Tohoku J. Exp. Med.

, 2012, 226, 11-17 Association of Nitrogen Compounds with ESCC 11

Association of Nitrogen Compounds in Drinking Water with

Incidence of Esophageal Squamous Cell Carcinoma in Shexian,
China
Nan Zhang,1 Cao Yu,2 Denggui Wen,2 Jun Chen,1 Yiwei Ling,1 Kenshi Terajima,1
Kohei Akazawa,1 Baoen Shan2 and Shijie Wang2
1
Department of Medical Informatics, Niigata University Medical and Dental Hospital, Niigata, Japan
2
Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang, P.R. China

The incidence of esophageal squamous cell carcinoma (ESCC), which is the eighth most common
malignancy worldwide, is highest in China. The purpose of this study was to investigate the association
between nitrogen compounds in drinking water with the incidence of ESCC by geographical spatial
analysis. The incidence of ESCC is high in Shexian county, China, and environmental factors, particularly
nitrogen-contaminated drinking water, are the main suspected risk factors. This study focuses on three
nitrogen compounds in drinking water, namely, nitrates, nitrites, and ammonia, all of which are derived
mainly from domestic garbage and agricultural fertilizer. The study surveyed 48 villages in the Shexian
area with a total population of 54,716 (661 adults with ESCC and 54,055 non-cancer subjects). Hot-spot
analysis was used to identify spatial clusters with a high incidence of ESCC and a high concentration of
nitrogen compounds. Logistic regression analysis was used to detect risk factors for ESCC incidence.
Most areas with high concentrations of nitrate nitrogen in drinking water had a high incidence of ESCC.
Correlation analysis revealed a significant positive relationship between nitrate concentration and ESCC (P
= 0.01). Logistic regression analysis also confirmed that nitrate nitrogen has a significantly higher odds
ratio. The results indicate that nitrate nitrogen is associated with ESCC incidence in Shexian county. In
conclusion, high concentrations of nitrate nitrogen in drinking water may be a significant risk factor for the
incidence of ESCC.

Keywords: esophageal squamous cell carcinoma; geographic information system; nitrate nitrogen; spatial analysis;
water pollution
Tohoku J. Exp. Med., 2012, 226 (1), 11-17.  © 2012 Tohoku University Medical Press

The incidence of esophageal squamous cell carcinoma
(ESCC), which is the eighth most common malignancy
worldwide (Stoner and Gupta 2001), is highest in China
and its geographic variation in incidence is striking. In
northern China, the southern part of Hebei province, the
eastern part of Shanxi province, and the northern part of
Henan province have a particularly high incidence of ESCC
(Yang 1980; Qiao et al. 2001; Gao et al. 2009; Zhang et al.
2011; Song et al. 2011). Shexian county, which is located
in the southern Taihang Mountains, has one of the highest
incidences of ESCC in the world, with a rate of 210.5-
325.8/10,000 in men and 117.5-185.7/10,000 in women
(Zhang et al. 2011). These rates are significantly higher
than the Chinese national average of 17.0/10,000 (Wu et al.
2006).
Environmental, biological, and genetic factors have all
been implicated in the pathogenesis of esophageal cancer.
The factors include low consumption of fresh fruit and
Received October 12, 2011; revision accepted for publication November 15, 2011. doi: 10.1620/tjem.226.11
Correspondence: Nan Zhang, Department of Medical Informatics, Niigata University Medical and Dental Hospital, Asahimachi-Dori
1-754, Niigata 951-8520, Japan.
e-mail: zhangnan@med.niigata-u.ac.jp
green vegetables, insufficient micronutrient intake, nitrosa-
mine, smoking, and alcohol consumption (Stoner and Gupta
2001). In addition, some studies have shown that
environmental factors such as drought, high wind speed,
normalized difference vegetation index, and elevation are
associated with esophageal cancer incidence (Kmet and
Mahboubi 1972; Wu and Li 2007; Wu et al. 2008).
Drinking water was found to be an important putative envi-
ronmental factor of the high ESCC incidence in the Linzhou
area, which is near the southern part of Shexian county (Han
et al. 2007). Consequently, researchers have suspected that
drinking water plays the same important role in ESCC
pathogenesis in the Shexian area. However, few studies
have examined the association between chemical com-
pounds in drinking water and the incidence of ESCC in
Shexian. Therefore, the Department of Medical Informatics
of Niigata University and the Fourth Affiliated Hospital of
Hebei Medical University started a cooperative research

