COMMENTARY N -Nitrosodimethylamine in the Kashmiri Diet and Possible Roles in the High Incidence of Gastrointestinal Cancers

The Kashmiri population is culturally distinct with special dietary features owing to the temperate climatic conditions of Kashmir valley. This has habituated the population to preserve food in smoked, pickled and sundried forms which include considerable amounts of N -nitroso compounds (NOCs). These are known to cause cytotoxicity, DNA damage, mutation, unscheduled DNA synthesis and DNA methylation. All of these changes at molecular level are known to contribute to the pathogenesis of cancer. One of the prominent NOCs found in Kashmiri food is N -Nitrosodimethylamine (NDMA). Here we review the occurrence of NDMA in sundried foods, dried fish, kehwa, traditional pickle, Brassica oleracia and tobbaco . We also discuss its possible role in the high prevalence of gastrointestinal cancers in Kashmir.


Introduction
Nitroso compounds (NOCs) have been classified as potent human carcinogens by the International Agency for Research on Cancer (IARC 2010). Human exposure to NOCs occurs almost entirely through food and endogenous nitrosation (A process of converting organic compounds into nitroso derivatives) of its precursors in the gastrointestinal tract (Tricker 1997) following absorption into the bloodstream. NOCs may also have deleterious effects in tissues of direct and indirect contact. Epidemiological studies have indeed associated human (endogenous) NOC exposure to several types of cancers including esophagus, stomach, colorectal and bladder cancer (Mirvish et al., 1995;Bingham et al., 1996;Magee et al., 1989). The Kashmiri food contains significant amount of NOCs and NDMA is one of the most prominent Nitroso compounds in this food .
NDMA the simplest and most widely occurring nitrosamine, is reported to be acute hepatotoxic and potentially carcinogenic in various animal species including humans (Lai et al., 1980;Robbiano et al., 1996;IARC 1998). Studies have described the ability of NDMA to induce DNA damage in the primary culture of human hepatocytes (Martelli et al., 1985). It is commonly inferred that nitrosamines are related to cancer induction through the formation of mutagenic DNA methyl adducts in important genes involved in carcinogenesis (Goto et al., 1999). There is mounting evidence that DNA alkylation, especially the formation of O 6 -alkylguanine (O 6 -alkG) is involved in mutagenesis and cancer (Doniger et al., 1985;Singer et al., 1991). O 6 -Methylguanine (O 6 -meG), for instance, has been detected in the DNA of esophageal tissues collected from people living in an area of China where the risk of esophageal cancer is considerably high (Huh et al., 1985).
The aim of this review was to look in to the possible etiology of high incidences of gastrointestinal cancer in Kashmir considering their dietary habits.

Gastrointestinal Cancers in Kashmir
Various studies have been done on incidence rate and distribution of gastrointestinal cancers in Kashmir (Azra & Jan, 1990;Khuroo et al., 1992;Dhar et al., 1993;Makhdoomi et al., 2005;Qurieshi et al., 2011). These studies have shown that cancers of oesophagus and stomach are common in Kashmir and together account for more than 60% of all the cancers prevalent in the valley. In a study conducted by Dhar and his team (1993), it was observed that oesophago-gastric malignancies constitute around 57.5% of total malignancies. In other study (Makhdoomi et al., 2005 ) based on the biopsy samples of patients collected at Department of Pathology, Govt. Medical College, Srinagar -Kashmir, over a period of 15 years (1984)(1985)(1986)(1987)(1988)(1989)(1990)(1991)(1992)(1993)(1994)(1995)(1996)(1997)(1998), showed that out of a total of 5892 patients with various malignancies 43.43% were afflicted by gastrointestinal malignancies and of these 76% had oesophago-gastric malignancies. This report shows an alarmingly high percentage of gastric and esophageal cancers in the population under study.

