MALAWIMEDJOURNAT;I60 ):6-8 March 2004 . ;{r.
Pharmacokinetics of biliary excretion of N-nitrosodimethylaminein ratsfed dietscontaininglevelsof protein AlanemeFO,MaduagwuEN Departmentof Biochemistry, collegeof Medicine,Universityof lbadan,lbadan,Nigeria. correspondingAuthor: ProfessorE N Maduagwu,Departmentof Biochemistry,college of Medicine,universityof Malawi,p,/Bag360, Chichiri,Blantyre3, Malawi.E-mail:[email protected]
ABSTRACT
\
Albino Wistar rats (Rattus nomegius) fed semi-purified diets ssnf qini n g 3.5 Vo,8 Vo,27 Vo, and 64 Vo casein,respectively, as the protein source, were poisoned with an intraperitoneal dose of 20mg N-nitrosodimethylamine (NDMA/kg, following cannulation of the bile duct, in vitro, under urethane anaesthesia Bile exudates was collected at designated time intervals and analysed for unchangetl NDMA using thin layer chromatography and gas liquid chromatography methods. Rats on 64Vo htgh protein diet (HpD) were the highest excretors of NDMA, followed by rats on the 3.SVo
kwashiorkorigenicdiet (KWD), 8Volow protein diet (LpD) and,27Vonormal protein diet (NDP) as the leastexcretors,in that order. The correspondingvaluesfor culmulative excretions of NDMA were4.38Vo,2.74Vo,2.96Vo and.4.ll7o, and, for elimination rate contentsthey were 54.05Kh",23.01Kh-', 23.76Kh" and 48.88Kh'', while the respectiveelimination half-life valueswere 0.013h,0.031h,0.029hand 0.014h.The toxicologicaland pharmacologicalimplication of the pharmacokineticfindings are discussed.
lntroduction
nutritional groups, namely; the peasantry who susbsistor marginal protein diets, and the well-to-do, who have affordable high protein meals. In view of this wide difference in dietary habits, and the dependenceof drug metabolizing enzymes of liver and other tissues on the quality and quaatity of protein, it would seem desirable to examine the biliary excretion of Nnitrosaminesin relation to dietary protein availability. The aim of this study was to establish, comparatively whether or not there are differences in the kinetics and mode of biliary elimination of NDMA (a representative N-nitrosamins 416 u prime suspectin the causation of urban cancers)as between the nutritional statesof chronic protein malnourishment, a cute protein deprivation, protein sufficiency and protein over nourishment using model experimental rats.
The excretion of N-nitrosamines (including NDMA) in the bile of humansl and of animalsz3has been reported,but the influence of the nutritional status, which is an important denominator in the metabolism and disposition of foreign compounds (xenobiotics) in this regard, especially with respect to dietary protein energy malnourishment (PEM) and over nourishment, has not been previous investigated. Volatile and non-volitile N-nitrosamines have been found to be toxica, and versatile carcinogens5in animals, and they contaminate the human environment,tt, food stuff, urban air, water and soil. In addition, they can be mutagenicr3,and a small dose of a nitrosamine, known to be capable of producing a malignant tumour in rats5,can be formed from simple substancesof plant and animal origin, such as nitrates, nitrites and secondary arnines.The elaboration of N-nitrosamines, in vitro and in vivo, occurs either spontaneouslyin acid medium, or are mediated by appropriate enzymes. These compounds require prior metabolic transformation'' for their activity, and they can have cumulative effects which are attributable to alkylating entities.r5-r7 NDMA is the only N-nitrosamine that has been constantly detected in the human environment. Like other nitrosamine species,it is organ specific in its toxicity and carcinogenicity, causinghaemorrhagiccentrilobular cell necrosisof the liver. The latter lesion is specific and a characteristicproperty which distinguishes the action of N-nitrosamines from other hepatotoxic chemicals, such as carbon tetrachloride the aflatoxins. The elemination of unchanged NDMA through the expired air, faeces and body fluids including urine and bile, is of immense pharmacological and toxicological importance, since certain drugs in clinical use are secondary amines and are known to form nitrosamines,lz vivo,,8 andthe excretion of pure NDM is a detoxication mechanism. In particular, the toxicity and carcinogenicity of a given nitrosamine is bound to be enhancedby its prolonged presenceinthe blood circulatory system through a recycling process,in the event of enterohepaticcirculation of the compound. In the tropics of Africa, Asia and Latin America, well known for their their high incidences of PEM, the population that would be exposed to nitrosamines through frequent ingestion of contaminated food, bverages, water and drugs can be divided into two Malawi Medical Journal
Material and Methods Pure N- Nitrosodimethylamine (NDMA) >99Vo,mol. fi. 74.08, and boiling point (b.p.) 1530was obtained from our stock in the Biochemical Toxicology laboratory of the Department of Biochemistry, College of Medicine, University of Ibadan, Ibadan (Nigeria). Urethane (ethylcarbamate) and other chemicals reagentsand solvents were of analytical grade and purchases from either May & Baker Ltd, or BDH Chemical Ltd. London. Animals: Albino Wistar rats (littermates) weaned at22-24 days of age were obtained from pre-clinical animal house of Ibadan College of Medicine, and they weighed between 30g and 40g. The rats were allocated on the basis of weight and litter origin to groups of 8 rats each. They were housed in warm cages (250C) and fed on semi-purified test diets containing different levels of casein as protein source (Tablel), and drinking water, for a period of 30 days, ad libitum. This dietary regimen establishedthe model test animals. Induction of kwashiorkor in rats: Weanling rats given the kwashiorkorigenic diet'e for 30 days were established using anatomical prognostic indicators and the biological statusesof kwashiorkor rats.20These parameters were monitored in the model animals against control animals on the normal protein diet.
