Detection of methylating activity due to nitrosamide in some nitrosated nigerian foodstuffs

Fd Chem. Toxic. Vol. 34, No. 2. pp. 14%151, 1996

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Research Section

Detection of Methylating Activity due to Nitrosamide in some Nitrosated Nigerian Foodstuffs* S. E. ATAWODI~':I:, P. M E N D E , R. P R E U S S M A N N a n d B. S P I E G E L H A L D E R Division of Toxicology and Cancer Risk Factors, German Cancer Research Centre, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany (Accepted 27 June 1995)

Abstract--Popular vegetables, condiments and some Nigerian staple foods were evaluated for their relative methylating potential due to nitrosamide formation following nitrosation under standardized conditions. Methylating activity of nitrosated foodstuffs, expressed as N-nitroso-N-methylurea equivalents, was determined by gas chromatography-thermal energy analysis. In positive samples (detection limit I0 pg/kg) methylating activity detected was in the range of 50-1200/~g/kg, the highest activity being found in Telfairia occidentalis (ugwu). This value constitutes the highest amount ever detected for a fresh vegetable. The data suggest that some commonly consumed local foodstuffs might contribute to overall human burden of environmental carcinogens in Nigeria.

INTRODUCTION N-Nitroso compounds are toxic and carcinogenic in many animal species, including primates (Magee and Barnes, 1967; Bartsch and Montesano, 1984; Bogovski and Bogovski, 1981; Preussmann and Stewart, 1984) with a high degree of organ specificity (Preussmann and Wiessler, 1977). The possible toxicity of nitrosamines in human subjects has been reported (Fussganger and Ditschuneit, 1980); these reports suggest strongly that N-nitroso compounds may be carcinogens in humans. Broadly, N-nitroso compounds may be divided into two classes--nitrosamines and nitrosamides (Preussmann and Eisenbrand, 1984). Nitrosamide as a class has relevance in human cancer (Preussmann and Stewart, 1984). The group includes nitrosoguanidine, nitrosourethane and nitrosourea. As a group, nitrosamides require no metabolic activation *Dedicated to Miss Winnie Ojochogwu Atawodi on the occasion of her fifth birthday. tAuthor for correspondence at: Unit of Environmental Carcinogenesis, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon Cedex 08, France. ,Permanent address: Biochemistry and Applied Molecular Biology Dept., National Veterinary Research Institute, Vom, Nigeria. Abbreviations: GC = gas chromatography; MNU = Nnitroso-N-methylurea; NTBAPA = N-nitroso-N-tertbutylaminopropionic acid; NTBG = N-nitroso-N-tertbutylglycine; TEA = thermal energy analysis.

before their tumorigenic and carcinogenic activities are elicited (Preussmann and Stewart, 1984). Their ability to alkylate different proteins has been documented (Ohgaki et al., 1991), while their possible importance in the aetiology of human gastric cancer has been a subject of many epidemiological studies and reviews (Mirvish, 1983; Zhang et al., 1991). Previous investigators have established that Asian and European foods may produce methylating activity due to nitrosamide when treated with nitrite under acidic conditions (Kumar et al., 1990; Mende et al., 1989). For Nigeria and other countries in Africa, such data are lacking. In earlier investigations, we demonstrated that Nigerian foodstuffs may contain preformed volatile nitrosamines (Atawodi et al., 1993) and N-nitroso precursors (Atawodi et al., 1992) that are indeed capable of undergoing nitrosation under gastric conditions to produce carcinogenic nitrosamines (Atawodi et al., 1991). Because of the incidence of many cancers (Ihekwaba and Solanke, 1984; Obafunwa, 1990) of poorly defined aetiology in Nigeria, it was considered important to extend these studies by assessing the relative potential of popular Nigerian foodstuffs to produce methylating activity due to nitrosamide when subjected to nitrosating conditions.

