Chem.-Biol. interactions, 13 (1976) 237-241 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

TRANSFER OF METHYL GROUPS FROM P:-DIMETHYLNITROSAMINE TO GLYCEROLIPIDS IN RAT LIVER

FRED SNYDER

and BOYD MALQNE

Medical and Health Sciences Division. Oak Ridge Associated Universities, Oak Ridge, Tenn. 37830 (U.S.A.) (Received September 8th, 197 5) (Revision received November 12th. 1975) (Accepted November 14th, 1975)

SUMMARY

We have examined the in vivo labeling of lipids after a single intraperitoneal injection of the carcinogen, [ *%I dimetbylnitrosamine, into rats. Liver was most active in incorporating [14C] methyl groups into lipids (0.91% of the injected dose) and 80% of the activity appeared in sn-3-phosphatidylcholine. Chromatographic analysis of the products (and derivatives) formed after treatment of the [“‘C] phosphatidylcholine with phospholipase A2 (EC 3.1.1.4) and phospholipase C (EC 3.1.4.3) demonstrated that 89% of the radioactivity was in the choline moiety. These results indicate the transfer of methyl groups to lipids occurred via the lipid methylation pathway that converts phosphatidyletbanolamine to phosphatidylcholine.

INTRODUCTION

Although the alkylation of proteins and nucleic acids by nitrosamines is well known [I] , the transfer of the alkyl moieties from carcinogens and other biohazards to membrane constituents such as lipids has not been explored. Takayama and Muramatsu [ 23 found that 50-80% of the radioactivity from [ 3H] dimethylnitrosamine incorporated into the microsomal and mitochondrial fractions of mouse liver could be extracted with lipid solvents; they concluded that the dimethylnitrosamine was readily soluble in lipid-containing membranes, without considering that the 1,abeled methyl groups could have been transferred via the l-carbon pool into covalent linkage with glycerolipids in the membranes. The importance of phosphatidylethanolamine and phosphatidylchollne as membrane constituents [ 31 and the methylation of phosphatidylethanol-

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amine to form phosphatidylcholine via methyl transferases that utilize Sadenosyl methionine [4] is well documented. Therefore, we set up an experiment to test whether methyl groups from IV-dimethylnitrosamine could be used in the lipid methylation pathway and found that indeed the methyl groups of this carcinogen could be incorporated into 3-sn-phosphatidylcholine in rat liver. Although such results might be anticipated, they have never been documented in earlier studies, and moreover, they correct the erroneous assumption that the lipid-soluble radioactivity encountered in experiments with ‘H- and 14(I’J-labeleddimethylnitrosamine is the unmodified labeled carcinogen. MATEItIALS AND METHODS

[Methyl-*4C]-labeled IV-dimethylnitrosamine (4.816 mCi/mmole) was purchased from New England Nuclear. Three male Fischer CDF rats were injected intraperitoneally with 20 PCi of the carcinogen (0.3 mg in 100 ~1 sterile water) and killed 4 h later; water and food were not available during this period. The tissues (brain, lung, heart, liver, kidney, and testes) were removed and homogenized (2 g per 5 ml water) in a glass vessel with four strokes of a Teflon pestle attached to a ‘Lourdes tissue homogenizer operating at a setting of 50 (approx. 7400 rpm). Total lipids were extracted with chloroform-methanol [5] containing 2% glacial acetic acid; aliquots of the purified extract were chromatographed on Silica. Gel HR layers in chloroform-methanol--acetic acid-water (50 : 25 : 8 : 3,, v/v) or on Silica Gel G layers in hexane-dietbyl ether-acetic acid (70 : 30 : 1, v/v). The phesphatidylcholine was isolated from one sample by preparative thin-layer chromatography on the HR layers and then treated with phospholipase AZ (EC 3.1.1.4) [6] or phospholipase C (EC 3.1.4.3) [7] ; products were resolved in the thin-layer chromatography systems described above. We isolated phosphocholine by ion exchange chromatography using t.he method of Weinhold and Rethy [8). The phosphocholine was then hydrolyzed [9] in 6 N HCl for 16 h at llO”, the choline chloride formed was analyzed by thin-layer chromatography in methanol-acetic acid (80 : 20, v/v). The distribution of radioactivity on thin-layer chromatograms was d:etermined by 5-mm zonal analysis [lo] using a Packard liquid scintillation spectrometer (Model 3320). RESULTS AND DISCUSSION

Recovered radioactivity in the total lipids of the major organs analyzed 4 h &afterinjection of [*4C]dimethylnitrosamine was 0.91% for liver, 0.02 !ung, 0.02% for kidney, 0.01% for brain, 0.01% for testes, and 0.00 heart; all percentages are mean values expressed as per cent of the injected radioactivity found in the lipids of the tissues analyzed. Only the labeled lipids isolated from the liver were characterized in detail since the other tissues contained so little 14C. 238

