139

Cancer Letters, 65 (1992) 139- 143 Elsevier Scientific Publishers Ireland Ltd.

Effect of dietary nitrate on endogenous humans

nitrosation

of piperazine

in

Rajiv Kumara Maqsood Siddiqib, Zia Fazilib, Claus-D. Wackerc, Bertold Spiegelhalder” and Rudolf Preussmannc ‘Departments %stitute

of Pharmaceutical

for Toxicology

Sciences and bBiochemisty, Uniuersity of Kashmir, Srinagar- 190006, J & K (India); German Cancer Research Centre, Heidelberg, (Germany)

and Chemotherapy,

(Received 1 January 1992) (Accepted 12 May 1992)

Summary

IntrodrctIon

The effect of dietary nitrate on endogenous nitrosation of a therapeutic dose of piperazine has been described in fioe human uolunteers who acted as their own controls. The urinary excretion of endogenously formed N-nitrosomonopiperazine (MNPZ) ranged between 9.2 and 80.1 pg/24 h on a normal uncontrolled diet which increased from 25.7 to 163.7 pg/24 h when the diet was supplemented with 250 mg nitrate. The corresponding urinary nitrate was 63.0- 122.7 mg/24 h and 119.2 - 322.0 mg/24 h, respectiuely . The dinitioso deriuatiue of piperadne was detected only in trace amounts and no detectable increase in ifs excretion was obserued during high nitrate exposure. The unchanged piperazine (range 294 - 784 mg/24 h) in urine showed a decrease under high nitrate regimen (range 185 - 399 mg/24 h).

Piperazine (1,4-diazocyclohexane) is a widely used drug for the treatment of ascariasis in many countries of Asia and Africa. It is known to form both IV-mononitrosopiperazine (MNPz) and NJ’-dinitrosopiperazine (DNPz) on in vitro nitrosation [ 141. In animal experiments, while MNPz has been shown to be noncarcinogenic [8], DNPz induces tumours of the esophagus, nasal cavity and liver in rats [4,1 l] and liver and lungs in mice [6]. The intragastric formation and excretion of MNPz in urine has been shown in piperazine dosed human subjects whereas its dinitroso derivative was not detected [l]. In a recent study we have reported the urinary excretion of MNPz in patients who were on therapeutic doses of piperazine citrate for antihelmintic treatment in Kashmir, India [14]. We also found 0.7 - 2.8 pg/day excretion of DNPz in the urine of some patients. Since it was the first reported detection of DNPz in human urine of piperazine dosed individuals, it was argued whether this could be a consequence of high nitrate diet generally taken by the natives of Kashmir [12]. Furthermore, there was a large inter-individual variation in the levels of MNPz which could also be due to varying dietary nitrate. It was therefore considered necessary to study the effects of dietary nitrate on the endogenous

Keywords: piperazine; endogenous formation of N-nitrosomonopiperazine and N,N’dinitrosopiperazine; dietary nitrate Correspondence to: Claus-D. Wacker, Institute for Toxicology and Chemotherapy, German Cancer Research Centre, Heidelberg, Germany.

0 1992 Elsevier Scientific Publishers Ireland Ltd. 0304-3835/92/$05.00 Printed and Published in Ireland

140

nitrosation of piperazine to further understand the mechanism of nitrosation of therapeutic doses of the drug in humans. In the present study we report the effect of high dietary nitrate on the excretion of nitrosated piperazine derivatives (MNPz and DNPz) in five human volunteers receiving therapeutic doses of piperazine. Materials

and Methods

Chemicals

Standard MNPz, DNPz, N-nitroso-N’butylpiperazine and N-butylpiperazine (NBuPz) were available from our standard reference compound library. All other reagents (purchased from Merck, Darmstadt or Sigma, FRG) were of analytical grade and used without further purification. Design of experiment

