Relevance to Human Cancer of N-Nitroso Compounds, 
Tobacco Smoke and My..otsecins. 
Ed, 1.K. O'Neill, J, Char and H. Bartsch 
Lyon, International Agen.y for Research on Cancer 
IARC, 1991 
EXPOSURE TO N-NITROSAMINES AM) OTHER RISK FACTORS FOR 
GASTRIC CANCER IN COSTA RICAN CHILDREN 
R. Sierra=, H. Ohshima2, N. Muiloz2, S. Teuchmann2, A.S. pena3, 
C. Malaveille2, B. Pignatelli2, A. Chinnocki, F. El Ghissassi2, 
C. Chen, A. Hautefeuille2, C. Gamboa' & H. Bartsch2 
'University of Costa Rica, San Josi, Costa Rica; 
nInternational Agency for Research on Cancer, Lyon, 
France; and 'Department of Gastroenterology, University 
Hospital, Leiden, The Netherlands 
The hypothesis that endogenous chemical nitrosation in the normal stomach in early life 
could play a crucial role in inducing chronic atrophic gastritis/intestinal metaplasia in 
later life was tested by applying the N-nitrosoproline (NPRO) test to 12-h urine samples 
from about 50 children (aged 8-14 years) living in high- and low-risk areas for stomach 
cancer. The median values of NPRO and the sum of four nitrosamino acids analysed were 
0.28-0.84 ag/12 h and 0.75-1.75 tig/12 h, respectively. The NPRO level after proline intake 
was significantly higher in children from a high-risk area than in those from a low-risk 
area (p < 0.04), and markedly reduced after ingestion of ascorbic acid and proline (p < 0.05). 
Urinary nitrate level was lower than that of adults. NPRO levels on the day of proline 
intake, however, correlated well with nitrate levels (p < 0.001), indicating that children in a 
high-risk area in Costa Rica have high endogenous nitrosation potential. Blood samples 
were also collected from about 300 children (aged 7-20 years) and analysed for antibodies 
against Campylabacter pylori, a suspected gastritis-causing bacteria. About 71% of children 
in both high- and low-risk areas for stomach cancer had antibodies. In addition, raw and 
cooked beans, which are consumed very frequently in Costa Rica, were collected from 
families in both areas and analysed for levels of nitrite/nitrate, total N-nitroso compounds 
and genotoxicity in the SOS chromotest. Mean levels of total N-nitroso compounds in an 
aqueous extract (pH 2) of cooked bean samples from high- and low-incidence areas were 
similar (0.4-0.6 nmol/g of cooked beans). Acid-catalysed nitrosation of the same aqueous 
extracts produced levels up to 2.4 pmol/g of cooked beans. There was no difference in mean 
levels of nitrosation-dependent total N-nitroso compounds between samples from the two 
areas. Only two out of 11 extracts from the low-incidence area and two out of 14 from the 
high-incidence area showed weak direct genotoxicity. After acid-catalysed nitrosation, all 
samples were genotoxic at similar levels. 
Stomach cancer is the most common cause of death from cancer in Costa Rica: in 1984, 
it caused 33% of deaths from cancer in males and 22% in females. For the period of 
1980-83, the age-adjusted incidence rate was 58.8 per 100 000 for men and 25.2 per 
-162- 
X25"  
Risk factors for gastric cancer in Costa Rica 	 163 
100 000 for women. Although these incidence rates are declining, they are the highest in 
Latin America, and internationally they are second only to those observed in Japan. There 
is, however, great variation in rates in this small country (51 000 km2) which has a relatively 
homogeneous population: for men, the rate varies from 842 per 100 000 in the highlands 
of the centre of the country to 25.4 in the coastal areas (Sierra et al., 1989). These data 
suggest that environmental factors and habits in the different regions could be important 
risk factors. 