of biliarVexcretion I Pharmacokinetics were as follows: High The dietary animal models established (NPD), low protein (LPD) and ini'U, Normal protein ;;;"i" kwashiorkor (KWD) rats'
Figure1 @$c€orAst{GLE90sE ';; L:*::: *#; t* tle oF RArsrEDIXrrERsNr
' oF A FoLtowNG i p ADMlNlsrRATloN ' g 9 O S EO F 2 0 m g N O M A l K "'it^it'i'*otii*S I N G L E
animals were Cannulation of bile duct: The experimental solution given intraperianaesthesizedwith a 257o utethane midline abdominal tonealy (i.p.) at a rate of 1'5glkg''' A small at its iuncexposed was duct bile the and incision w^ascreated, glass tube clean in collected was Bile tion with the duodeneum' inserted into the ttroogft a suitable polythene cannula surgically junction." The cannuuil" iro about 1.5cm from the duodenum to avoid its dislodgela was held in position with a tied thread
E _g
zv
in
ment.
2 z
Administrationof DNMS
4q
Fourtestratsfiomeachdietarywereused.NMDAwasinjected a l0-minute collection of i.f. u, ttt" rate of Z}mgkgfoliowing bile was bile for baseline evaluation' Subsquently' pit" t**-f) intervals until the antcollected at 10, 20, and finally in 30 min' were immediately mal became moribund' ffre Uite samples As much as possianalysed' 4aC,until stored in a retrigerator aI of collection of 24hrs within performed ble, analysis of bile was
s':*
.--.,i 100
the fluid.
?00
300
I
4.00
- ]ise {nin}
Analysisof the bile
of NDMA, uslng The bile samPles were extracted to lml on an concentrated then dichloromethaneas solvent, and Extrelut column.t' in the dichloromethane extract Qualitative analysis of NDMA chromatography(t'l'c') with layer Joncentrateof bile was by thin two^recommended" spray to positive response(purple spot) for the presence of test u, un id"ntification i""g"*t diethylether: n-hexane: nitr"osamines.The solvent system was exposed first were plates Jichlorometh ane(4:3:2)' The treated photochemical undergo to to shortwave UV light ior 3 minutes' denitrosationbefore their development' was by qou*lo,iu" estimation for unchanged NDMA content ionisation flame using (g'l'c')25 gas liquid chromatography standard reference comietection (F.I.D) and the appropriate
Figure2 $D$A: Ttlll€ t'gLATlOltSHlP lN .2: sg!,/ll{o€ PLOTlf''l BILE FXCREItD rlc" 'n"ira"rnr,-aua* A SINGLEI p oF DIETARYPRorElNs oF RAT. GI'EN DOSEoF mmgNd'tA/Xg
1000
9
100
E z
oounds. Computation of kenetic parameters from the semiElimination rate consiant (K) was calculated using the relationship time log plot of bile excretedNDMA: -Kl2'303 equation:SloPe= derived using the Biologicat tratf-tife (tLl2) pfNDMA was = constant equationIll2 = 0.6931Kwhere K rate
Results
high protein diet (HPD) The data in Table 2 suggestthat rats on of unchanged excretors and normal (NPD) weie the highest and the low (KWD) rats NnfnlA in bile, while the kwashiorkor in compound' the of protein rats (LPD) were the least excretors (t'l') half-life biological that order. The correspondingvalues for and for eliminating were 0.013h, 0.014hr,0'031hr,and 0'029hr and23"l6I{h-''This rateconstant,54.05Kh',48'8Kh', 23'0lKh' is eliminated in rat ndma ,"fot Outushows that the rate at which not appear to does but lii" it dependenton the protein status' protein depdietary of discriminate between the varying degrees rivation or Protein sufficiencY'
Hr.