The experiment was conducted on five healthy human volunteers (two males and three females, age 22-31 years) who were administered a therapeutic dose of piperazine phosphate (1.9 g piperazine) 2 h after evening meals and 24 h urine was collected. A week later the same five volunteers were given a similar dose of piperazine, after a normal meal supplemented with extract of Hak, (a locally grown Brassica vegetable) containing 250 mg of nitrate and urine was collected. The volunteers participating in the experiment were not put on any dietary restriction during the experiment. Collection of urine samples

The 24-h urine samples were collected in 2-I polyethylene bottles containing 10 g NaOH for preservation and to prevent artefactual nitrosation of piperazine. Urine samples were also spiked with 50 mg morpholine to monitor post-sampling artefact formation. The total volume of urine was recorded and a 100-ml aliquot from each sample was frozen at -2OOC. All samples were analysed within 4 weeks of collection.

Analysis of nitrate and nitrite in urine

Nitrate and nitrite concentrations in urine samples were determined using a dual channel continuous flow analyser as described earlier u41. Analysis urine

of

MNPz,DNPz and

piperazine in

MNPz and DNPz were analysed as described earlier [14]. Briefly, alkaline sample aliquots (15 ml) were spiked with NBuPz (200 ng, internal standard) and applied to a column containing Extrelut 20 (Merck, Darmstadt, FRG). After equilibration for 20 min, the column was eluted with dichloromethane (40 ml) into a Kuderna-Danish evaporator for concentration to 5 ml at 80°C and finally under a stream of nitrogen for analysis by GC-TEA using an 0V17 column. Piperazine was analysed according to the method of Skarping et al. [13], by two phase extraction and derivatisation with ethylchloroformate to form piperazine carbamate esters which were measured on GC-TEA as described earlier [14]. Determination of MNPz piperazine formulation

and

The commercially available granule formulation used in the dissolved in water and the volume 500 ml. Appropriate aliquots were analysis of piperazine, MNPz and

DNPz

in

piperazine study was made up to used for the DNPz.

ResUlti

The urine samples were stabilized (pH > 10) and no sample showed artefactual nitrosation as indicated by the absence of NMOR. The data for urinary excretion of piperazine, MNPz, DNPz and nitate is presented in Table I. These results have been corrected to recovery percentages (piperazine and butylpiperazine, >95%; MNPz, 84%, S.D. = 4%, n = 5; NBuPz, 8646, S.D. = 3%, n = 5; and DNPz, 8946, S.D. = 3%, n = 5) obtained from analysis of sterile urine samples spiked with standard reference compounds.

141

Tabh

1.

Effect of dietary nitrate on the urinary excretion

(24 h) of nitrate, piperazine

and N-nitrosopiperazines

after a dose of

1.9 g plperaztne. Volunteer

Urine vol.

Nitrate

Piperaztne

MNPz

DNPz

fmf)

(mg)

fmg)

kg)

(Irg)

a

b

a

b

a

b

a

b

a

1

1500

1700

122.7

137.0

784

352

67.5

163.7

2

1400

1500

64.4

119.2

443

399

12.2

70.7

b

tr

tr

n.d

tr tr

3

2000

2000

104.2

196.6

479

258

15.5

25.7

n.d

4

2000

1900

73.5

208.5

294

185

9.2

39.2

n.d

n.d

5

1700

2000

63.0

322.0

521

347

80.1

121.0

tr

0.3

a: Diet without supplemented

nitrate. b: Diet supplemented

with 250 mg nitrate

tr: trace (less than 0.1 @g/l). n.d.: not detected (less than 0.05

pgg/l).

