The age-related occurrence of wax esters in the mouse preputial gland tumour

The age-related occurrence of wax esters in the mouse preputial gland tumour

157 Biochimica ef Biophysics Acta, 431 (1976) 157-164 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA56753 T...

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157

Biochimica ef Biophysics Acta, 431 (1976) 157-164 @ Elsevier Scientific Publishing Company, Amsterdam

- Printed

in The Netherlands

BBA56753

THE AGE-RELATED OCCURRENCE PREPUTIAL GLAND TUMOUR

OF WAX ESTERS

IN THE MOUSE

M.R. GRIGOR Biochemistry Research Group (Medical Council of New Zealand) and Department chemistry, University of Otago, Dunedin (New Zealand) (Received

October

of Bio-

7th, 1975)

Summary The mouse preputial gland tumour (ESR-586) accumulates wax esters from between 20 and 25 days after transplantation until they become the most abundant lipid class. Prior to this time wax esters are not detectable. The process occurs in both male and female host mice and appears to be determined by a response of the host to the tumour, rather than by a property of the tumour itself. The most abundant fatty alcohol present in the wax esters is hexadecanol. This contrasts with the greater proportions of the CzO to Cz4 chains found for the alkyl portion of the alkyldiacylglycerols, for which the precursors are fatty alcohols.

Introduction The mouse

preputial

gland tumour, ESR-586 [l], (also known as WS-586 for studies in lipid metabolism. Kandutsch and Russell [3--51 have studied the sterols found in this tumour and were able to demonstrate that these were all intermediates of a pathway from lanosterol to cholesterol. Snyder and co-workers [6-91 have used the tumour to study the synthesis of ether-linked lipids. Properties of the enzyme system responsible for the esterification of cholesterol have also been described [lo]. During the course of this latter study it was noted that tumours obtained 7 weeks after transplantation contained considerable quantities of wax esters (Grigor and Snyder, unpublished observations) which contrasted with the traces only of wax esters in tumours obtained 3 weeks after transplantation [6]. In this report the term wax ester is used to describe the simple monoester of a fatty alcohol with a fatty acid. This report describes the changes in lipid composition of the preputial gland tumour during its growth. [ 2 J) has been used extensively

158

Experimental Animals

and tumours

Mice of the C57BL strain were bred locally from mice obtained from Massey University, Palmerston North. Our original mice containing the preputial gland tumour, ESR-586, were made available by Dr. F. Snyder of Oak Ridge Associated Universities, Oak Ridge, Tennessee, who obtained the initial tumour-bearing mice from the Jackson Laboratories, Bar Harbor, Maine. For each transplantation, approx. 1 g of tumour tissue was minced using a razor blade in about 5 ml of saline. Between 0.3 and 0.5 ml of this suspension was injected subcutaneously along the dorsal midline of recipient mice which had been anaesthetised with diethyl ether. This procedure was used instead of the Trocar method and appeared to result in a more even rate of tumour growth between animals. In some experiments the tumour suspension was innoinnoculated subcutaneously above the thigh muscle. This did not appear to alter the rate of tumour growth. To harvest the tumours the host mice were lightly anaesthetised with diethyl ether, decapitated, and the tumours excised. The tumour tissue was homogenised in water using a Silverson Laboratory mixer and a portion of the homogenate was frozen for subsequent deoxyribose determination [ll] while the lipid was extracted from the remainder using the method Bligh and Dyer [ 121. Lipid analysis

