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[CANCER RESEARCH 49, 3117-3121, June 1, 1989]
Endogenous Nitrosation in Relation to Nitrate Exposure from
Drinking Water and
Diet in a Danish Rural PopulationHenrik Moller,' Jannik Landt,
Erling Pedersen, Per Jensen, Herman Autrup, and Ole Mrtllcr
Jensen
Danish Cancer Society, Danish Cancer Registry, Institute of
Cancer Epidemiology, Rosenvaengets Hovedvej 35, P. O. Box 839,
DK-2100, Copenhagen [H. M., O. M.J.], Danish Cancer Society, The
Fibiger Institute, Copenhagen fJ. L., H, A.], National Food Agency,
Stborg [E. P.], Institution of Medical Health Officers,Nordjyllands
Amt [P. J.], Denmark
ABSTRACT
Increasing levels of nitrate in drinking water is of concern due
to thepossibility of an associated increase in long-term exposure
to endoge-nously formed carcinogenic A-nitroso compounds. Excretion
of .V-nitro-soproline in 12-h overnight urine after intake of
500-mg L-proline wasused to quantify the rate of endogenous
nitrosation in 285 individuals inan area in northern Denmark with
large variation in nitrate concentrationof the drinking water.
Nitrate intake was measured in a 24-h duplicatediet sample. The
crude association between nitrate concentration indrinking water
and rate of endogenous nitrosation in individuals is onlyweakly
positive and not quite statistically significant (/' = 0.08). The
risk
of having detectable nitrosation increases significantly with
total nitrateintake and tobacco smoking. In nonsmokers, nitrosation
is determined bynitrate intake. Smokers have increased nitrosation
which does not dependon nitrate intake. Effect modification through
dietary factors was evaluated and indicated a protective effect of
tea consumption, while the effectof eating vegetables was not
clear-cut. The experimental design (12-hurine sample; proline dose
taken in the evening) is likely to underestimatethe effect of
nitrate in drinking water relatively to nitrate in the diet.
INTRODUCTION
The average concentration of nitrate (NOj) in ground waterused
as drinking water in Denmark increased from 3.0 mgNOj/liter to 13.3
mg/liter over the period 1940 to 1983 (1).The maximum nitrate
concentration in Denmark should notexceed 50 mg/liter and it is
recommended that water with morethan 25 mg/liter is not used as
drinking water. However, by1983 some 6-7% of the Danish population
had water supplieswith more than 50 mg/liter, and 13-14% used water
with morethan 25 mg/liter (1). Health hazards related to a high
intake ofnitrate include the rare, acute toxic effect
methaemoglobinae-mia in bottle-fed infants (2), and the possibility
of increasedexposure to endogenously formed carcinogenic
¿V-nitrosocompounds (3-8). I has been suggested that gastric
cancer is associated with nitrate intake through this mechanism
(9-15), although the more recent evidence, especially from Britain,
donot show this association (16-19).
In a previous paper we demonstrated that drinking watermay be a
major source of total nitrate intake (20). With 50 mgnitrate per
liter drinking water, the average Danish consumeris exposed to 90
mg nitrate per day, more than half of which isderived from the
drinking water. Consumption of vegetables isthe other important
source of nitrate and provides on averageabout 40 mg per day. The
purpose of the present study was toexamine whether a high-nitrate
source of drinking water isassociated with increased exposure to
endogenously formed N-nitroso compounds, and to examine the
association betweentotal nitrate intake and endogenous
nitrosation.
Received 9/30/88; revised 2/13/89; accepted 2/21/89.The costs of
publication of this article were defrayed in part by the
payment
of page charges. This article must therefore be hereby marked
advertisement inaccordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
' To whom requests for reprints should be addressed.
MATERIALS AND METHODS
Endogenous nitrosation was investigated in a Danish rural
population through measurement of urinary excretion of the nontoxic
andnoncarcinogenic compound NPRO2 in 12-h urine samples
following
oral intake of 500 mg L-proline. This method is a modification
of theassay developed by Ohshima and Bartsch (4) which has been
usedintensively to quantify the potential for endogenous
nitrosation inhumans (21-22).