The mean urinary nitrate of volunteers on normal diet was 85.5 f 23.7 mg/24 h which increased to 196.6 A 71.2 mg/24 h when the diet was supplemented with 250 mg of nitrate. The excretion of unchanged piperazine was found to decrease from 504.0 * 159.0 mg to 308.0 f 76.0 mg/24 h on high nitrate diet. The mean urinary excretion of MNPz was 36.9 * 30.4 pg in 24 h on a normal diet which increased to 84.0 A 51.6 pg when the diet was supplemented with additional nitrate. DNPz was only detected in trace amounts in some samples and did not show any proportional increase on nitrate supplementation. The commercial piperazine formulation used in the study was found to contain 4.2 pg MNPz/g piperazine and no detectable DNPz. Diacadon Piperazine, a cyclic secondary amine, pK, 5.57, 9.8 [9], undergoes in vitro nitrosation over a range of physiological pH indicating the possibility of formation of MNPz as well as DNPz [14]. Earlier studies in this direction have shown the urinary excretion of MNPz following administration of sub-therapeutic doses of piperazine [l], after low level exposure to piperazine in air [2] and occupational exposure through air-borne particulates

of piperazine [3]. In our previous study we had shown varying levels of MNPz excretion in urine from piperazine dosed individuals and had also detected low quantities of DNPz, although these results could not be validated due to absence of its major metabolites in urine t71. The present study where five volunteers acted as their own controls (see Methods), shows not only formation of MNPz under normal dietary conditions but the extent of nitrosation was found to increase 2 -6-fold when piperazine was given under a high nitrate regimen (Table I). These data clearly demonstrate a dietary nitrate dependent formation of MNPz in the human body. It is interesting to note that all the volunteers showed endogenous nitrosation of piperazine and its increase under a nitrate supplemented diet. Since the diets were not controlled, variation in dietary nitrate is expected. However, no correlation was observed in the levels of urinary nitrate and ingested nitrate as well as MNPz and nitrate excretion. This clearly underlines the complexity of endogenous nitrosation and could partly be a reflection of the individual variation observed in the proportion of ingested nitrate which is circulated and reduced to nitrite in the human body [lo]. MNPz has been previously shown to be

142

converted to N-nitroso-3-hydroxy pyrrolidine (NHPYR) in the human body [14]. Thus, while MNPz formation may not provide any specific health hazard per se, its metabolite (NHPYR) is a known rat liver carcinogen [5]. Therefore, the present results call for applying caution in repeated therapeutic use of piperazine especially in populations where the staple diet contains high nitrate contents as in Kashmir [ 121. Urinary excretion of unchanged piperazine ranged between 15.4% and 41.2% of the administered dose when volunteers were on a normal diet and it decreased from 9.7% to 21.0% under a high nitrate regimen (Table I) However, in the present study, DNPz could only be detected in trace amounts in most samples and quantitation was possible just in one case (0.3 pg/day). In our earlier studies, 7 of 14 patients showed detectable urinary excretion of DNPz although no metabolites could be detected. In the current study no metabolites were determined, however, its trace level appearance in urine even under high nitrate regimen tends to point out that endogenous nitrosation of piperazine results mainly in the formation of MNPz with small amounts of DNPz which possibly metabolises through a different path than observed in experimental animals [ 151. Acknowledgements

The authors would like to thank Mrs. I. Kling and Mrs. C. Ditrich for their help in the nitrate/nitrite analysis. Support received from the Indian Council of Medical Research, New Delhi and GSF, Munchen under Indo-German Scientific programme is gratefully acknowledged. R. Kumar would also like to thank DKFZ, Heidelberg for financial support during his stay at the Institute for Toxicology as a visiting scientist. References 1

Bellander, T., Gsterdahl, B.-G. and Hagmar, L. (1985) Formation of N-mononitrosopiperasine in the stomach and