Kashmiri Diet and Nitroso Compounds
Kashmiri population is ethnically very distinct due to their special dietary intakes. Owing to the temperate climatic conditions of Kashmir valley, the population is habituated to preserve foods in smoked, pickled and sundried form. In a study  it was found that these food products contain considerable amounts of NOCs. Some of the prominent NOCs found in the local food are N-Nitrosodimethylamine (NDMA), Methyl Nitrosourea (NMU), N-Nitrosopyrrolidine (NPYR), N-Nitrosopiperidine (NPip) and N-Nitrosoproline (NPro). Further the authors showed that under stimulated gastric conditions there is formation of high concentration of NOCs in the gastric fluids and NDMA being the major one of all, is formed from intake of dried fish (20 μg/kg), dried and pickled vegetables (35.6 μg/kg and 7.3 μg/kg), locally grown Brassica oleracea ('Hak') leaves (69.9 μg/kg), and the traditional tea 'Kehwa' (9.2 μg/kg). Brassica, a green vegetable from Kashmir, which is a major constituent of the local diet, when cooked contains around 11 μg/kg of NDMA (Kumar et al., 1990). According to the ATSDR (1989) report, LD 50 of NDMA in rats is 37mg/kg body weight. Repeated intake of food containing high amounts of NDMA could lead to adverse effects of this toxic compound and its metabolites in various organs leading to favorable milieu for cancer especially malignancies of alimentary canal. Table 1. shows the common Kashmiri food products and different types of NOCs present in these diets.
Several researchers (Khuroo et al., 1992;Dhar et al., 1993;Murtaza et al., 2006;Qurieshi et al., 2011) have concluded a probable role of dietary factors in high incidence of gastrointestinal cancers in Kashmir. Khuroo with his colleagues (1992) were the first to link special dietary habits of Kashmiris with high incidence of esophageal cancer. Dhar et al. (1993) concluded that preponderance of esophageal cancer was attributable to the local practice of drinking boiling hot salted tea. Murtaza et al. (2006) reviewed the dietary habits of three groups consisting of 51 individuals each, in Kashmiri population. In this study Group I represented patients of esophageal cancer; Group II represented control with familial history of gastric cancer and Group III were control with no history of gastric cancer. A significant agreement of dietary habit and varying degree of incidence of esophageal cancer post consumption of locally grown Brassica vegetable (Hakh), red chillies, hot salty soda tea, and local baked bread was observed. A very recent survey by Qurieshi and co-workers (2011) on 81 patients of gastric carcinoma in Government Medical College, Srinagar -Kashmir, reported that 87.35% of these patients consumed sun dried vegetables; 48% had sun dried fish; 66.7% consumed pickles and 35.8% of them had smoked fish in their regular diets. All of these consumables are known to contain variable amounts of NDMA .

Possible Role of NDMA in Etiology of Cancers
N-Nitrosodimethylamine is a symmetric N-nitrosamine and hydroxylation of either of the two carbons will yield methylating agent -Alpha hydroxyl NDMA. For this it undergoes enzymatic oxidation through Cytochrome -P 450 to convert into a precursor (α-hydroxyl NDMA) which finally metabolizes into an ultimate carcinogen -methyl diazonium ion (CH 3 N 2 + ) (Magee et al., 1962;Wang et al., 2002;Agnew et al., 2004). In vivo studies on rat liver showed alterations of DNA methylation patterns by NDMA (Singer & Grunberger, 1983) and it was found that 6.6% of O 6 position in guanine is methylated. Methylation at the guanine O 6 (O 6 -methyl-guanine -O 6 meG) position confers a high mutagenic and carcinogenic susceptibility. DNA polymerases insert thymine opposite O 6 -methylguanine with a similar frequency of normal cytosine opposite guanine (Warren et al., 2006). Flow chart (Figure   DOI:http://dx.doi.org/10.7314/APJCP.2012.13.3.1077 N-Nitrosodimethylamine in the Kashmiri Diet -Possible Roles in a High Incidence of Gastrointestinal Cancers 1) shows a diagrammatic representation of molecular events taking place during NDMA induced mutagenesis and thus leading to onset or progression of cancer.

Conclusion
To validate the role NDMA in the etiology of gastrointestinal cancers in Kashmir valley a high throughput Genome wide association studies (GWAS) would give a better understanding to the molecular mechanisms contributing to gastric cancers. Looking at GC -AT transitions per se in promoter regions and exon of important genes contributing to gastrointestinal cancers would help us explore novel target genes for early diagnosis and therapeutics. Families with history of gastrointestinal cancers should totally evade the use of food rich in NOCs. The most important factor working towards the control of gastrointestinal cancer in Kashmiri population is to bring awareness of dietary intakes and reduce the intake of NOC containing food products.