The Population. In the rural area of west Himmerland in
northernJutland, Denmark, age- and sex-stratified samples of 18
persons wereselected from the population being served by each of 21
waterworks,i.e., altogether 378 persons. The 21 water supplies had
deliberatelybeen selected to provide high variation in nitrate,
concentration in thedrinking water of the participants in the
study. Eight supplies had 0-5mg NOï/liter, five had 35-59
mg/liter, and eight had 60 mg/liter ormore. Thirty-nine persons
were permanently or temporarily living awayfrom the "permanent"
address obtained from the Danish Central Pop
ulation Register, and were considered not eligible for the
study. Of 339eligible persons, 294 (87%) participated in the study.
Of these, 281provided sufficient information to be included in all
statistical analyses.The participation rate was uniform over the
three groups of watersupplies.
Data Collection. A detailed description of the data collection
procedure has been given elsewhere (20). In short, the following
informationand samples were collected: (a) a questionnaire on
smoking habits,history of gastric disease, and dietary habits; (hi
a 24-h duplicate portionof the total diet on the day of
participation from early morning to latenight. Separate containers
were provided for beverages (including tapwater) and more solid
dietary items; (c) a record of all dietary itemsconsumed during the
24-h period of collection of the duplicate portion;(if) a 12-h
overnight urinary sample collected from l h after the eveningmeal
to (and including) the first void in the following morning.
Capsuleswith 500-mg L-proline were taken by the participants at the
start of theperiod of urine collection. An aliquot of the urine
sample was conservedwith NaOH and stored at -20"C; (e) two samples
of tap water from
the residence. The data and biological samples were collected
fromSeptember 1986 to March 1987.
Chemical Analyses. Nitrate in drinking water samples, duplicate
dietportions, and urine samples were measured following
homogenizationand extraction (20, 23). Urinary creatinine was
determined by thealkaline picrate method (24).
The method used for measurement of NPRO was based on the workof
Ohshima and Bartsch (4, 25). NPIC and NPRO was kindly suppliedby
Dr. Bartsch, IARC, Lyon, and by the Danish Institute of
ProteinChemistry, H0rsholm. Other chemicals were analytical
grade.
To 15ml of thawed, centrifuged urine in a separating funnel
wereadded 2 ml 20% ammonium sulfamate solution in 1.8 M sulphuric
acid,4 g sodium chloride, and 247 ^g of NPIC as internal standard.
Theurine was extracted three times with 25 ml dichloromethane
containing10% methanol, and the organic phase was filtered though
anhydroussodium sulfate into a 100 ml pear-shaped evaporation
flask. The sodium sulphate was washed with extraction solvent, the
combined extracts were evaporated in rotary evaporator at 35°Cand
dissolved in 1
ml ethylacetate.A diazomethane generator was mounted in the
flask and cooled in
an ice bath. Diazomethane was generated from approx. 40 mg
N-methylnitrosourea by addition of 300 n\ 6 N NaOH. After
methylation
2The abbreviations used are: NPRO, ¿V-nilroso-L-proline;NPIC,
iV-nitroso-L-
pipecolic acid.
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ENDOGENOUS NITROSATION IN RELATION TO NITRATE EXPOSURE
for l h the yellow extracts were transferred into 2-ml
autosampler vialsclosed with teflon-lined septa. Ten M' extract was
injected on a 4-mmi.d. stainless steel column packed with 10%
Superox 4 on ChromosorbW with the following program: 140°Cisotherm
for 5 min, 4°C/minto220"C, which was kept for 2 min. The separated
nitrosocompounds
were detected with a Thermal Energy Analyser model 502 using
apyrolyzer temperature at 475'C. The retention times of NPIC
and
NPRO were 22.0 min and 23.7 min.Contents of NPRO was calculated
from peak heights corrected for
recovery of NPIC using a standard curve with NPIC and
NPROmethylated in the same way as the urine extracts. The standard
curvewas linear from 0 to at least 300 ng NPRO/ml extract. The
detectionlimit was better than 1.0 MgNPRO/12 h.