11
12 N. Zhang et al.
project to investigate such associations in southern Hebei
Province. In this paper, we attempt to show the relationship
between each risk factor and the incidence of ESCC in the
study area, as these findings have the potential to help local
governments establish effective cancer prevention policies.
In the past, researchers used paper maps to evaluate
and describe cancer distribution, but such maps have limita-
tions. For example, it is difficult to combine layers, and
doing so is costly and time-consuming. Recently, geo-
graphic mapping has been increasingly used for
epidemiology studies, particularly after the development of
geographic information system (GIS) technologies that
enable data analysis by means of spatial strategies and more
comprehensive visual representation of information. GIS is
commonly used in environmental epidemiology to assess
exposure to pollutants in air (McEntte and Ogneva-
Himmelberger 2008), water (Wycisk et al. 2003; Paliwal et
al. 2007), and soil (Jarup 2004).
However, only a few reports have posited a relation-
ship between nitrogen compounds in drinking water and the
incidence of ESCC in regions with a high incidence of
ESCC. This study investigates the association between the
incidence of ESCC and the concentration of nitrogen
compounds in drinking water of Shexian, China, an area of
high ESCC incidence. Spatial distribution of the
concentration of nitrogen compounds in the area should
increase the understanding of their role in ESCC pathogen-
esis and lead to the postulation of hypotheses regarding the
mechanisms of each compound on the high incidence of
ESCC.
Materials and Methods
Fig. 1 shows the location of Shexian county in Hebei province.
The period of epidemiological survey was from January to December
2010. We surveyed 52 villages in three areas (Lutou, Gucheng, and
Hezhang) of Shexian county. Of the villages, two in Lutou were so
close to each other that it was not possible to distinguish a geographic
boundary, so both villages were considered a single village. Another
four villages share a single well and are within 500 m of each other in
the Guxing area, so these villages were also considered a single vil-
lage. Finally, we collected case data of the 48 villages, which are
distributed across three districts. Patient data were obtained from the
Shexian Cancer Registry, a population-based registry established in
1974, which is used to register local cancer patients and residents.
All esophageal cancer patients were coded according to the
International Classification of Diseases. There were 19 cancer
patients without ESCC that were excluded. As a result, there were
661 adults with ESCC and 54,055 non-cancer subjects. We then
extracted the age-adjusted annual incidence rates of ESCC in 2010.
Water samples were extracted from wells, rivers, and cisterns.
In the study area, wells serve all residents in a village, which nor-
mally owns one or two wells, although some large villages own four
or five wells. Some families obtain water directly from the
Qingzhanghe or Zhuozhanghe rivers because their houses are nearer
to these rivers that to wells. In all cases, water was sampled from the
locations where villagers obtained their daily drinking water. Some
villages were located at a high elevation (> 700 m) and were therefore
far from surface water. It is difficult and expensive to sink wells at
high elevations. Therefore, drinking water cannot be obtained from
wells or rivers, so such villages build a large cistern to store rain
water as drinking water for residents. The altitude and global
positioning system (GPS) coordinates of the water sampling loca-
tions, as well as depth for wells, were recorded for each source.
Domestic garbage and agricultural fertilizer were considered as
two possible sources of nitrogen contamination in areas of Hebei
province with high ESCC incidence (Zhang et al. 2003). We
surveyed 86 water sampling points from 60 wells, 10 cisterns, and 16
water points along the Qingzhanghe or Zhuozhanghe rivers. Water
samples were extracted twice during 2010; during the low water
period (March), and during the high water period (September).
Concentrations of nitrogen compounds were measured within 24 h
after sampling. The average concentration of nitrate nitrogen, nitrite
nitrogen, and ammonia nitrogen, compounds which constitute the
principal suspected risk factors for ESCC in the study area
(Yokokawa et al. 1999), was recorded and used for analysis (Table 1).
Statistical and Spatial analysis
R software (R 2.13.1) was used for statistical analysis (Crawley
2007). Spearman’s correlation was used to analyze the correlation
between variables. Logistic regression analysis was performed to
obtain risk factors for incidence of ESCC. The standardized
incidence ratio for ESCC was calculated for each village and mapped
to digital map. To detect the high incidence of ESCC and the high
concentration of nitrogen compounds in the study area, hot-spot
analysis was performed using Getis-Ord Gi* cluster analysis in
ArcGis spatial software (ArcGis 9.1.3, Redlands, CA). We used a
fixed distance band of 2 km for cluster analysis of incidence. All
villages with significant Z scores were identified. Negative values
indicate villages with 2-km radius clusters of lower than expected
cancer incidence rates, and high Z scores indicate villages with higher
than expected cancer incidence rates within a 2-km radius. The
digital map was generated by ArcGIS (9.3.1, Redlands, CA).
Results
Esophageal squamous cell carcinoma clusters
The standardized incidence ratio for ESCC was
calculated for each village. Results show a statistically
significant cluster of villages with a high incidence of
ESCC in the southwest region. Low-incidence ESCC
clusters occurred predominantly in the northern region. A
small low-incidence cluster occurred in the southeast region
(Fig. 2).
Nitrogen compound clusters
Villages with a high concentration of nitrate nitrogen
(NO3) were distributed in the southwest region of the study.
Conversely, large clusters of villages with low
concentrations of NO3 were distributed in the northern
region, along the Qingzhanghe and Zhuozhanghe rivers
(Fig. 3). Overall, the concentration of nitrite nitrogen (NO2)
was low in study area. Only a small cluster of villages with
a high concentration of NO2 were distributed in the north-
west region (Fig. 4). The high concentration of ammonia
nitrogen (NH4) clusters appeared in the northwest region
(Fig. 5). In contrast to the distribution of NO3, a low
 Association of Nitrogen Compounds with ESCC 13