DtscussloN the varrof the biliary excretion of NDMA by il; ,ir"" thal show 1) (Figure """rses investigated ous dietary protein rat models plot ol A semi-log case' each in Ulffruri" kinetics is operational a possible.-pharmacokinet ifri, tin",i", (Figure 2) supports on biliary excretlon o. protein ciassification of effect of dietary gfoups' namely; fas distinct -exihibited nitrosaminesinto two significantly well-nourished rat the by and slow excretors as (KWD & LPD) animals t"tD A NPD) and the malnourished
respectivelY. with protein nourish si;l"gi.A idf-life (t'l') values decreased constant (K)' Th rate for case the conversewas the the time coursesof bil- ment, while respectively' rate c show, 2 clearance Figure 1 and slowest Figure rats exhibited the plot of the data for the malnourished semi-log the toxt the and xllra o] ameliorates diet protein iuiyXOVIA. It is known that a low "^"r",ion in this study' used rats model protein JifLrent-dietary
Miilawi Medical Joun
8
Pharmacokinetics of biliary excretion
effects of NPMA by slowing doul heparic merabolism of compound into harmful products. and invariabll.enhancing its elimilation from the body. Therefore. the results of this srudy which show that a protein depleteddiet diminished the rate of elimination of NDMA via bile, and increaseshalf-life of the compound, would suggestthat the biliary route of excretion of nitrosamines, on the one hand, and the urinary route. on the other hand, could be mutually exclusive. There is also the possibiliry that excretion of such a small but highly polar molecule as NDMA in bile, in apparentviolation of the rule.6that only large molecular weight compounds (molecular weigh >250-300) areexcretableto any large extent, could entail a supportiverole ofhigh protein diet in the biliary elimination of nitrosamines. The biphasic kinetics (Figure 1) in the eliminarion of NDMA in bile in protein malnourishedrats is in line with the pharmacokinetics2128 of many chemical compoundsin biological fluids. The study emphasizesthe need for the dtermination of the nutritional status of humans in the pharmacokinetic evaluation of drugs and other environmental chemicals,before a correct toxicological and pharmacological assessmentof a xenobiotic can be obtained.
Table 1: Composition of experimental diets Diet
Amounts of component in exDerimental diet%
Components
Kwashiorkorigenic
Low protein
Nomal protein
Highproteln
(KwD)
(LPD)
(NPD)
(HPD)
3.5
8.0
27.0
64.0
81.5
77.0
58.0
2t.0
Casein Corn.starch VegetableOil
8.0
8.0
8.0
8.0
Saltmixture
4.0
4.0
4.0
4.0
All vitamin
3.0
3.0
3.0
3.0
Supplement
References 1. Allen-Mersh T G, Marshail C E, Smith R L, Walters C L. Nitrosamine in b l l e . L a c n c e t1 9 8 1 ;1 : 8 3 5 - 8 3 6 . 2. Bonfanti M, Magafnotti C, Fanelli R, Airoldi L. Chem. Biol. Interacrions 1986:56:203-210 3. Atawodi SE, Maduwagwu EN. Interraction of N-nitrosdiphenylargine with ratblle. WestAfric. J. Biol. Appl. Chem. 7987;32:26-29. 4. Barnes JM, Magee PN. Some toxic properties of dimethylnitrosamine. Br J. Indust. Med. 19541'fI:167 -114 5. Magee PN, Barnes JM. Coarcinogenic nitroso compounds.Adv. Cancer Res.1967: 10:163-246 6. Ender F, Ceh L. Occurrence of nitrosamines in food stuffs for human and animal consumption. Food Cosmet. Toxicol 1968; 6: 569-57ir. 7. Bassir O, Maduagwu EN. Occurrence of nitrate, nitrite, dimethylamine, and dimethylnitrosamine in some fermented Nigerian beverages.J. Agic. Food Chem. I9'78: 26: 200-203. 8. Gough TA, Webb KS, Coleman RF. Estimation of the volatile nitrosamine c o n t e n to f U K f o o d .N a t u r e 1 9 7 8 . 2 1 2 : 1 6 i - 1 6 3 . 