MATERIALSANDMETHODS Chemicals and standards

N-Nitroso-N-methylurea (MNU) was synthesized from the corresponding alkylurea by nitrosation and 147

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S.E. Atawodi et al.

recrystallization from methanol. N-Nitroso-N-tertbutylglycine (NTBG) was prepared according to the protocol previously described by Mende et al. (1989), while N-nitroso-N-tert-butylaminopropionic acid (NTBAPA) was a gift from G. Eisenbrand (University of Kaiserslautern, Germany). Other reagents and solvents were obtained from commercial sources in analytical grade.

Samples and sample preparation Spices, condiments and other foodstuffs were purchased from retail outlets in Bukuru or Jos, Plateau State, Nigeria. Fresh vegetables were purchased from Lagos and Ibadan, Nigeria. Foodstuffs were selected to reflect those items that are consumed, or are components of diets that are taken, at least once a day, by virtually all Nigerians. Garri (a fermented cassava product) and maize are staple foods in Nigeria, while vegetables such as Telfairia occidentalis, Amaranthus hybridus and Corchorus olitorius are very popular throughout Nigeria. Onions and pepper are ingredients of virtually all cooked foods in Nigeria, and Digitaria exilis is a staple food in Plateau State, Nigeria. In general, samples were prepared as previously described (Atawodi et al., 1991), but most vegetables were analysed fresh. Fresh samples were ground to a smooth paste with dry ice in a laboratory mortar. These were analysed immediately or stored at -20°C. The smooth paste was used for analysis without further extraction. Two independent samples were analysed in duplicates.

solution). Each experiment was carried out with two controls. In one control, nitrite was omitted and the sample was spiked with MNU (5/~g) after complete incubation. This served as an external standard for the quantification of MNU equivalents. An additional control was nitrosated, but without addition of any scavenger reagent. This was used to evaluate the nitrosamide background of nitrosated samples. The methylated derivative of NTBG with a retention time of 4.8 min could interfer with other peaks in this region. Therefore, another scavenger reagent (NTBAPA), which forms a methylated derivative with a much higher retention time (8.7 min), was also used. Sample aliquots (1 ml each) of nitrosated extract, dissolved in dichloromethane, were applied to Extrelut columns and eluted with dichloromethaneether (9: i; v/v) to yield a final volume of 4 ml. Each determination was performed in duplicate. The eluates were concentrated to 1 ml under a stream of nitrogen and 10-pl aliquots of the eluates were then analysed by gas chromatography-thermal energy analysis (GC/TEA) (Mende et al., 1991). The Foodmaterial nitrosation,extractionand elutionwithCH2Cl2 CH3. O~ R

Nitrosamide

Nitrosation experiments A sample of smooth vegetable paste (5 g) or dried foodstuffs (2 g) was mixed with 20 ml sodium nitrite solution (final concentration 100 mM) and the pH was adjusted to 1.5 with concentrated HCI. The mixture was incubated at 37°C for 1 hr. Nitrosation was stopped by addition of 1 g solid ammonium sulfamate. To stabilize the nitrosourea compounds formed, the pH was adjusted to 3-4 with solid sodium hydrogen carbonate under vigorous stirring. Sticky samples were centrifuged (3000 rpm, 10 min) before extraction, and the pellets were resuspended in 10 ml water and centrifuged again. After washing the aqueous solution twice with 10ml n-hexane to remove fat, the hexane phase was re-extracted with 10mi water. The combined aqueous phases were applied to an Extrelut column (Merck AG, Darmstadt, Germany) and eluted with dichloromethane to yield a final volume of 50 ml (Fig. 1). The eluate was evaporated to dryness in vacuo and dissolved in 4 ml dichloromethane (Mende et al., 1989 and 1991).

Analysis of nitrosamides by their alkylating activity Nitrosamides formed in nitrosated foodstuffs were then determined after releasing diazoalkanes by treatment with 5 ml 1 M NaOH and trapping the aikylating agents with a scavenger reagent (200/~1 of a 15 mg/ml

N- - N O

I Releaseofdiazoalkaneby treatmentwith NaOH CH2N2

+ CH3 - - ~ - - N - - - ' C H 2 ----COOH

[CH3~qO

NTBG

or

c.3

CH3NO

NTBAPA

(Trapping the alkylatingagent with a scavengerreagent)

1 CHINO

Methylated N'rBAPA(ormethylatedNTBG)

Clean-up on silicagel by CH2CI2:Et20 elution

GC - TEA analysis

Fig. I. Analytical scheme for estimation of alkylating activity in foodstuffs.