Statistical Analysis. First, the three groups of persons with
watersupplies with low, intermediate, and high nitrate
concentration in thedrinking water were compared. The Wilcoxon rank
sum test was usedto evaluate differences between medians in NPRO
excretion and othervariables. Secondly, the crude association of
NPRO excretion withnitrate concentration in drinking water and with
total nitrate intakewere evaluated, and x2 test for trend was used.
The relative risk of
having NPRO excretion exceeding 1 jug/day was used as the
measureof association. Thirdly, by means of multivariate maximum
likelihoodlogistic regression analysis (26) using the CATMOD
program in theSAS-package (27), the determinants of endogenous
nitrosation measured as urinary excretion of NPRO was further
evaluated. Due to theextremely skewed distribution of urinary NPRO,
this variable wasanalyzed as a discrete outcome phenomenon. One ng
NPRO or morein the 12-h urinary sample was considered a positive
outcome, whileurinary excretion of less than 1 Mgwas considered a
negative outcome.Similarly, separate regression models were
evaluated to identify factorsinfluencing the risk of exceeding 5
¿igand 1U¿¡gNPRO in the 12-hurine sample. The effect
modification of dietary factors on the association between nitrate
intake and endogenous nitrosation, was furtherevaluated among
nonsmokers. Tobacco smoking was quantified as"cigarette equivalents
per day": one cigar = one pipe-full = 2.5 cigarillos
= five cigarettes.
RESULTS
Table 1 shows the marked variation in the nitrate concentration
of drinking water reflecting the selection of the three groupsof
persons. The nitrate content of the 24-h duplicate dietportion
(including beverages) and urinary nitrate varies significantly with
the measured nitrate concentration in drinkingwater. Urinary
creatinine is identical in the three groups. Themedian of urinary
excretion of NPRO is highest in the groupwith the highest nitrate
concentration in the drinking water,
but none of the pairwise differences between median
NPROexcretion in the three groups are statistically
significant.
Fig. 1 shows the distribution of NPRO in individual
urinesamples. About half of the samples contained less than 0.5
/¿gNPRO while the other half contained from 0.5 to 119
/tg.Consumption of cured meat products during the 24-h periodbefore
urine collection is associated with a high urinary level ofNPRO.
The odds-ratio for excreting more than 1 ¿tg/12hassociated with
consumption of cured meat is 3.68 (1.27-10.67)for having more than
1 ^g NPRO/12 h, which increases to 4.63(1.54-13.87) and 9.30
(2.04-42.40) for excreting more than 5ng/12 h and more than 10
ng/12 h, respectively. This meansthat consumers of cured meat
contribute markedly to the uppertail of the distribution of urinary
NPRO measurements (Fig.1). It is well known that cured meat may
contain up to severalgram NPRO per kilo meat product (28). Like
NPRO formedin the stomach, ingested NPRO is not metabolized but is
rapidlyexcreted in the urine. It is thus likely that the increased
urinaryNPRO associated with consumption of cured meat
representingested NPRO rather than intragastric formation.
Tables 2 and 3 show a positive association between
nitrateconcentration in drinking water and nitrate intake
respectivelyon the prevalence of NPRO excretion exceeding 1 ng in
personswho do not smoke tobacco. NPRO excretion increases
withnitrate concentration in drinking water in nonsmokers, although
not statistically significant (x2 test for trend; P = 0.08)(Table
2) or in smokers and nonsmokers combined (P = 0.08).NPRO excretion
increases with total nitrate intake in non-smokers (x2 test for
trend; P < 0.01) (Table 3) and in smokersand nonsmokers combined
(x2 test for trend; P < 0.01).
Results of the logistic regression analysis of these
individualdata are shown in Table 4. From the model including sex,
age,history of gastric disease, batch of NPRO analysis,
urinarycreatinine concentration, and consumption of cured meat it
canbe estimated that the odds-ratio for excretion of 1 ng NPRO
ormore is 1.9 for an increase in nitrate intake of 50 mg/dayamong
nonsmokers; this is highly significant (95% confidenceinterval =
1.39-2.58). Nitrate intake was in this model evaluated as a
continuous variable with a unit = 50 mg/day. Amongsmokers of 1-20
cigarette-equivalents per day, nitrosation issignificantly
increased compared with nonsmokers and nitrateintake is not a
strong determinant within this group. In thegroup of heavy smokers
(21+ cigarette-equivalents) nitrosationis also increased compared
with nonsmokers, but the effect of
Table 1 Quantitative information from samples of drinking water,
24-h duplicate dietary samples, and overnight urinary samples in
three drinking water categories
Number of samples examined given in brackets.