Samples of 200-300 mg of the tumour lipid were applied to columns of 15 g Florisil [13] and these were eluted successively with 50-ml portions of hexane; 3, 4.5, 6,10, 15 and 25% diethyl ether all in hexane; and finally with methanol. Fractions of 10 ml were collected and their content was monitored on 250~pm layers of silica gel H developed in hexane/diethyl ether/acetic acid (80 : 20 : 1, by vol.). Fractions containing the same components were pooled and determined gravimetrically. The content of the combined wax plus sterol esters, and alkyl- and alk-lenyldiacylglycerols, and the triacylglycerols were determined by photodensitometry of thin-layer chromatograms of individual tumour extracts. For these analyses layers contained 5% (w/w) (NH,),SO, as an internal charring agent. A standard mixture containing these three fractions was prepared from fractions obtained from Florisil chromatography. To assay the relative amounts of wax and of steryl esters the relative amounts of fatty alcohols and sterols in the non-saponifiable fraction obtained from the combined wax plus sterol ester fraction were determined. Portions of the combined fraction (up to 10 mg) obtained either from Florisil chromatography or by preparative thin-layer chromatography were saponified in sealed tubes containing 0.5 g KOH and 5 ml of methanol at 100°C for 4 h. The non-saponifiable fraction was extracted into hexane/diethyl ether (4 : 1, v/v), evaporated to dryness, silylated with N,O-bis-(trimethylsilyl)-trifluoroacetamide (Regisil, supplied by Regis Chemical Co., Morton Grove, Ill.) and analysed by gas-liquid chromatography. A Pye 104 chromatograph containing a column of 1% (w/w) SE-30 on Gas Chrom Q 100-120 mesh (Applied Science Laboratories, Inc.) was used. The temperature was programmed from 150 to 250°C at 6°C per min.

159

Peaks were identified by cochromatography with known standards (hexadecanol, cholesterol and lanosterol), analyses of samples before and after hydrogenation; and the use of semilogarithmic plots of the retention times determined for isothermal analyses. The retention times of the sterols were also determined relative to 5a-cholestane using isothermal analyses at 250°C. The alkyl- and alk-l-enyldiacylglycerols were heated with 2 ml of 6% HCl in methanol in a selaed tube at 100°C for 30 min. After cooling the products were extracted into hexane and the alkylglycerols were separated from the methyl esters using preparative thin-layer chromatography in the same solvent system as that described previously. Silyl derivatives of the alkylglycerols were prepared and analysed using gas-liquid chromatography. The same column was used as for the sterols except the temperature range was altered to MO-280°C. Standard selachyl alcohol (1-octadecenylglycerol) was also silylated and chromatographed. The chromatograms were quantitated by determining peak areas after triangulation. The mass response ratio of cholesterol/hexadecenol was 1.12. The standards were purchased from Applied Science Laboratories Inc., State College, Pa. or Analabs Inc., North Haven. Conn. Statistics. Linear regression lines were fitted to all the data obtained for tumour growth and tumour lipid contents to test for significant changes with age of the tumour. Results The rate of tumour growth in both male and female mice is shown in Fig. 1. After a lag period there was rapid growth and in some cases tumours of more

8

100

1”

Fig. 1. Growth and lipid content of preputial gland turnours. Growth was determined as tumour mass. g wet weight (A), or tumour DNA measured after acid hydrolysis as mg deoxyribose (B). The content of DNAltumour mass, mg deoxyriboselg wet weight is shown in C and D and E show the lipid content, respectively. as mg lipid/g wet weight and mg lipid/mg deoxyribose. l, tumours grown in male mice, and 0, tumours grown in female mice.

160 TABLE

I

LIPID CLASSES

OF MOUSE

PREPUTIAL

GLAND

TUMOUR

Lipid classes were determined gravimetrically after separation by chromatography using Florisil sorbant. For each assay a pooled sample from at least four tumours grown in male mice was used. __ _~__ -_--~ Lipid content

(me/g

as ab-

Age to tumour

wet weight)

22-27 35-59

days (percent

37-45 days 44-97 recovered as)

Wax and steryl esters Alkyldiacylglycerols * Triacylglycerols Free fatty acids and diacylglycerols Sterols ** Phospholipids

16.4 6.7 21.7 4.6 9.8 35.7

38.2 8.6 12.7 6.0 8.5 24.0

Total recovered

94.9

98.0

* Contains as well alk-lenyl-diacylglycerols. ** Only cholesterol detectable by gas-liquid chromatography.

than 9 g wet weight were obtained. The host mice were likely to die any time after 35-40 days. DNA content measured as deoxyribose after acid hydrolysis was also determined as an index of tumour growth. The ratio of deoxyribose to tumour mass decreased significantly with age (P< 0.001) consistent with the presence of larger cells in the older tumours. The lipid content measured as either mg lipid/g wet weight tumour or mg lipid/mg deoxyribose increased with the age of the tumour. In each case the trend was highly significant. No statistically significant difference could be detected in either the growth rate or lipid content of tumours grown in male or female mice. The composition of pooled lipid samples from tumours of different ages is shown in Table I. The most marked difference in these analyses was the increased amount of the combined wax plus sterol ester fraction in the older tumours. These were eluted as a single fraction from the Florisil columns. When A 40

0

0

C

Fig. 2. Content of wax and steryl esters, triacylglycerols and alkyldiacylglycerols in preputial gland turnours. A. Band C show the content of the wax and steryl esters, the triacylglycerols and the alkyldiacylglycerols, respectively. as mg/mg deoxyribose in individual preputial gland tumours of different ages.