9. SpiegelhalderB, Eisenbrand G, PreussmannR. Contamination of beer with trace quantitive of N^nitrosodimethylamine. Food Cosmet. Toxicol. 19'19 I7:29-31. 10. Ayanaba A, Alexander M. Microbial formation of nitrosamine in vitro. Appl., Microbiol. 7973; 25: 862. 11. Hotchkiss JH. Preformed N-nitrosocompounds in foods and beverages.In Forman D, Shuker D (eds), Nitrate, Nirate and Nitroso compounds in Human Cancer. Cancer Survey 1989: 8: 295-321. 12. Fan SDH, Fine R, Ross DP, Roundbehler A, Silvergled A, Song L. Determination of N-nitroso compounds in air, water and soi1.Paper presentedat the 172"dACS National Meeting, 1976, August 29 September 3. San Franscisco, California, USA. 13. Dobo KL, Easmond DA, Grosovsky AJ. The influence of cellulat apoptotic capacity on N-nitrosodimethylamine induced loss of heterozygosity mutations in human cells. Carcinogenesis 1997 l8: 1'701-1101. 14. Magee PN, Vandekar M. Toxic liver injury: the metabolism of dimethylnitrisamine in vitro. Biochem. "f. 1958; 70:600. 15 Dutton AH, Heath DF. Demethylation of dimethylnitrosamine in rats and mice. Narurc. )956; 178:644. 16. Magee PN, Faber F. Toxic liver injury and carcinogenesis:Methylation of rat liver nucleic acids by dimethylnitrosamine in vivo. Biochem. J. 1962: 83:l14-124 t ] Haggerty HG, Holsapple MP Role of metabolism in dimethylamine induced immunosuppression.Review Toricol. 1990; 62:l -2.
1 8 . Lijinsky W, Greenblatt M. Carinogenic dimethylnitrosamine produced in vivo form nitrite and aminopyrine.Nature (New Biol.) 1972.-177-178_
Table 2: Effects of dietary protein insufficiency and oyer-sufficiency on the pharmacokinetics of *excretion of N-nitrosodimethylamine in rat bile. Dietry
Culmulative
Protein
NDMA NDMA peak (ug) excretion(ug) Excretion(7o)
Regimen
CulmulativeExcretion
t9. Bydd EM, Carsky E. Kwashiorkorigenic diet and diazonium ro\icir)..{cr.r Pharmacol. Taxicol. 1969',21 :284.
20 Olowookere JO. Some energy implications of protein energl. malnutrition in the rat. Ph.D Thesis. University of Ibadan, Ibadan, 1980.
2 l Amure BO, Omole A. Comparativ study of intra gastrin activity in some Peak Elimination
Biological
rime rare (min) Constant (Kh-1)
halfJifeft'/r) (hr)
3.4Ea(.KWD)82.33
2.74
18.49
50
23.Q1
0.03
8Va(LPD)
118.28
2.96
27.14
50
23.16
0.029
277o(NPD) 164.19
4.t1
31.19
90
48.8
0.014
64Vo(HPD) 175.20
4.38
33.09
90
54.05
0.013
22
L).
24. 25
+values ue arithmetic means of 3 successful cannulations. zo.
l" t:
21
Acknowledgement The authors are grateful Science, Alagbon-Irtgos, szs.
Malawi Medical Journal
to Mr Eliaz Uzoemeka (DSP) of the Police Forensic Nigeriafor arranging the gas chromatographic analy-
28
mammals.Br J. Phatmacol. l91l:41:629-693. Abou-El-'Makareem MM, Miilburn P, Smittr RL, William RT. Biliary excretion of foreign compounds: Benzene and its derivatives in the rat. Biochem J. 19671105: 1289-1293. SpiegelhalderB, Eisenbrand G, PreussmannR. In: Environmental Carcinogenesis;SupplementVol. IARC. Sci. Pub., 1983; No. 45. PreussmannR, Daiber D, Hengy H. A sensitive colour reaction for nitrosamines on thin layer chromatograms.Nature 1964;201: 502-503. Alaneme FO, Maduagwu EN. N-nitrosation of the juicy extract of some tropical edible leafy vegetables.Malawi Med. J., 2004 (In Press). Smith RL. The Excretory Function of Bile. 1973; Chapman and Hill, London. Maduagwu EN, Umoh IB. Bilary excration of linamarin in the Wistar rat after a single oral dose.Biochem.Pharmacol. 1986; 35:3003-3006. Meyer DFK, Weitering JG, Vermeer A. Pharmacokinetics of biliary excretion in man. V: Dibromosulfophthalein. El;r J. CIin. Pharmacol. 1983; 24: 249-556.