Drinking water category/' value of pairwise comparison of
medians
Low nitrate (L)eight water supplies (0-5 mg/li
ter)Intermediate
(I)five water sup
plies (35-59 mg/liter)High
nitrate (H)eight water supplies
(60+mg/liter)L&II&HL&HNitrate
concentration in drinking water(mg/liter)Mean±standard
deviation(n)Median;
quartilerangeTotalnitrate in 24-h duplicate portion(mg)Mean
±standard deviation(n)Median;quartilerangeNitrate
in urinary sample(mg)Mean±standard deviation(n)Median;
quartilerangeUrinarycreatinine(mM)Mean±standard
deviation(n)Median;
quartilerangeUrinaryNPRO (>ig/l 2h)Mean±standard
deviation(n)Median;
quartile range0.3
±0.7(115)0.0;0.0-0.046.5
+43.6(114)37.2;17.5-58.336.3
+23.8(112)31.6;22.0-44.39.6
±4.7(115)8.2;6.3-11.62.90
±9.15(112)0.00;0.00-3.0546.5
±6.9(76)45.3;41.0-53.0102.4
±46.3(76)89.3;72.6-123.054.9
+26.9(71)50.3;35.8-65.69.9
±5.0(73)9.0;5.7-14.31.86
±2.85(74)0.69;0.00-2.3584.4
±19.2(103)80.0;69.0-96.0143.6
±79.8(103)122.8;87.5-187.572.6
+38.0(100)63.7;44.4-86.19.7
±4.8(103)9.0;6.3-11.73.75
±12.57(99)0.83;0.00-3.270.00010.00010.00010.460.260.00010.010.010.980.810.00010.00010.00010.340.19
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ENDOGENOUS NITROSATION IN RELATION TO NITRATE EXPOSURE
URINARY NPRO ( ug/12h)
Fig. 1. Distribution of NPRO in 285 overnight urine samples,
ng/12 h. TheNPRO analysis was run in three batches with an
estimated detection limit of atleast 1.00 MgNPRO/12 h. Four samples
were out of the range 0-20 MgNPRO/12 h: 21, 30, 90, and 119 Mg/12
h.
nitrate appears to be reversed although the confidence
intervalsare very wide.
Table 5 shows the results of further analyses of
dietarydeterminants of nitrosation in nonsmokers. When
informationon consumption of selected foods is added individually
to thebaseline model given in Table 4, increased nitrosation is
seenfor green vegetables, cabbage, and other vegetables,
althoughonly the effect of green vegetables is statistically
significant.Consumption of tea and vegetables on sandwiches
providessignificant protection against nitrosation.
The 24-h duplicate portions were collected with use of separate
containers for beverages and solid dietary items. Nitrate inthese
subsamples were measured separately. It is thereforepossible to
estimate the effect of nitrate in the liquids (mainlyfrom drinking
water but also from fruit juices etc.) and in theof more solid
dietary items [mainly from vegetables but alsofrom drinking water
used in cooking (20)]. An odds ratio of1.88 (1.37-2.57) among
nonsmokers for 50 mg total nitratebreaks down to 1.48 (0.95-2.32)
for 50 mg nitrate in the liquidssubsample and 2.41 (1.46-3.96) for
50-mg nitrate in the sub-sample of solid dietary items, when these
are considered simultaneously in the regression model.
DISCUSSION
It was the purpose of the present study to examine
theassociation between nitrate intake and endogenous
nitrosation
in view of the hypothesized relationship between nitrate,
nitrosation, and gastric cancer. Examination of the general
population exposed to various levels of nitrate is of particular
importance in view of the often high and increasing exposure
tonitrate from drinking water which result from intensive
agricultural practices.
