reverse mutation but did not induce micronucleus formation in the bone marrow or peripheral erythrocytes of mice treated in vivo, consistent with previous results.
A few studies on the endogenous formation of N-nitroso compounds after intake of nitrite or nitrate and of nitrosatable compounds (amines and amides) and the possible association with cancer were available. In addition, one long-term study of toxicity and carcinogenicity was reviewed, in which rats were fed fishmeal at various concentrations concomitantly with sodium nitrite. Dose-dependent increases in the incidences and multiplicity of atypical renal tubules, adenomas and renal-cell carcinomas were found. The concentration of N-nitrosodimethylamine in the stomach contents after 4 weeks of treatment with 64% fishmeal plus 0.12% sodium nitrite was twice that measured after administration of 8% fishmeal plus 0.12% sodium nitrite. However, the diets used in this study were considered to be nutritionally inappropriate for the rat: they had a high protein content, resulting in abnormally high renal nitrogen clearance. These studies do not provide additional insight for the safety evaluation of nitrite.
A number of epidemiological studies of the relationship between the intake of nitrite and cancer risk have been published since the forty-fourth meeting. At its present meeting, the Committee ranked the study designs according to their capacity to provide evidence of a relationship. In the descriptions below, relative risk estimates are given for those studies in which levels of intake of nitrite were provided. Four of the studies were cross-sectional, involving measurement of nitrite in, e.g., salivary or gastric juice in cancer patients and healthy subjects. Because cross-sectional studies do not take into account the time between exposure and disease, any observed differences in biomarkers of exposure might also be a consequence of the disease; therefore, these studies cannot contribute to a causal interpretation of the results of studies of nitrite and cancer risk.
Nine (longitudinal) case–control studies on previous nitrite intake and various cancer types were reviewed. For oral and laryngeal cancer, no association was found with nitrite intake. One study conducted in the USA reported a positive association with oesophageal cancer, with ORs of 1.0 (reference category), 1.2 and 1.6 for persons with a daily nitrite intake of < 1.1 mg, 1.1–1.6 mg and > 1.6 mg, respectively. The ORs and the trend across ORs were not statistically significant, however. The association between nitrite intake and oesophageal cancer was stronger, and it was significant for persons with a history of canker sores. Another study in the USA, however, found no association between nitrite intake and oesophageal cancer, nor with the subtypes adenocarcinoma and squamous-cell carcinoma; a positive association was found only with gastric cancer other than of the cardia. A positive association with gastric cancer was also reported in an Italian case–control study (average consumption, 2.4 mg/day), while no association was found in a French study (average consumption, 1.9 mg/day).
An association of borderline significance was found between nitrite intake and urinary bladder cancer in men but not women of Japanese descent, nor in whites of either sex, in Hawaii, USA. Although a positive association was reported from a study in the USA between brain tumours in children and their mothers’ consumption of processed meat, no association was found with nitrite intake during gestation or in childhood in a recent case–control study from Israel. One study on nasopharyngeal cancer among Taiwanese reported no association with nitrite intake in adulthood (as reported by cases and controls), but a positive association was found with childhood nitrite intake as recalled by the mothers of the cases and controls. The validity of recall of remote dietary intake is questionable, however.
Two prospective cohort studies have been conducted on nitrite intake and cancer risk. A cohort study from the Netherlands, with 6 years of follow-up, on dietary nitrite and gastric cancer risk reported relative risks of 1.0 (reference category), 1.2, 1.2, 0.9 and 1.4 for increasing mean quintiles of nitrite intake of 0.01, 0.04, 0.09, 0.16 and 0.35 mg/day, respectively. Neither the relative risks nor the trend was significant. A Finnish cohort study, with 24 years of follow-up, reported no association with the incidence of stomach, colorectal, or head-and-neck tumours. The average nitrite intake by this cohort was reported to be 5.3 mg/day.
Thus, some studies indicated increased risks for oesophageal and gastric cancer; however, other studies – particularly prospective cohort studies – revealed no such association. The results for brain tumours in children and for urinary bladder cancer in adults were equivocal. Wide variation between the studied populations in the recorded intake of nitrite was noted. In none of these studies was a possible interaction between nitrite and nitrosatable amines evaluated in respect of cancer risk. The results of these studies and those of the epidemiological studies considered by the Committee at its forty-fourth meeting do not provide evidence that nitrite is carcinogenic to humans. In addition, studies on nitrate intake and cancer risk (of relevance because of the conversion of nitrate to nitrite) also did not provide evidence of a positive association.
A 6-week study was performed in young adult and old rats to determine whether older animals are more sensitive to the toxic effects of nitrite, particularly with regard to the known effects on the kidney and on the functions of some hormones (insulin, thyroxine). No age-related differences were found. The hypertrophy of the zona glomerulosa in the adrenals of rats of this strain was seen only at an intake equivalent to 50 mg/kg bw per day, expressed as nitrite ion.
Several studies were available on the mechanism of action of nitrite on the vascular system, blood pressure and the adrenals in rats. Doses of 50 mg/kg bw or higher, expressed as nitrite ion, are known to lower blood pressure by causing vasodilatation. The Committee calculated that an oral dose equivalent to 160 mg/kg bw of nitrate ion induced a reduction in blood pressure of 15–20 mm Hg.
Administration of nitrite to rats resulted in minimal hypertrophy of the zona glomerulosa of the adrenal gland and secretion of aldosterone, which was reversible after 60 days. Inhibition of angiotensin-converting enzyme indicated that the effect was produced indirectly via stimulation of the renin–angiotensin axis.
The Committee concluded that the minimal hypertrophy reflected physiological adaptation to small fluctuations in blood pressure and should not be considered a direct toxic action on the adrenals. This conclusion implies that the safety evaluation should not be derived from the NOEL for minimal hypertrophy of the adrenal zona glomerulosa, used by the Committee at its forty-fourth meeting, but on NOELs for other end-points. The NOEL of 5.4 mg/kg bw per day (expressed as nitrite ion) was therefore considered to be no longer relevant, as it was based on the indirect effect on the adrenals described above. A NOEL of 6.7 mg/kg bw per day was identified in a 2-year study in rats, in which effects on the heart and lungs were observed at the next higher dose .
The Committee established an ADI of 0–0.07 mg/kg bw, expressed as nitrite ion, on the basis of the NOEL of 6.7 mg/kg bw per day for effects on the heart and lung in the 2-year study in rats and a safety factor of 100.
Nitrite causes methaemoglobinaemia. As this might occur after a single dose, it would be appropriate to establish an acute reference dose for nitrite. The data available for review at the present meeting related primarily to its long-term toxicity and were not relevant for establishing an acute reference dose. The Committee recommended that the acute toxicity of nitrite be reviewed at a future meeting.
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See Also: Toxicological Abbreviations Nitrite (WHO Food Additives Series 35) NITRITE (JECFA Evaluation)