161

the content of this fraction was assayed in lipid from individual tumours, this increase was particularly marked. Using either the deoxyribose content of the tumour (Fig. 2) or the tumour mass as a base the accumulation of this lipid fraction with age was statistically very significant (P < 0.001). Similarly significant increase in the content of the alkyldiacylglycerols with tumour age were also observed. A small increase in the amount of triacylglycerol per mg deoxyribose was noted (Fig. 2; 0.05 > P > 0.01) but no change in the triacylglycerol content per g tumour could be detected. The non-saponifiable portion from the combined wax plus sterol ester fraction was then analysed using gas chromatography. In the material from older tumours two groups of compounds were readily detected, a low molecular weight group made up of fatty alcohols, and a higher molecular weight group made up of sterols. The proportion of fatty alcohols was considerably lower in younger tumours being barely detectable in material from tumours obtained IO or 14 days after transplantation. Hexadecanol was the major alcohol accounting for 35-4076 of the total fatty alcohols (Table II), while cholesterol was the major sterol making up 50% of the total sterols in the sterol ester fraction (Table III). TABLE IX DISTRIBUTION OF ALKYL CHAINS OF FATTY ALCOHOLS DIACYLGLYCEROLS FROM PREPUTIAL GLAND TUMOURS

FROM WAX ESTERS

AND ALKYL

Analyses were performed of trimethylsilyl derivatives of fatty alcohols and alkylglycerols obtained from a pooled lipid extract from several turnours. Results are expressed as percent of total. Fatty alcohols

14:1* 14:o Unidentified * * 15: Obr 15:o 16:1+16:Obr 16 : 0 17 : Obr 17 : 0 18:1+18:Obr 19 : 0 19 : Obr 19 : 0 20:1+20:Obr 20:o 21: Obr 21 : 0 22:1+22:0 23 : Obr 23 : 0 24:o 25:Obr+25:0 26:0

1.3 5.5 6.1

0.9 6.8 2.2 34.7 2.5 4.4 1.3 10.6

1.9

-

0.7 1.7 3.4 1.6 3.8 1.8 0.9 4.6 0.9 1.6

AIkyl chains of alkyidiacylglycerols

-

I.8 4.6

3.2 2.2 9.8 3.2 4.9 3.4 12.0 0.8 3.8 3.2 9.4 3.9 2.0 14.5 2.8 3.1 10.2 0.8 0.3

* Chain length: number of double bonds, br, branched chain {these peaks were not removed by hydrogenation). ** Carbon number when anaIysed isothermally using SE-30 column 14.35 (branched chain aicohofs had carbon number n -0.35).

162

TABLE

III

COMPOSITION

OF

STEROLS

FROM

STERYL

ESTER

FRACTIONS

OF

PREPUTIAL

GLAND

TUMOURS Retention sterol

time

(Applied

relative Science

to Labs)

5a-cholestane consisted

Retention

determined of a 3

time

:

5

:2

in isothermal mixture

Percent

of total

Cholesterol

1.94

50.6

f 15.0

A

2.18

19.7

i

B

2.46

C

2.65

D

2.85 * Mean

? S.D.

of 10

estimations

6.6

from

paired

analyses

of sterols

sterols

at 25O’C.

B. C and

*

Commercial

lano-

D.

-

8.6

+

5.9

14.9

2

8.2

1.7

i

5.2

tumours.