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M e t h y l a t i n g a c t i v i t y in f o o d s t u f f s Table 1. M N U equivalents in some nitrosated Nigerian foodstuffs Foodstuff

Botanical name

TelJ~iria occidentalis Corcorus olitorius Vigna sp. savi Capsicum annuum Hib;scus esculentus Allhtm cepa Glycine max Ses~rnum indicum Zea mays Manihot sp. Irvingia gabonensis Digitaria exilis Amaranthus hybridus Capsicum frutescens Lycopersicon esculentum Cuc~rneropsis manii Capsicum sp.

M N U equivalent (/~g/kg) determined with:

C o m m o n Nigerian/ English name

Nature of sample

Ugwu Ewedu Green beans Red pepper Okra Onions Soya beans Beniseed Maize Garri Bush mango Acha Spinach Tatache Tomatoes Melon/egusi Pepper

Fresh Fresh Fresh Fresh Fresh Fresh Dried Dried Dried Dried Dried Dried Dried Dried Dried Dried Dried

NTBG 1100 + 80 + 30 + -nd 50 _ nd * * nd nd * -nd nd * *

55 7 5

3

NTBAPA 1200 __+50 90 _+ 7 50 + 7 240 + 20 nd 50 + 5 50 _+ 8 * * nd nd * 100 + 20 nd nd * *

- - = nat determined nd = not detectable ( < 10/~g/kg) Nitrosation was carried out with 100 mM nitrite at pH 1.5, incubation 1 hr at 37°C. Values are means _+ standard deviation (2 x 2 analyses). *Not determined due to peak overlapping; qualitatively considered positive for methylating activity.

gas chromatograph was fitted with a 1.2 m x 2 mm i.d. glass column packed with 10% OV-17 on Chromosorb WHP. Helium was used as carrier gas (18 ml/min) and the injection port temperature was maintained at 200°C. The GC oven was programmed from 120 to 190°C at 5°C/min. Under these conditions, retention times of methyl derivatives of NTBG and NTBAPA were 4.8 and 8.7 min, respectively. The recoveries for M N U ranged from 62 to 95%. RESULTS

Of the 17 foodstuffs analysed, six (35.3%) had no detectable methylating activity. Mean M N U equivalents in the range of 50 to 1200/~g/kg could be quantified in six samples (35.3 %), while, for five other samples (29.4%), I~INU equivalents could not be quantified owing to overlapping of peaks (Table 1). The use of two scavenger reagents, NTBG and NTBAPA, as quality control guarantee against such chromatographic int,:rference was not sufficient for these five samples. Qualitatively, the five samples were considered po~;itive for methylating activity. Generally, a larger ~umber and greater amounts of M N U equivalents were detected using NTBAPA as scavenger reagent. Hence, this set of data was selected for use in our discussion. DISCUSSION

Methylating activity due to nitrosamide (expressed as M N U equivalent,,;) of 110-4840 #g/kg has been detected in nitrosated meat and fresh and dried fish, while M N U equivalents of up to 170 # g/kg have been detected in various plant products (Mende et al., 1991). These investigators showed that, when subjected to chemical nitrosation, fresh meat and fish of German origin may produce M N U equivalents of 100-440 #g/kg, while boiled broccoli and cauliflower