Pharmacokinetics of biliary excretion of N-nitrosodimethylaminein ratsfed dietscontaininglevelsof protein AlanemeFO,MaduagwuEN Departmentof Biochemistry, collegeof Medicine,Universityof lbadan,lbadan,Nigeria. correspondingAuthor: ProfessorE N Maduagwu,Departmentof Biochemistry,college of Medicine,universityof Malawi,p,/Bag360, Chichiri,Blantyre3, Malawi.E-mail:[email protected]
ABSTRACT
\
Albino Wistar rats (Rattus nomegius) fed semi-purified diets ssnf qini n g 3.5 Vo,8 Vo,27 Vo, and 64 Vo casein,respectively, as the protein source, were poisoned with an intraperitoneal dose of 20mg N-nitrosodimethylamine (NDMA/kg, following cannulation of the bile duct, in vitro, under urethane anaesthesia Bile exudates was collected at designated time intervals and analysed for unchangetl NDMA using thin layer chromatography and gas liquid chromatography methods. Rats on 64Vo htgh protein diet (HpD) were the highest excretors of NDMA, followed by rats on the 3.SVo
kwashiorkorigenicdiet (KWD), 8Volow protein diet (LpD) and,27Vonormal protein diet (NDP) as the leastexcretors,in that order. The correspondingvaluesfor culmulative excretions of NDMA were4.38Vo,2.74Vo,2.96Vo and.4.ll7o, and, for elimination rate contentsthey were 54.05Kh",23.01Kh-', 23.76Kh" and 48.88Kh'', while the respectiveelimination half-life valueswere 0.013h,0.031h,0.029hand 0.014h.The toxicologicaland pharmacologicalimplication of the pharmacokineticfindings are discussed.
lntroduction
nutritional groups, namely; the peasantry who susbsistor marginal protein diets, and the well-to-do, who have affordable high protein meals. In view of this wide difference in dietary habits, and the dependenceof drug metabolizing enzymes of liver and other tissues on the quality and quaatity of protein, it would seem desirable to examine the biliary excretion of Nnitrosaminesin relation to dietary protein availability. The aim of this study was to establish, comparatively whether or not there are differences in the kinetics and mode of biliary elimination of NDMA (a representative N-nitrosamins 416 u prime suspectin the causation of urban cancers)as between the nutritional statesof chronic protein malnourishment, a cute protein deprivation, protein sufficiency and protein over nourishment using model experimental rats.
The excretion of N-nitrosamines (including NDMA) in the bile of humansl and of animalsz3has been reported,but the influence of the nutritional status, which is an important denominator in the metabolism and disposition of foreign compounds (xenobiotics) in this regard, especially with respect to dietary protein energy malnourishment (PEM) and over nourishment, has not been previous investigated. Volatile and non-volitile N-nitrosamines have been found to be toxica, and versatile carcinogens5in animals, and they contaminate the human environment,tt, food stuff, urban air, water and soil. In addition, they can be mutagenicr3,and a small dose of a nitrosamine, known to be capable of producing a malignant tumour in rats5,can be formed from simple substancesof plant and animal origin, such as nitrates, nitrites and secondary arnines.The elaboration of N-nitrosamines, in vitro and in vivo, occurs either spontaneouslyin acid medium, or are mediated by appropriate enzymes. These compounds require prior metabolic transformation'' for their activity, and they can have cumulative effects which are attributable to alkylating entities.r5-r7 NDMA is the only N-nitrosamine that has been constantly detected in the human environment. Like other nitrosamine species,it is organ specific in its toxicity and carcinogenicity, causinghaemorrhagiccentrilobular cell necrosisof the liver. The latter lesion is specific and a characteristicproperty which distinguishes the action of N-nitrosamines from other hepatotoxic chemicals, such as carbon tetrachloride the aflatoxins. The elemination of unchanged NDMA through the expired air, faeces and body fluids including urine and bile, is of immense pharmacological and toxicological importance, since certain drugs in clinical use are secondary amines and are known to form nitrosamines,lz vivo,,8 andthe excretion of pure NDM is a detoxication mechanism. In particular, the toxicity and carcinogenicity of a given nitrosamine is bound to be enhancedby its prolonged presenceinthe blood circulatory system through a recycling process,in the event of enterohepaticcirculation of the compound. In the tropics of Africa, Asia and Latin America, well known for their their high incidences of PEM, the population that would be exposed to nitrosamines through frequent ingestion of contaminated food, bverages, water and drugs can be divided into two Malawi Medical Journal
Material and Methods Pure N- Nitrosodimethylamine (NDMA) >99Vo,mol. fi. 74.08, and boiling point (b.p.) 1530was obtained from our stock in the Biochemical Toxicology laboratory of the Department of Biochemistry, College of Medicine, University of Ibadan, Ibadan (Nigeria). Urethane (ethylcarbamate) and other chemicals reagentsand solvents were of analytical grade and purchases from either May & Baker Ltd, or BDH Chemical Ltd. London. Animals: Albino Wistar rats (littermates) weaned at22-24 days of age were obtained from pre-clinical animal house of Ibadan College of Medicine, and they weighed between 30g and 40g. The rats were allocated on the basis of weight and litter origin to groups of 8 rats each. They were housed in warm cages (250C) and fed on semi-purified test diets containing different levels of casein as protein source (Tablel), and drinking water, for a period of 30 days, ad libitum. This dietary regimen establishedthe model test animals. Induction of kwashiorkor in rats: Weanling rats given the kwashiorkorigenic diet'e for 30 days were established using anatomical prognostic indicators and the biological statusesof kwashiorkor rats.20These parameters were monitored in the model animals against control animals on the normal protein diet.