In all 28 tumours paired by age were thus examined. The fatty alcohols were absent or barely detectable in tumours obtained up to 20 days after transplantation, but increased in content from around 25 days until by 40 days they equaled or exceeded the sterols. These results are consistent with these tumours accumulating wax esters after a lag period of 20-25 days, so that at 40 days or more they became one of the major lipid components of the tumour. In contrast to the distribution of the chain lengths of the fatty alcohols found in the wax esters, the distribution of the alkyl chains found in the alkyldiacylglycerols showed the presence of considerable quantities of longer chains (Table II). The distribution of the chain lengths of the fatty alcohols, the alkyl chains, and the distribution of the sterols all appeared to be independent of the age of the tumour. In a series of experiments two tumours were transplanted into the same mice at intervals of around 14 days. 21 days later the hosts were killed and the tumours were obtained. Thus for each mouse there was a tumour of approx. 35 days and one of 21 days. The ratio of the fatty alcohols to sterols was then determined in the combined wax and sterol ester fraction from these tumours. For each mouse the values obtained were similar for both turnours, but lower than that expected for a single 35-day tumour. Discussion The change in lipid composition with growth reported here for the mouse preputial gland tumour appears to be unique. No similar observations appear to have been made during studies of other tumours [14]. With the initiation of wax ester synthesis and the accumulation of this lipid class the lipid composition of the tumour tends more to that of the normal preputial gland from which the tumour developed. Wax esters make up almost half the lipid of the preputial gland [ 151. Although the gland contains only small amounts of sterol esters (less than 10% of the total lipid) these appear to contain the same sterols as the tumour. The lipid content of the excised glands is very high amounting to several times that observed for the most lipid-rich tumours (Grigor, unpublished results). The pattern of sterols observed here is similar to those observed for the seba-

163

ceous lipid of many species [ 161. Although positive identification of many of these was not possible, it is probable that they represent the intermediates of the ‘Kandutsch-Russell’ pathway for cholesterol synthesis [3-51. As with the sebaceous lipid the free sterols have a much simpler pattern and in the tumour only cholesterol could be detected (Table I). As well as being incorporated into the wax esters, fatty alcohols are also precursors of the alkyl and alk-1-enyl chains of the ether-linked lipids [ 171. The dissimilarity in the distribution of chain lengths observed for the fatty alcohols from the wax esters and the alkyl chains from the alkyldiacylglycerols suggests that there might be different pools of fatty alcohols present in the tumour. This is consistent with the further observation that although the content of the alkyldiacylglycerols increased with the tumour age, measurable amounts of this lipid class were present in younger tumours in which wax esters could not be detected. In the tumour fatty alcohols are formed by the reduction of fatty acyl-CoA’s by a NADPH-linked enzyme [7] and the wax esters are formed by a microsomal acyltransferase where a fatty acyl-CoA is the acyl donor [ 181. There is now considerable evidence that the activity of both the sebaceous gland and the preputial gland in rodents is controlled by hormones. In particular these activities are stimulated by androgens and depressed by oestrogens [19-211. For this reason the parameters of tumour growth and tumour lipid production have been presented with respect to the sex of the host mice. In no cases were there any detectable sex-related differences. The experiments in which mice were innoculated with two tumours were designed to test whether the change in lipid composition observed for the tumour was determined by the tumour itself or by the host. If the tumour was the determining factor, the 35-day tumours would have had considerable amounts of wax esters while the 21-day tumours would have contained only trace amounts. However, if the host determined the change in composition then both tumours would have contained similar amounts of wax esters. That the latter was observed suggests that the change in lipid composition was determined by the host. The wax esters accumulate in the tumours during the period in which the host animals are likely to die, and the destruction of host tissues might lead to an altered supply of precursors to the tumour. Acknowledgements I wish to thank Dr. F. Snyder for donating the tumour bearing mice, Dr. C.M. Goodall for helpful discussions, and Mr. D.W. Mackie for technical assistance. References 1 Green. E.L. (1968) Handbook on Genetically Standardized JAX mice, p. 62, The Jackson Laboratory Bar Harbor 2 Fekete. E. and Kent, E.L. (1955) Transplant. Bull. 2, 61-62 3 Kandutsch. A.A. and Russell, A.E. (1959) J. Biol. Chem. 234, 2037-2042 4 Kandutsch, A.A. and Russell, A.E. (1960) J. Biol. Chem. 235.2253-2255 5 Kandutsch. A.A. and Russell. A.E. (1960) J. Biol. Chem. 235, 2256-2261 6 Snyder. F., Malone, B. and Blank, M.L. (1970) J. Biol. Chem. 245,1790-1799 7 Snyder, F. and Malone. B. (1970) Biochem. Biophys. Res. Commun. 41.1382-1397

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