from the same country produced M N U equivalents of 4 and 20/~g/kg, respectively. Relevant data for other European foods are lacking. On a dry weight basis, M N U equivalents of 1200/~g/kg have been reported in dried Brassica oleracea plant from areas of Kashmir, India with a high incidence of oesophageal and gastric cancer (Kumar et al., 1990). The detection of 1200/~g/kg in fresh Telfeiria occidentalis (one of the most popular Nigerian vegetables) as far as we are aware constitutes the highest M N U equivalent ever reported in literature for any plant-based foodstuff: it is about three times the methylating activity found in the caffeinated natural stimulants, kola nuts (Atawodi et al., 1995). It is noteworthy that other widely consumed Nigerian vegetables, namely, Amaranthus hybridus and Corchorus olitorius, also produced as much as 100 and 90/~g M N U equivalents/kg, respectively. T. occidentalis, A. hybridus and C. olitorius are leafy vegetables which, when fresh or dried, may be cooked and eaten alone or as vegetable soup in combination with foods prepared from cereals and tuber crops such as cassava (Manihot spp.) and yam (Dioscorea spp.). Consumption of these vegetables has increased in recent years because of the general belief that they are rich in vitamins and mineral elements. Although Capsicum annuum produced a relatively high level of M N U equivalents, the overall contribution of this spice to endogenous exposure to methylating agent may be relatively insignificant, since only a few grams are used for each recipe. In western Nigeria, however, consumption of all species of Capsicum can be relatively high. Previous investigators have suggested that precursors for direct-alkylating N-nitroso compounds such as nitrosamides might include primary amines and amides (Boido et al., 1980; Mende et al., 1989; Shephard and Lutz, 1989), creatinine (Mirvish et al.,

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1982), pyroilidon-(2)-one (Mende et al., 1994) or, as in the case of B. oleracea, caffeidine and caffeidinic acid (Kumar et al., 1992). At present, the exact nature of the nitrosamide precursor in Nigerian vegetables is unknown. However, it is remarkable that T. occidentalis, which in this investigation produced the highest methylating activity, was previously shown to contain a much higher level of primary amines, particularly methylamine, than any of the other vegetables or spices examined (Atawodi et al., 1992). Nitrosamides are mutagens that, on spontaneous decomposition, are capable of binding directly to cellular macromolecules without prior metabolic activation (Preussmann and Eisenbrand, 1984; Preussm a n n and Stewart, 1984). Ohgaki et al. (1991) have reported the methylation of D N A in the digestive tract of rats chronically exposed to N-nitroso-Nmethylurea, while other workers (Mirvish, 1983) have implicated nitrosamides as a very important aetiological factor in gastric carcinogenesis. A positive correlation has, indeed, been demonstrated between nitrosamide exposure and gastric cancer in China (Zhang et al., 1991). The detection of high methylating activity in some nitrosated Nigerian foodstuffs must therefore be considered in relation to their potential contribution to the aetiology of certain human cancers in Nigeria. Owing to the absence of relevant epidemiological data in Nigeria, no association can be made between food-related endogenous exposure to nitrosamides and gastric or other forms of cancer at this time. Moreover, compared with physiological nitrite exposure, the present investigation used a relatively high nitrite concentration in order to obtain information on the relative potential of these foodstuffs to form nitrosamides. The nitrite concentration in the normal fasting stomach is much less, although prevailing tropical diseases of bacterial and parasitic origin, such as bilharzia and schistosomiasis, are known to increase the endogenous nitrite burden (Leach et al., 1987; Tricker et al., 1989). In addition, the high nitrate concentration of these vegetables (Atawodi et al., 1992) and in the water supply of this population (Gbodi and Atawodi, 1987) could lead to higher endogenous exposure to nitrite and, consequently, could increase the potential for endogenous formation of direct-alkylating nitrosamides. Despite these limitations, the results reported here suggest that some commonly consumed Nigerian vegetables may contribute to the endogenous burden of direct-alkylating nitrosamides; their possible aetiological role in human carcinogenesis in this population should, therefore, be further explored. More studies are also required to elucidate the exact chemical nature of the nitrosamide precursor(s) in these vegetables.

Acknowledgements--S.E.A. would like to thank the Ger-

man Academic Exchange Service (DAAD) for financial

support during his stay at the Division of Toxicology and Cancer Risk Factors, DKFZ, Heidelberg as a guest scientist.

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