of biliarVexcretion I Pharmacokinetics were as follows: High The dietary animal models established (NPD), low protein (LPD) and ini'U, Normal protein ;;;"i" kwashiorkor (KWD) rats'
Figure1 @$c€orAst{GLE90sE ';; L:*::: *#; t* tle oF RArsrEDIXrrERsNr
' oF A FoLtowNG i p ADMlNlsrRATloN ' g 9 O S EO F 2 0 m g N O M A l K "'it^it'i'*otii*S I N G L E
animals were Cannulation of bile duct: The experimental solution given intraperianaesthesizedwith a 257o utethane midline abdominal tonealy (i.p.) at a rate of 1'5glkg''' A small at its iuncexposed was duct bile the and incision w^ascreated, glass tube clean in collected was Bile tion with the duodeneum' inserted into the ttroogft a suitable polythene cannula surgically junction." The cannuuil" iro about 1.5cm from the duodenum to avoid its dislodgela was held in position with a tied thread
E _g
zv
in
ment.
2 z
Administrationof DNMS
4q
Fourtestratsfiomeachdietarywereused.NMDAwasinjected a l0-minute collection of i.f. u, ttt" rate of Z}mgkgfoliowing bile was bile for baseline evaluation' Subsquently' pit" t**-f) intervals until the antcollected at 10, 20, and finally in 30 min' were immediately mal became moribund' ffre Uite samples As much as possianalysed' 4aC,until stored in a retrigerator aI of collection of 24hrs within performed ble, analysis of bile was
s':*
.--.,i 100
the fluid.
?00
300
I
4.00
- ]ise {nin}
Analysisof the bile
of NDMA, uslng The bile samPles were extracted to lml on an concentrated then dichloromethaneas solvent, and Extrelut column.t' in the dichloromethane extract Qualitative analysis of NDMA chromatography(t'l'c') with layer Joncentrateof bile was by thin two^recommended" spray to positive response(purple spot) for the presence of test u, un id"ntification i""g"*t diethylether: n-hexane: nitr"osamines.The solvent system was exposed first were plates Jichlorometh ane(4:3:2)' The treated photochemical undergo to to shortwave UV light ior 3 minutes' denitrosationbefore their development' was by qou*lo,iu" estimation for unchanged NDMA content ionisation flame using (g'l'c')25 gas liquid chromatography standard reference comietection (F.I.D) and the appropriate
Figure2 $D$A: Ttlll€ t'gLATlOltSHlP lN .2: sg!,/ll{o€ PLOTlf''l BILE FXCREItD rlc" 'n"ira"rnr,-aua* A SINGLEI p oF DIETARYPRorElNs oF RAT. GI'EN DOSEoF mmgNd'tA/Xg
1000
9
100
E z
oounds. Computation of kenetic parameters from the semiElimination rate consiant (K) was calculated using the relationship time log plot of bile excretedNDMA: -Kl2'303 equation:SloPe= derived using the Biologicat tratf-tife (tLl2) pfNDMA was = constant equationIll2 = 0.6931Kwhere K rate
Results
high protein diet (HPD) The data in Table 2 suggestthat rats on of unchanged excretors and normal (NPD) weie the highest and the low (KWD) rats NnfnlA in bile, while the kwashiorkor in compound' the of protein rats (LPD) were the least excretors (t'l') half-life biological that order. The correspondingvalues for and for eliminating were 0.013h, 0.014hr,0'031hr,and 0'029hr and23"l6I{h-''This rateconstant,54.05Kh',48'8Kh', 23'0lKh' is eliminated in rat ndma ,"fot Outushows that the rate at which not appear to does but lii" it dependenton the protein status' protein depdietary of discriminate between the varying degrees rivation or Protein sufficiencY'
Hr.