It has been postulated that N-nitroso compounds, in particular N-nitrosamides, are 
formed intragastrically by bacteria colonized in the stomach, on the basis of studies of 
patients with chronic atrophic gastritis (a precancerous condition of the stomach; Correa 
et al., 1975; Correa, 1988). However, it is a matter of controversy whether the level of 
N-nitroso compounds formed in the stomach of such patients is higher than that of normal 
subjects (Bartsch a al., 1989); patients with chronic atrophic gastritis did not excrete more 
N-nitrosoproline than patients with a normal stomach after ingestion of proline and nitrate 
(Crespi et al., 1987). On the basis of these results and epidemiological data, in particular 
that from studies of migrants, it has been hypothesized that N-nitroso compounds formed 
by acid-catalysed chemical nitrosation in the normal stomach in early life could play a 
crucial role in gastric carcinogenesis (Crespi et al., 1987; Ohshima a al., 1988). 
We have postulated that bacterial colonization in the stomach, especially by the 
bacterium Campylobacterpylori, recently identified in human stomach mucosa as a possible 
etiological agent of gastritis, may contribute to gastric carcinogenesis by inducing chronic 
irritation or inflammation. We tested these hypotheses in Costa Rican children living in 
high- and low-risk areas for stomach cancer, using the N-nitrosoproline test and serological 
examination for C. pylori infection to assess exposures. 
One community in a high-risk area (Turrubares; age-adjusted incidence rate of gastric 
cancer, 61.3/100 000) and one in a low-risk area (Hojancha; 18.7/100 000) for stomach 
cancer were selected on the basis of the following criteria: rural community, a large enough 
population of school children, similar ethnic characteristics and accessibility. A list was 
produced of all children aged 8-14 years in each community, and 25 subjects were selected 
at random from each list. Two samples of 12-h overnight urine were collected from children 
after they had ingested 500 mg proline and 200 mg ascorbate or 500 mg proline alone 1 
h after the evening meal. The urine samples were analysed for N-nitrosamino acids and 
nitrate as exposure markers, as described previously (Ohshima et al., 1987). 
Blood samples were also collected from about 200 children aged 7-13 years and from 
about 80 volunteers aged 14-20 in the same communities and analysed for antibodies to 
C. pylori, using a method described by Pena et al. (1990). 
Levels of N-nitrosamino acids and nitrate 
The results of analyses for N-nitrosoamino acids and nitrate are given in Table 1. The 
levels of N-nitrosamino acids were much lower (1/5-1/10) than those detected in adult 
urines collected in Japan, China, Poland and other countries (Bartsch et a1., 1989). It is not 
known whether the low levels in Costa Rican children are due to their low body weight or 
to other factors related to age, or to geographical, environmental and dietary factors, such 
164 
	 R. Sierra et al. 
Table 1. Median (95% confidence intervals) amounts of N-nitrosandno acids (µg/I2 h) and 
nitrate (mmo1/12 h) excreted in 12-h urine' 
Urinary 
component' 
Low-risk area High-risk area 
Day 1 (proline plus 
vitamin C) 
Day 2 (proline) Day 1 (proline plus 
vitamin C) 
Day 2 (proline) 
Volume (ml) 415.00 (395-542) 445.00 (396-559) 327.00 (293-473) 377.00 (286-460) 
N-Nitrosamino 
adds 
NPRO 0.28 (0.21-0.51) 0.54 (0.43-1.09) 0.66 (0.44-1.29) 0.84 (0.89-2.44) 
NTCA 0.48 (038-1.27) 0.52 (037-1.48) 0A0 (0.33-1.64) 0.77 (0.64-1.80) 
NMTCA 0.05•(0.05-0.10) 0.05 (0.04-0.51) 0.05 (0.04-0.40) 0.05 (0.04-030) 
Sum 0.75 (0.73-1_80) 1.25 (1.08-2.82) 1.62 (1.00-3.13) 1.75 (1.67-4.44) 
Nitrate 0.23 (0.22-0.38) 0.20 (0.21-0.39) 0.29 (0.26-0.66) 0.23 (0.17-0.87) 
26 children (19 boys and 7 girls) and 25 children (15 boys and 10 girls) from high- and tow-risk areas for stomach 
cancer, respectively, participated in the study; their ratan agcy were 1L3 and 10.5 years. and mean body weights were 36 
and 34 kg. respectively 
NPRO, N-nitresoproline; NTCA, N-nitrosothiazolkline 4-carboxylic acid; NIMTCA. N-nitroso-2-methythiazolidine 