Fig. 1. Study area (highlighted in orange) in Hebei province.

concentration of NH4 occurred in the southern region.
Correlation between incidence of esophageal squamous cell
carcinoma and nitrogen compounds
There was a weak negative correlation between EC
incidence and nitrite nitrogen concentration, ammonia
nitrogen concentration and elevation (−0.15, −0.39 and
−0.50, respectively), and a positive correlation between EC
incidence and nitrate nitrogen concentration (0.38) (Table
2).
Logistic regression analysis was employed to estimate
the risk of ESCC incidence. Table 3 presents the estimated
coefficient and odds ratio for each candidate risk factor.
The risk factor of NO3 was significantly associated with the
14 N. Zhang et al.

Table 1. Concentration of nitrogen compounds by water source.

Nitrate nitrogen Nitrite nitrogen Ammonia itrogen
Water source Sample points
(mg/L) (mg/L) (mg/L)
Well 60 8.08 ± 4.39 0.01 ± 0.05 0.12 ± 0.09
River 16 5.59 ± 2.39 0.01 ± 0.03 0.12 ± 0.07
Cistern 10 4.55 ± 2.60 0.04 ± 0.05 0.10 ± 0.07

Fig. 2. Spatial clustering of villages by incidence of esopha-

geal squamous cell carcinoma (per 10,000 population). Fig. 3. Spatial clustering of villages by nitrate concentration.

incidence of ESCC in the study area, and the odds ratio was
46.29. NO2, NH4, and elevation had no significant associa-
tion with the incidence of ESCC.
Discussion
Biological, genetic, and environmental factors are
thought to play a critical role in ESCC. Although many
compounds might be involved in the pathogenesis of ESCC,
nitrogen compounds are thought to play the major role in
this process. Recent study has shown that nitrogen
compounds are not risk factors for cancer (Ward et al.
2008). In contrast, the present study showed that ESCC
was influenced by nitrogen compounds in drinking water,
especially NO3; high ESCC incidence areas were mostly
areas with high concentrations of NO3.
The mechanism of how NO 3 affects cancer is
complicated and is likely indirect. Although NO2 can be
converted to nitrosamine in the stomach (McKnight et al.
1999), the concentration of NO2 in nature is too low to pose
a problem in this respect (Table 1). Therefore, it is hard to
consider environmental NO2 as a risk factor, since even
high-incidence ESCC areas do not have high concentrations
of NO2 (Fig. 4). Logistic regression analysis confirmed that
NO2 was not a significant risk factor. A high concentration
of NO3, on the other hand, is a primary risk factor. van
Loon et al. (1998) showed that NO3 has no harmful effect,
even when intake is high. However, when NO 3 is
assimilated in the body, it is converted to NO 2 by
denitrifying bacteria, and NO 2 can subsequently be
converted to nitrosamine in the stomach (McKnight et al.
1999). Consequently, a high concentration of NO3 will lead
to a high incidence of ESCC. Hot-spot analysis and logistic
 Association of Nitrogen Compounds with ESCC 15

Fig. 4. Spatial clustering of villages by nitrite concentration. Fig. 5. Spatial clustering of villages by ammonia concent­
ration.

