Pergamon Press
Life Sciences Vol . 17, pp . 1599-1606 Printed in the U .S .A .
INVESTIGATIONS INTO THE HEPATIC METABOLISM OF DIMETHYLNITROSAMINE IN THE RAT Brian G . Lake, Margaret J . Minski, John C . Phillips, Sharat D, Gangolli and Alun G . Lloyd The British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey SM5 4DS, UK . (Received in final form October 23, 1975)
Summary In this paper we report the detection and identification of methanol as an intermediate formed during both the in vivo and the in vitro metabolism of dimethylnitrosamine (DMN) in the rat . Methanol was formed in both hepatic 10,000 g av . supernatant and washed microsomal fractions ovér a wide range of nitrosamine substrate concentrations . Furthermore the total amounts of methanol and formaldehyde formed largely accounted for the metabolic fate of both methyl moieties of DMN . Although a number of inhibitors of alcohol metabolism profoundly inhibited the hepatic metabolism of DMN they had little effect on the activities of two mixed function oxidase dependent enzymes . The results suggest that DMN and possibly other dialkylnitrosamines are degraded by enzymic pathway s) not dependent on cytochrome P-450 . The hepatocarcinogen dimethylnitrosamine (DMN) has been shown to be metabolised in vivo in the rat to C02 (1) . Additional studies in vitro (2,3,4,5) have shown that hepatic subcellular preparations in the presence of NADPH and molecular oxygen convert DMN to formaldehyde . It has been proposed (6) that this process proceeds by the oxidative demethylation of the nitrosamine to monomethylnitrosamine which may subsequently form nitrous acid, diazomethane or methyl carbonium ions . Although it has been suggested (4,7) that the metabolism of DMN to formaldehyde is mediated by a microsomal demethylase enzyme dependent on cytochrome P-450, previous studies in our laboratory (8) have shown several marked differences between the properties of "DMN demethylase" and typical enzymes representative of the microsomal mixed function oxidase system . These observations led us to re-examine the metabolism of this nitrosamine in the rat in terms of the metabolites formed and we now report the formation of methanol as a result of DMN biodegradation . Methods DMN was purchased from Eastman Organic Chemicals, Kirkby, Liverpool, U,K . and was shown to be >99% pure by g .l .c . (9) . NADP, DL-isocitric acid and isocitric dehydrogenase were obtained from the Sigma Chemical Company, London, U,K, Gas chromatograph column packing materials were purchased from the Field Instrument Company, 1599
Hepatic Metabolism of DMN
1600
Vol . 17, No . 10
Richmond, Surrey, U.K . Preparation of hepatic subcellular fracti ons Male Wistar Albino rats (80-150 g) were used for all studies . They were allowed free access to laboratory diet and water . Livers were homogenised in 0 .154 M KC1 containing 50 mM Tris-HC1 buffer pH 7 .4 and centrifuged at 10,000 g av . for 20 min to obtain the postmitochondrial supernatant fractions . Microsomes were sedimented by centrifuging the 10,000 g av supernatants at 105,000 g av . for 60 min . The pellets-were resuspended in fresh homogenising medium and again centrifuged at 105,000 g av . for 60 min to obtain washed microsomal fractions . Enzyme assays The in vitro metabolism of DMN in either 10,000 g av . supernatant or washed microsomal fractions was determined in a medium containing 0 .5 mM NADP, 7 .5 mM DL-isocitric acid, 5 .0 mM MgS04, 7 .5 mM semicarbazide and 0 .05 mg/ml isocitric dehydrogenase in 50 mM Tris-HC1 buffer pH 7 .4 . Formaldehyde was determined in deproteinised tissue incubates by the method of Nash (10) . Methanol was determined by using a Pye series 104 gas chromatograph equipped with a flame ionisation detector and employing a Chromosorb 102 (80-100 mesh) column at 105°C . DMN was similarly measured using a 10~ Carbowax 20 M (on 100-120 mesh acid washed Celite) column at 120oC . The NADPH-dependent metabolism of methanol to formaldehyde was determined using the incubation system as described above for DMN with a methanol substrate concentration of 5 mM . Assays of ethylmorphine N-demethylase (11) and aniline 4-hydroxylase (12) were performed on 10,000 g av . supernatant fractions . Results In vitro stud ies with hepatic subcellular fractions
When DMN was incubated with either 10,000 g av . or washed microsomal fractions the formation of methanol could be detected by g .l .c . analysis of the deproteinised tissue incubates . The methanol produced from the metabolism of DMN had identical retention times to that of authentic methanol on three separate gas chromatograph columns (Table 1) . TABLE 1 The Determination of Methanol by G .L .C . in Rat Plasma Extracts and Deproteinised Tissue incubates Retention time(min) Temperature ethanol formed from Column Packing °C Authentic Methanol DMN Porapak Q 70 6 .92 (4) 6 .98 (4) 11 .87 (7) Porapak S 100 11 .87 (7) 105 3 .67(10) 3 .67(10) Chromosorb 102 Nitrogen carrier gas flow rates were 70 ml/min for the Porapak Q and S columns and 50 ml/min for the Chromosorb 102 column . Retention times are expressed as means, with the number of determinations in parentheses .
Vol . 17, No . 10
Hepatic Metabolism of DMN
1601
Further, the chemical identity of the methanol formed was confirmed by mass spectrometry . When either 10,000 g av .supernatant or washed microsomal fractions were incubatedwith an NADPH-generating system but without the nitrosamine, no methanol was formed (detection limit ~osi~ a .o~~ a
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Life Sciences Vol . 17, pp . 1599-1606 Printed in the U .S .A .