4-carboxylic acid 
as a high intake of ascorbic acid (dietary data obtained in these areas are currently being 
analysed; Sierra et al., in preparation). All comparisons of levels of N-nitrosoproline were 
statistically significant. Thus, the level was reduced by ingestion of ascorbic acid with proline 
compared with that seen with proline alone in both areas (low-risk area, p < 0.01; high-risk 
area, p < 0.05; Wilcoxon test); and the level was significantly higher in children in the 
high-risk area than in those in the low-risk area after ingestion of ascorbic acid plus proline 
(p < 0.02) and after ingestion of proline alone (p < 0.04; both Mann-Whitney test). Thus, 
the level of N-nitrosoproline after proline intake was higher in children from the high-risk 
area than in those from the low-risk area and markedly reduced after ingestion of ascorbic 
acid and proline. The nitrate level was also lower (1/2-1/3) than that in adult urines; 
however, the N-nitrosoproline levels on the day of proline intake correlated well with the 
nitrate levels (p < 0.001). These results indicate that children living in this high-risk area 
in Costa Rica have a higher potential for endogenous nitrosation than those living in the 
low-incidence area, although the absolute values are lower than those of adult subjects in 
other countries. 
Infection with Campylobacter pylori 
As shown in Table 2, seropositivity for C. pylori in young subjects was 62.6-71.7% for IgG 
antibody and 38.7-73.0% for IgA antibody. There was no difference between subjects living 
in high- and low-risk areas; however, seropositivity and average antibody values were 
greater in some groups of children in the age range 14-20 years than in that of 7-13 years. 
These results indicate that the majority of the children studied were infected with C. pylori 
166 
	 R. Sierra et al. 
Table 3. Concentrations of total N-nitroso compounds (NOC) and genotosicity in the SOS 
ehromotest (SOS induction potency; SOSIP) of acidic aqueous extracts of cooked beans 
collected in high- and low-risk areas for stomach cancer in Costa Rica 
Area 	 No. of 	 Mean (range) of total NOC Mean (range) of SOSIP 	 SOSIP/iimol 
samples 	 (nnioI/g cooked beans) 	 (per g cooked beans) 	 total NOC 
Y ° 	  
Before 	 After 	 Before 	 After 	 (After 
nitrosation nitrosation nitrosation nitrosation nitrosation) 
High-risk 14/13 0.57 1231 4 96 58 
(0-1.7) (560-1600) (0-28) (38-162) (26-92) 
Low-risk 11/11 0.42 1322 8 80 45 
(0-1.8) (798-1810) (0-44) (22-189) (12-80) 
' Before (first figure) and after nitrosation (second figure). Nitrosation was carried out in vino at pH 2 at. 37•C for 60 min 
with 100 mmol/INaNOt. The reaction was stopped by adding an excess of sulfamic acid. Total NOC concentration was 
determined by the method of Pignatelli et el (1987) and genotoxicity by the SOS chromotest as described by MaEaveille er 
al. (1989). 
Conclusion 
We have demonstrated that Costa Rican children living in a high-risk area for stomach 
cancer have a greater potential for endogenous nitrosation than children living in a low-risk 
area; the levels of N-nitrosamino acids detected in their urine were, however, much lower 
than those in adults living in other countries. C. pylori infection, assessed by serological 
examination, was found to be very prevalent in children in both areas, indicating that 
gastritis occurs very early in Costa Rican children and persists for a long time. The 
genotoxicity and total concentrations of N-nitroso compounds in beans consumed very 
frequently in Costa Rica. before and after nitrosation in vitro, were similar for the two areas. 
Further studies are required to identify other factors that determine the risk for developing 
gastric cancer in the inhabitants of the high-risk area. 
Acknowledgements 
This study was undertaken during the tenure of an International Cancer Research 
Technology Transfer Programme (ICRETT) fellowship awarded to R.S. by the International 
Union Against Cancer. We thank Mrs E. Bayle for secretarial work. 
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