Table 2. Correlation of esophageal squamous cell carcinoma incidence

with concentration of nitrogen compounds and elevation.
Factor Correlation coefficient P value
Nitrate nitrogen  0.38  0.01
Nitrite nitrogen −0.15  0.31
Ammonia nitrogen −0.39  0.01
Elevation −0.50 < 0.001

Table 3. Regression coefficient (β), standard error (SEβ), odds ratio (OR), and 95% confidence intervals (CI) for
OR by logistic regression analysis.
Factors β SEβ OR 95% CI for OR P value
Intercept  5.77 0.32 < 0.001
Nitrate nitrogen  3.84 1.37 46.29    3.16 - 667.39  0.01
Nitrite nitrogen −1.24 0.90  0.29 0.05 - 1.68  0.17
Ammonia nitrogen −1.09 0.87  0.34 0.06 - 1.84  0.21
Elevation −0.19 0.88  0.82 0.15 - 4.59  0.83

regression implied this association in our study. In Ammonia nitrogen correlated negatively with the
particular, hot-spot analysis showed a high concentration of incidence of ESCC in this study. In the middle and
NO3 in the middle and southern parts of Shexian county; southern parts of the study area, villagers use rivers and
areas with a high incidence of ESCC (Fig. 3). wells as their main water source. Both sources have fresh
16
water because it is not stored for long periods. In contrast,
northern villages use cisterns to store water for long periods
because it is difficult to obtain drinking water from rivers
and wells. In this case, stored drinking water is at higher
risk of contamination with organic matter than flowing
water, and the concentration of NH4 is higher in the
northern villages than in the middle and southern villages.
Despite these findings, the high incidence of ESCC was in
the middle and southern parts, while the northern parts had
a relatively low incidence of ESCC (Fig. 5). Therefore, in
this case, the correlation between NH4 and the incidence of
ESCC is negative. A more accurate correlation between
ESCC and NH4 should be confirmed using a high number
of water samples and comparing them with areas of high
and low incidence of ESCC.
The incidence of many cancers is associated with
elevation (Krain 1991). Wu et al. (2008) suggested that
high altitude might decrease the incidence of EC. Our
study confirmed this negative correlation between elevation
and ESCC incidence. Spearman’s correlation test showed
that the correlation coefficient of elevation and incidence
was −0.50 (P < 0.001) (Table 2). Hot-spot analysis showed
that high elevated areas have a low incidence of ESCC.
The present study has several limitations. This study
focused on drinking water in residential areas, but water
sampling was not comprehensive as, for example, water
samples were taken only from the middle and lower reaches
of rivers making it impossible to determine the source of
river pollution. Furthermore, we focused mainly on nitro-
gen compounds in drinking water and did not collect infor-
mation on land use (e.g., agricultural versus industrial).
Thus, there is no way of determining whether surface water
and groundwater were polluted by industrial or agricultural
wastewater. In addition, other known ESCC risk factors,
such as biological and genetic factors, were not investigated
in the present study. Thus, further studies are needed to
address potential risk factors, preferably by using individual
level data on health.
In conclusion, we have established an association
between nitrogen compounds in drinking water and the
incidence of ESCC; consequently, high concentrations of
nitrate nitrogen in drinking water may increase the risk of
ESCC. Furthermore, residents living in the southwest part
of Shexian county were found to carry a particularly high
risk of ESCC.
Acknowledgments
We appreciate the participation of all subjects and persons
in this study. We would like to express our gratitude to David
Kipler for proofreading on our manuscript.
Conflict of Interest
All authors declare no conflict of interest.
N. Zhang et al.
References
Crawley, M.J. (2007) The R Book, John Wiley & Sons Ltd: British
Press.
Gao, Y., Hu, N., Han, X., Giffen, C., Ding, T., Goldstein, A. &
Taylor, P. (2009) Family history of cancer and risk for