500

400

P 4 u e

300

200

100

4

0

0

16

12

20

24

28

32

ZONE NUMBER

Fig. 1. Distribution of radioactivity after thin-layer chromatography of a total lipid extract from the liver of a rat injected intraperitoneally with [L4C]dimethylnitrosamine. Chromatography was done on Si!iaa Gel HR layers developed in chloroform-methanol-acetic acid-water (50 : 25 : 8 : 3, V/V).Phosphatidylcholine is located between zones B-13; the quantity of 14C between zones 30-32 is associated with the neutral lipid fraction.

I- CY' acy1 -

I

b

-chol ine

phosphatidyichoiine

I

Phospho;

+ RCOOH

acyi-

000%

‘“c)

I

+ P-choline

L-0,

-choi Ine lyso-phosphatldylchoilne

i pase C

Fatty

acid

(0% 14c)

phosph atidylcholine Fig. 2. Analysis of “C-labeled peritoneal administration of [ “Cldimethylnitrosamine

diacylglycerois (Ii%

i4c)

Phosphochoiine

(89% 14c)

isolated from liver after to rats.

the intra-

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The zonal scan depicted in Fig. 1 for the total liver lipids indicates that approx. 80% of the radioactivity was associated with the phosphatidylcholine fraction. To prove that the radioactivity was mainly in the choline moiety, we treated one sample of the phosphatidylcholine (purified by preparative thin-layer chromatography) with phospholipase Al and another with phospholipase C and then analyzed the products formed. These data are summarized in Fig. 2. After phospholipase AZ treatment of the phosphatidylcholine, all of the 14Cwas in the lyso-phosphatidylcholine, whereas after the phospholipase C treatment less than 11% of the radioactivity was associated with the lipids. 92% of the radioactivity in the water phase after phoapholipase C treatment was recovered as phosphocholine on an AG-l.OH--X8, 100-200 me& ion exchange resin [8]. Acid hydrolysis of the phosphocholine removed the phosphate moiety, yielding a radioactive product that had an RF identical with choline chloride on the Silica Gel HR layer developed in methanol-acetic acid (80 : 20, v/v). These resuRs firmly establish that most of the radioactivity (derived from dimethylnitrosamine) in the phosphatidylcholine of rat liver is associated with the choline moiety. Radioactivity from dimethylnitrosamine .Hasnot incorporated into sphingomyelin, another important membrane constituent and the only other cholinecontaining phospholipid in addition to phosphatidylcholine found in liver. Thus the transfer of methyl groups from dimethylnitrosamine to lipids appears to occur only on intact phosphatidylethanolamine. Since methyl groups from dimethylnitrosamine are metabolized to formate [ 11) , it is not surprising that lipids, like proteins and nucleic acids [l] , are methylated. However, this note calls attention to the fact that most of the radioactivity in lipid extracts from livers of animals injected with labeled dimethylnitrosamine is specifically located in 3sn-phosphatidylcholine. ACKNOWLEDGEMENT This work was supported by the Energy Research and Development Administration, the Environmental Protection Agency, and the American Cancer Society (BC-7OF). REFERENCES P.N. Magee and J.M. Barnes, Carcinogenic nitroso compounds, Adv. Cancer Res., 10 (1967) 163. S. Takayama and M. Muramatsu, Incorporation of tritiated dimethylnitrosamine into subcellular fractions of mouse liver after long term administration of dimethylnitrosamine, 2. Krebsforsch., 73 (1969) 172. L.L.M. van Deenen, Some structural and dynamic aspects of lipids in biological membranes, Ann. N.Y. Acad. Sci., 137 (1966) 717. J. Bremer and D.M. Greenberg, Methyl transferring enzyme system of microsomes in the biosynthesis of lecithin (phiosphatidyIcholine), Biochim. Biophys. Acta, 46 (1961) 205. E.G. Bligh and W.J. Dyer, A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol., 37 (1959) 911.

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one, P. 1 lor the

membrane

heim and FSnyder, Urethan-induced pulmonary of surfactant biosynthesis, Cancer Res., 33 (1973)

Bell and P.R. Vagelos, Effect of phospholipase C hydrolysis of phoapholipids on membranous enzymes, J. Biol. Chem., 247 (1972) 2835. tion, and characterization of ocheaistry, 13 (1974) 5135. Modification of glycerolipid ammo-alcohol, N-isopropylethanolmmun., 62 (1976) 963. ion and computer ma1 N-demethylation

and the effect of the into the proteins

a, 19 (1960) 467.

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