DtscussloN the varrof the biliary excretion of NDMA by il; ,ir"" thal show 1) (Figure """rses investigated ous dietary protein rat models plot ol A semi-log case' each in Ulffruri" kinetics is operational a possible.-pharmacokinet ifri, tin",i", (Figure 2) supports on biliary excretlon o. protein ciassification of effect of dietary gfoups' namely; fas distinct -exihibited nitrosaminesinto two significantly well-nourished rat the by and slow excretors as (KWD & LPD) animals t"tD A NPD) and the malnourished
respectivelY. with protein nourish si;l"gi.A idf-life (t'l') values decreased constant (K)' Th rate for case the conversewas the the time coursesof bil- ment, while respectively' rate c show, 2 clearance Figure 1 and slowest Figure rats exhibited the plot of the data for the malnourished semi-log the toxt the and xllra o] ameliorates diet protein iuiyXOVIA. It is known that a low "^"r",ion in this study' used rats model protein JifLrent-dietary
Miilawi Medical Joun
8
Pharmacokinetics of biliary excretion
effects of NPMA by slowing doul heparic merabolism of compound into harmful products. and invariabll.enhancing its elimilation from the body. Therefore. the results of this srudy which show that a protein depleteddiet diminished the rate of elimination of NDMA via bile, and increaseshalf-life of the compound, would suggestthat the biliary route of excretion of nitrosamines, on the one hand, and the urinary route. on the other hand, could be mutually exclusive. There is also the possibiliry that excretion of such a small but highly polar molecule as NDMA in bile, in apparentviolation of the rule.6that only large molecular weight compounds (molecular weigh >250-300) areexcretableto any large extent, could entail a supportiverole ofhigh protein diet in the biliary elimination of nitrosamines. The biphasic kinetics (Figure 1) in the eliminarion of NDMA in bile in protein malnourishedrats is in line with the pharmacokinetics2128 of many chemical compoundsin biological fluids. The study emphasizesthe need for the dtermination of the nutritional status of humans in the pharmacokinetic evaluation of drugs and other environmental chemicals,before a correct toxicological and pharmacological assessmentof a xenobiotic can be obtained.
Table 1: Composition of experimental diets Diet
Amounts of component in exDerimental diet%
Components
Kwashiorkorigenic
Low protein
Nomal protein
Highproteln
(KwD)
(LPD)
(NPD)
(HPD)
3.5
8.0
27.0
64.0
81.5
77.0
58.0
2t.0
Casein Corn.starch VegetableOil
8.0
8.0
8.0
8.0
Saltmixture
4.0
4.0
4.0
4.0
All vitamin
3.0
3.0
3.0
3.0
Supplement
References 1. Allen-Mersh T G, Marshail C E, Smith R L, Walters C L. Nitrosamine in b l l e . L a c n c e t1 9 8 1 ;1 : 8 3 5 - 8 3 6 . 2. Bonfanti M, Magafnotti C, Fanelli R, Airoldi L. Chem. Biol. Interacrions 1986:56:203-210 3. Atawodi SE, Maduwagwu EN. Interraction of N-nitrosdiphenylargine with ratblle. WestAfric. J. Biol. Appl. Chem. 7987;32:26-29. 4. Barnes JM, Magee PN. Some toxic properties of dimethylnitrosamine. Br J. Indust. Med. 19541'fI:167 -114 5. Magee PN, Barnes JM. Coarcinogenic nitroso compounds.Adv. Cancer Res.1967: 10:163-246 6. Ender F, Ceh L. Occurrence of nitrosamines in food stuffs for human and animal consumption. Food Cosmet. Toxicol 1968; 6: 569-57ir. 7. Bassir O, Maduagwu EN. Occurrence of nitrate, nitrite, dimethylamine, and dimethylnitrosamine in some fermented Nigerian beverages.J. Agic. Food Chem. I9'78: 26: 200-203. 8. Gough TA, Webb KS, Coleman RF. Estimation of the volatile nitrosamine c o n t e n to f U K f o o d .N a t u r e 1 9 7 8 . 2 1 2 : 1 6 i - 1 6 3 . 