esophageal and gastric cancer in Shanxi, China. BMC Cancer,
9, 269.
Han, J.Y., Xu, Z.X., Xing, H.P., Tan, J.J., Wang, M.S., Zhang, S.H.,
Kong, F.J., Si, J.H., Li, X.M., Tang, T.N. & Tian, P.C. (2007)
A historical cohort study on the effect of drinking water
improvement to the mortality of esophageal carcinoma among
Linzhou residents. Zhonghua Liu Xing Bing Xue Za Zhi, 28,
515-516.
Jarup, L. (2004) Health and environment information systems for
exposure and disease mapping, and risk assessment. Environ.
Health Perspect., 112, 995-997.
Kmet, J. & Mahboubi, E. (1972) Esophageal cancer in the Caspian
littoral of Iran: initial studies. Science, 175, 846-853.
Krain, L.S. (1991) Aviation, high altitude, cumulative radiation
exposure and their associations with cancer. Med. Hypotheses,
34, 33-40.
McEntee, J.C. & Ogneva-Himmelberger, Y. (2008) Diesel
particulate matter, lung cancer, and asthma incidences along
major traffic corridors in MA, USA: A GIS analysis. Health
Place, 4, 817-828.
McKnight, G.M., Duncan, C.W., Leifert, C. & Golden, M.H.
(1999) Dietary nitrate in man: friend or foe? Br. J. Nutr., 81,
349-358.
Paliwal, R., Sharma, P. & Kansal, A. (2007) Water quality
modeling of the river Yamuna (India) using QUAL2E-
UNCAS. J. Environ. Manage., 83, 131-144.
Qiao, Y.L., Hou, J., Yang, L., He, Y.T., Liu, Y.Y., Li, L.D., Li, S.S.,
Lian, S.Y. & Dong, Z.W. (2001) The trends and preventive
strategies of esophageal cancer in high-risk areas of Taihang
Mountains, China. Zhongguo Yi Xue Ke Xue Yuan Xue Bao,
23, 10-14.
Song, P.I., Liang, H., Fan, J.H., Wei, W.Q., Wang, G.Q. & Qiao,
Y.L. (2011) Long-term survival after esophagectomy for early
esophageal squamous cell carcinoma in Linxian, China. J.
Surg. Oncol., 104, 176-180.
Stoner, G.D. & Gupta, A. (2001) Etiology and chemoprevention
of esophageal squamous cell carcinoma. Carcinogenesis, 22,
1737-1746.
van Loon, A.J., Botterweck, A.A., Goldbohm, R.A., Brants, H.A.,
van Klaveren, J.D. & van den Brandt, P.A. (1998) Intake of
nitrate and nitrite and the risk of gastric cancer: a prospective
cohort study. Br. J. Cancer, 78, 129-135.
Ward, M.H., Heineman, E.F., Markin, R.S. & Weisenburger, D.D.
(2008) Adenocarcinoma of the stomach and esophagus and
drinking water and dietary sources of nitrate and nitrite. Int. J.
Occup. Environ. Health, 14, 193-197.
Wu, K. & Li, K. (2007) Association between esophageal cancer
and drought in China by using Geographic Information
System. Environ. Int., 33, 603-608.
Wu, K.S., Huo, X. & Zhu, G.H. (2008) Relationships between
esophageal cancer and spatial environment factors by using
Geographic Information System. Sci. Total. Environ., 393,
219-225.
Wu, M., Zhao, J.K., Hu, X.S., Wang, P.H., Qin, Y., Lu, Y.C., Yang,
J., Liu, A.M., Wu, D.L., Zhang, Z.F., Frans, K.J. & van’t Veer,
P. (2006) Association of smoking, alcohol drinking and
dietary factors with esophageal cancer in high- and low-risk
areas of Jiangsu Province, China. World J. Gastroenterol., 12,
1686-1693.
Wycisk, P., Weiss, H., Kaschl, A., Heidrich, S. & Sommerwerk, K.
(2003) Groundwater pollution and remediation options for
multi-source contaminated aquifers (Bitterfeld/Wolfen,
 Association of Nitrogen Compounds with ESCC
Germany). Toxicol. Lett., 140-141, 343-351.
Yang, C.S. (1980) Research on esophageal cancer in China: a
review. Cancer Res., 40, 2633-2644.
Yokokawa, Y., Ohta, S., Hou, J., Zhang, X.L., Li, S.S., Ping, Y.M.
& Nakajima, T. (1999) Ecological study on the risks of
esophageal cancer in Ci-Xian, China: the importance of
nutritional status and the use of well water. Int. J. Cancer, 83,
620-624.
Zhang, N., Wen, D., Shan, B., Wang, S., Zhang, L., Wei, L., Zou,
17
W., Kitsu, K. & Akazawa, K. (2011) Clustering and
geographic variation of upper gastrointestinal cancers in a
high-risk region of esophageal cancer in northern China.
Asian Pac. J. Cancer Prev., 12, 193-198.
Zhang, X.L., Bing, Z., Xing, Z., Chen, Z.F., Zhang, J.Z., Liang,
S.Y., Men, F.S., Zheng, S.L., Li, X.P. & Bai, X.L. (2003)
Research and control of well water pollution in high esopha-
geal cancer areas. World J. Gastroenterol., 9, 1187-1190.