INVESTIGATIONS INTO THE HEPATIC METABOLISM OF DIMETHYLNITROSAMINE IN THE RAT Brian G . Lake, Margaret J . Minski, John C . Phillips, Sharat D, Gangolli and Alun G . Lloyd The British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey SM5 4DS, UK . (Received in final form October 23, 1975)
Summary In this paper we report the detection and identification of methanol as an intermediate formed during both the in vivo and the in vitro metabolism of dimethylnitrosamine (DMN) in the rat . Methanol was formed in both hepatic 10,000 g av . supernatant and washed microsomal fractions ovér a wide range of nitrosamine substrate concentrations . Furthermore the total amounts of methanol and formaldehyde formed largely accounted for the metabolic fate of both methyl moieties of DMN . Although a number of inhibitors of alcohol metabolism profoundly inhibited the hepatic metabolism of DMN they had little effect on the activities of two mixed function oxidase dependent enzymes . The results suggest that DMN and possibly other dialkylnitrosamines are degraded by enzymic pathway s) not dependent on cytochrome P-450 . The hepatocarcinogen dimethylnitrosamine (DMN) has been shown to be metabolised in vivo in the rat to C02 (1) . Additional studies in vitro (2,3,4,5) have shown that hepatic subcellular preparations in the presence of NADPH and molecular oxygen convert DMN to formaldehyde . It has been proposed (6) that this process proceeds by the oxidative demethylation of the nitrosamine to monomethylnitrosamine which may subsequently form nitrous acid, diazomethane or methyl carbonium ions . Although it has been suggested (4,7) that the metabolism of DMN to formaldehyde is mediated by a microsomal demethylase enzyme dependent on cytochrome P-450, previous studies in our laboratory (8) have shown several marked differences between the properties of "DMN demethylase" and typical enzymes representative of the microsomal mixed function oxidase system . These observations led us to re-examine the metabolism of this nitrosamine in the rat in terms of the metabolites formed and we now report the formation of methanol as a result of DMN biodegradation . Methods DMN was purchased from Eastman Organic Chemicals, Kirkby, Liverpool, U,K . and was shown to be >99% pure by g .l .c . (9) . NADP, DL-isocitric acid and isocitric dehydrogenase were obtained from the Sigma Chemical Company, London, U,K, Gas chromatograph column packing materials were purchased from the Field Instrument Company, 1599
Hepatic Metabolism of DMN
1600
Vol . 17, No . 10
Richmond, Surrey, U.K . Preparation of hepatic subcellular fracti ons Male Wistar Albino rats (80-150 g) were used for all studies . They were allowed free access to laboratory diet and water . Livers were homogenised in 0 .154 M KC1 containing 50 mM Tris-HC1 buffer pH 7 .4 and centrifuged at 10,000 g av . for 20 min to obtain the postmitochondrial supernatant fractions . Microsomes were sedimented by centrifuging the 10,000 g av supernatants at 105,000 g av . for 60 min . The pellets-were resuspended in fresh homogenising medium and again centrifuged at 105,000 g av . for 60 min to obtain washed microsomal fractions . Enzyme assays The in vitro metabolism of DMN in either 10,000 g av . supernatant or washed microsomal fractions was determined in a medium containing 0 .5 mM NADP, 7 .5 mM DL-isocitric acid, 5 .0 mM MgS04, 7 .5 mM semicarbazide and 0 .05 mg/ml isocitric dehydrogenase in 50 mM Tris-HC1 buffer pH 7 .4 . Formaldehyde was determined in deproteinised tissue incubates by the method of Nash (10) . Methanol was determined by using a Pye series 104 gas chromatograph equipped with a flame ionisation detector and employing a Chromosorb 102 (80-100 mesh) column at 105°C . DMN was similarly measured using a 10~ Carbowax 20 M (on 100-120 mesh acid washed Celite) column at 120oC . The NADPH-dependent metabolism of methanol to formaldehyde was determined using the incubation system as described above for DMN with a methanol substrate concentration of 5 mM . Assays of ethylmorphine N-demethylase (11) and aniline 4-hydroxylase (12) were performed on 10,000 g av . supernatant fractions . Results In vitro stud ies with hepatic subcellular fractions
When DMN was incubated with either 10,000 g av . or washed microsomal fractions the formation of methanol could be detected by g .l .c . analysis of the deproteinised tissue incubates . The methanol produced from the metabolism of DMN had identical retention times to that of authentic methanol on three separate gas chromatograph columns (Table 1) . TABLE 1 The Determination of Methanol by G .L .C . in Rat Plasma Extracts and Deproteinised Tissue incubates Retention time(min) Temperature ethanol formed from Column Packing °C Authentic Methanol DMN Porapak Q 70 6 .92 (4) 6 .98 (4) 11 .87 (7) Porapak S 100 11 .87 (7) 105 3 .67(10) 3 .67(10) Chromosorb 102 Nitrogen carrier gas flow rates were 70 ml/min for the Porapak Q and S columns and 50 ml/min for the Chromosorb 102 column . Retention times are expressed as means, with the number of determinations in parentheses .
Vol . 17, No . 10
Hepatic Metabolism of DMN
1601
Further, the chemical identity of the methanol formed was confirmed by mass spectrometry . When either 10,000 g av .supernatant or washed microsomal fractions were incubatedwith an NADPH-generating system but without the nitrosamine, no methanol was formed (detection limit ~osi~ a .o~~ a
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