9. SpiegelhalderB, Eisenbrand G, PreussmannR. Contamination of beer with trace quantitive of N^nitrosodimethylamine. Food Cosmet. Toxicol. 19'19 I7:29-31. 10. Ayanaba A, Alexander M. Microbial formation of nitrosamine in vitro. Appl., Microbiol. 7973; 25: 862. 11. Hotchkiss JH. Preformed N-nitrosocompounds in foods and beverages.In Forman D, Shuker D (eds), Nitrate, Nirate and Nitroso compounds in Human Cancer. Cancer Survey 1989: 8: 295-321. 12. Fan SDH, Fine R, Ross DP, Roundbehler A, Silvergled A, Song L. Determination of N-nitroso compounds in air, water and soi1.Paper presentedat the 172"dACS National Meeting, 1976, August 29 September 3. San Franscisco, California, USA. 13. Dobo KL, Easmond DA, Grosovsky AJ. The influence of cellulat apoptotic capacity on N-nitrosodimethylamine induced loss of heterozygosity mutations in human cells. Carcinogenesis 1997 l8: 1'701-1101. 14. Magee PN, Vandekar M. Toxic liver injury: the metabolism of dimethylnitrisamine in vitro. Biochem. "f. 1958; 70:600. 15 Dutton AH, Heath DF. Demethylation of dimethylnitrosamine in rats and mice. Narurc. )956; 178:644. 16. Magee PN, Faber F. Toxic liver injury and carcinogenesis:Methylation of rat liver nucleic acids by dimethylnitrosamine in vivo. Biochem. J. 1962: 83:l14-124 t ] Haggerty HG, Holsapple MP Role of metabolism in dimethylamine induced immunosuppression.Review Toricol. 1990; 62:l -2.
1 8 . Lijinsky W, Greenblatt M. Carinogenic dimethylnitrosamine produced in vivo form nitrite and aminopyrine.Nature (New Biol.) 1972.-177-178_
Table 2: Effects of dietary protein insufficiency and oyer-sufficiency on the pharmacokinetics of *excretion of N-nitrosodimethylamine in rat bile. Dietry
Culmulative
Protein
NDMA NDMA peak (ug) excretion(ug) Excretion(7o)
Regimen
CulmulativeExcretion
t9. Bydd EM, Carsky E. Kwashiorkorigenic diet and diazonium ro\icir)..{cr.r Pharmacol. Taxicol. 1969',21 :284.
20 Olowookere JO. Some energy implications of protein energl. malnutrition in the rat. Ph.D Thesis. University of Ibadan, Ibadan, 1980.
2 l Amure BO, Omole A. Comparativ study of intra gastrin activity in some Peak Elimination
Biological
rime rare (min) Constant (Kh-1)
halfJifeft'/r) (hr)
3.4Ea(.KWD)82.33
2.74
18.49
50
23.Q1
0.03
8Va(LPD)
118.28
2.96
27.14
50
23.16
0.029
277o(NPD) 164.19
4.t1
31.19
90
48.8
0.014
64Vo(HPD) 175.20
4.38
33.09
90
54.05
0.013
22
L).
24. 25
+values ue arithmetic means of 3 successful cannulations. zo.
l" t:
21
Acknowledgement The authors are grateful Science, Alagbon-Irtgos, szs.
Malawi Medical Journal
to Mr Eliaz Uzoemeka (DSP) of the Police Forensic Nigeriafor arranging the gas chromatographic analy-
28
mammals.Br J. Phatmacol. l91l:41:629-693. Abou-El-'Makareem MM, Miilburn P, Smittr RL, William RT. Biliary excretion of foreign compounds: Benzene and its derivatives in the rat. Biochem J. 19671105: 1289-1293. SpiegelhalderB, Eisenbrand G, PreussmannR. In: Environmental Carcinogenesis;SupplementVol. IARC. Sci. Pub., 1983; No. 45. PreussmannR, Daiber D, Hengy H. A sensitive colour reaction for nitrosamines on thin layer chromatograms.Nature 1964;201: 502-503. Alaneme FO, Maduagwu EN. N-nitrosation of the juicy extract of some tropical edible leafy vegetables.Malawi Med. J., 2004 (In Press). Smith RL. The Excretory Function of Bile. 1973; Chapman and Hill, London. Maduagwu EN, Umoh IB. Bilary excration of linamarin in the Wistar rat after a single oral dose.Biochem.Pharmacol. 1986; 35:3003-3006. Meyer DFK, Weitering JG, Vermeer A. Pharmacokinetics of biliary excretion in man. V: Dibromosulfophthalein. El;r J. CIin. Pharmacol. 1983; 24: 249-556.