While no overall association is found between nitrosation
inthree groups of individuals with household water-supplies
with0.3, 46.5, and 84.4 mg nitrate/liter, respectively, nitrosation
ofproline is strongly associated with nitrate intake in
individualswho do not smoke tobacco. The nitrosation rate should
theoretically depend on the square of the concentration of
nitrate(29). Inclusion of the square of nitrate intake in the
regressionanalysis did not improve the goodness-of-fit. Ohshima
&Bartsch (4) found, in a nonsmoking individual, very low
nitrosation rates at nitrate intakes below 200 mg/day and
stronglyincreasing nitrosation with higher nitrate intake. Few
individuals in the present study had a nitrate intake of 200 mg/day
ormore, and it appears that nitrate intake increases
endogenousnitrosation in nonsmokers, also at the level of 20-150
mg/daytypically found in the Danish population.
The odds-ratio of excreting more than 5 ng NPRO/12 h and10 ng
NPRO/12 h for a change of 50 mg nitrate per day arenot
substantially different from the 1.90 obtained with a threshold at
1 fig NPRO/12 h. The effect of nitrate intake in non-smokers is
thus generally to increase the rate of endogenousnitrosation.
Endogenous nitrosation is associated with tobacco smokingand
apparently independent of nitrate intake in smokers. Thi-
ocyanate is a strong catalyst of nitrosation of amines,
andsmokers have three to four times higher levels of thiocyanatein
saliva than nonsmokers (30). Also, thiocyanate and nitratecompete
at the level of active transportation and salivary recirculation
(31). Increased salivary thiocyanate levels may therefore increase
the rate of endogenous nitrosation in the stomachand at the same
time, for any level of nitrate intake, reduce theflow of
recirculated nitrate in saliva. Our findings of an increased
excretion of NPRO in smokers is in line with previousobservations
(32).
Generally, vegetables are the major source of nitrate intake(15)
but in Danish communities with nitrate concentration indrinking
water exceeding about 50 mg/liter (20) this was notfound to be the
case. Vegetables contain nitrate which mayincrease endogenous
nitrosation, but vegetables also containascorbate, a strong
nitrosation inhibitor (33). Our results (Table5) show that
consumption of vegetables increases the endogenous formation of
NPRO. The effect is strongest for greenvegetables (OR = 2.37) but
the effects of cabbage (OR = 1.82),and other vegetables (OR = 1.43)
is in the same direction,although these effects are not
statistically significant. On theother hand, consumption of other
sources of ascorbate: vege-
Table 2 Prevalence of excretion of 1 ng NPRO or more in
overnight urine, in relation to nitrate concentration in drinking
waterNonsmokers0Amount
NPROinNitrate
concentrationin
drinkingwater(mg/liter)0-2425-4950-7475-99100+overnight
urinarysample>lMg22918107=
1Mg36142394%
alMg37.939.143.952.663.6Cruderate-ratio1.001.031.161.391.68Smokers*Amount
NPROinovernighturinary
samplealMg201111113
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ENDOGENOUS NITROSATION IN RELATION TO NITRATE EXPOSURE
Table 3 Prevalence of excretion of I /ig NPRO or more in
overnight urine, in relation to total nitrate
intakeNonsmokers"Total
nitrateintake
(mg/24h)0-49
50-99100-149150-199
200+Amount
NPRO inovernight urinary
sampleal
Mg
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ENDOGENOUS NITROSATION IN RELATION TO NITRATE EXPOSURE
that nitrate in drinking water can only be one of several
determinants of gastric cancer incidence in Denmark where
theincidence rate of this disease has decreased steadily for
severaldecades. A full understanding of the epidemiology of
gastriccancer in Denmark requires, within the framework of
Correa's
model, that changes in the intake of nitrate from other
sourcesthan drinking water and changes in the intake of
nitrosationinhibitors in this century are given consideration.
ACKNOWLEDGMENTS
The authors wish to thank the persons who participated in the
study,and the following individuals for their skillful technical
assistance: JetteAndersen, Niels Christensen, Charlotte Eriksen,
Pia Kristiansen, Marianne Lorentzen, Maibritt Mikkelsen, Bernard
Borys Szoymer, andVibe Roepstorff. The financial resources were
provided by the DanishCancer Society (Krzftens Bekasmpelse) and a
grant from Nordjyllandsamtskommune.
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