6

7

8

9

10

11

12

13

its excretion in the urine after oral intake of piperazine. Toxicol. Appl. Pharmacol., 80, 193- 198. Bellander, T., osterdahl, B.-G. and Hagmar, L. (1988) Excretion of N-mononitrosopiperazine after low level exposure to piperazine in air: effect of dietary nitrate and ascorbate. Toxicol. Appl. Pharmacol., 93, 281- 287. Bellander, T., Gsterdahl, B.-G., Hagmar, L. and Skerfving, S. (1988) Excretion of N-nitrosomonopiperazine in urine in workers manufacturing piperasine. Int. Arch. Occup. Environ. Health, 60, 25-29. Druckrey, H., Preussmann, R., Ivankovic, S. and Schmahl, D. (1967) Organotrope carcinogene Wirkung bei 65 verscheidenen N-nitroso-Verbindungen an BD-Ratten. 2. Krebsforsch., 67, 103-201. Eisenbrand, G., Habs, M., Schmahl, D. and Preussmann, R. (1980) Carcinogenecity of N-nitroso-3-hydroxypyrrolidine and dose response study with N-nitrosopiperadine in rats, pp. 657-666. In: N-Nitroso Compounds: Analysis, Formation and Occurrence. Editors: E.A. Walker, L. Griute, M. Castegnaro and M. B(jrzsBnyl. IARC Scientific Publication No. 31, IARC, Lyon. Greenblatt, M., Mirvish, S.S. and So, B.T. (1971) Nitrosamine studies. Induction of lung adenomas by concurrent administration of sodium nitrite and secondary amines in Swiss mice. J. Natl. Cancer Inst., 46, 1029 - 1034. Hecht, S.S., Morrison, J.B. and Young, R. (1984) Nnitroso(2-hydroxyethyl)glycine, a urinary metabolite of N,N’-dinitrosopiperazine with potential utility as a monitor for its formation in vivo from piperasine. Carcinogenesis, 5, 979-981. Love, L.A., Lijinsky, W., Keefer, L. and Garcia, H. (1977) Chronic oral administration of I-nitrosopiperazine in high doses to MRC rats. Z. Krebsforsch., 89, 69- 73. Mirvish, S.S. (1975) Formation of N-nitroso compounds: chemistry, kinetics and in vivo occurrence. Toxicol. Appl. Pharmacol., 31, 325-351. Packer, P. J., Leach, S.A., Duncan, S.N., Thompson, M.H. and Hill, M.J. (1989) The effect of different sources of nitrate exposure on urinary nitrate recovery in humans and its relevance to the method of estimating nitrate exposure in epidemiological studies. Carcinogenesis, 10, 1989 - 1996. SchmBhl, D. and Thomas, C. (1965) Erzeugung von Lungen-und Lebertumoren bei Mausen mit N,N’Dinitrosopiperazine. Z. Krebsforsch., 67, 11 - 15. Siddiqi, M., Tricker, A.R., Kumar, R., Fasili, Z. and Preussmann, R. (1990) Presence of N-nitroso compounds in commonly used foodstuffs from a high esophageal cancer area in Kashmir, India. In: N-Nitroso Compounds: Biology and Chemistry, pp. 19-25. Editors: S.V. Bhide and K.V. Rao. Omega Scientific Publications, New Delhi. Skarping, G., Bellander, T. and Mathiasson, L. (1986) Determination of pipersine in working atmosphere and in human urine using a derivatisation and capillary gas chromatography with nitrogen- and mass-selective detection. J. Chromatogr., 370, 245-258.

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Tricker, A.R., Kumar. R., Siddiqi, M., Khuroo, M.S. and Preussmann, R. (1991) Endogenous formation of Nnitrosamines from piperazine and their urinary excretion following antihelmintic treatment with piperazine citrate. Carcinogenesis, 12, 1595 - 1599.

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Tricker, A.R., Khlbe, T. and F’reussmann, R. (1991) Comparative metabolism and urinary excretion of Nmononttrosoptperazine and N,N’-dinitrosopiperaztne in the rat. Cancer Lett., 59, 165- 169.

Effect of dietary nitrate on endogenous nitrosation of piperazine in humans.

The effect of dietary nitrate on endogenous nitrosation of a therapeutic dose of piperazine has been described in five human volunteers who acted as t...
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