effect of a zinc-deficient diet on mitochondrial and microsomal lipid composition in tepc-183...
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BIOCHEMICAL MEDICINE AND METABOLIC BIOLOGY 37, 148-156 (1987)
Effect of a Zinc-Deficient Diet on Mitochondrial and Microsomal Lipid Composition in TEPC-183 Plasmacytoma
JAMES P. BURKE,’ KENNETH OWENS, AND MARILYN R. FENTON
Department of Physiological Sciences, Pennsylvania College of Podiatric Medicine, Eighth at Race Street, Philadelphia, Pennsylvania 19107
Received September 30, 1985
Studies with animals have suggested a relationship between zinc status and lipid metabolism. Zinc deficiency in male rats resulted in a marked increase in triglycerides and a smaller increase in serum phospholipid and cholesterol (1). On the other hand, it has been reported that total cholesterol is reduced in rats fed a zinc-deficient diet for 4 weeks (2,3), with the decrease primarily due to a drop in HDL-cholesterol. Such discrepancies may be due to the type of dietary regimens employed. Studies examining both zinc and essential fatty acid deficiency have concluded that there is a physiological interaction between the two, in terms of both growth rate and dermal lesions (4,5). Also, Clejan and co-workers having found increases in the cholesterol to phospholipid ratio in small intestine and liver microsomes from rats fed a zinc-deficient diet postulated that zinc deficiency alters the lipid composition of cellular membranes (6).
Tumor growth also alters lipid metabolism in the host. Plasma free fatty acids increase in tumor-bearing animals (7,8) and hyperlipidemia is seen in CBA mice bearing Ehrlich ascites carcinoma (9). Concerning the tumor cell, malignant transformation has been shown to lead to an alteration in the various lipid components of cellular membranes, such as the cholesterol to phospholipid ratio, phospholipid composition as well as enzymes involved in phospholipid metabolism (10).
We are interested in the relationship of zinc to both lipid metabolism and tumor growth for several reasons. First, we have demonstrated that the growth of the plasmacytoma TEPC-183 in BALB/c mice is inhibited by a zinc-deficient diet (11). Also, data from our laboratory indicate that a zinc-deficient diet causes an increase in the rate of lipid peroxidation in vitro in mitochondrial and microsomal membrane fractions (12). In addition, zinc deficiency combined with tumor resulted in alterations in lipid metabolism in the tumor-bearing host such as lower liver lipid levels and decreased serum phospholipid, triglycerides, and free fatty acids (13). These results have prompted us to determine if a zinc-deficient diet alters
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148 0885-4505/87 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
ZINC DEFICIENCY AND LIPID METABOLISM 149
TABLE 1 Composition of Diets
Ingredients
Egg white solids, spray-dried 200.0 Dextrose, monohydrate 634.3058 Corn oil 100.00 Nonnutritive fiber (cellulose) 30.0 Calcium phosphate 19.767 Sodium chloride 0.7781 Magnesium sulfate 2.4752 Manganese sulfate 0.1662 Ferrous sulfate 0.2 Potassium iodate 0.004 Cupric sulfate 0.0151 Vitamin mix, Teklad 10.0 Biotin 0.004
Note. Diets were prepared by Teklad (Madison, WI). Zinc carbonate was added to yield a final concentration of 100 ppm.
a Content of mix (g/kg): p-aminobenzoic acid, 11.0132; ascorbic acid, 101.6604; biotin, 0.0441;
. vttamm Blz, 2.9736; calcium pantothenate, 6.6079; choline dihydrogen citrate, 339.6916; folic acid; 0.1982; inositol, 11.0132; menadione, 4.9559; niacin, 9.119; pyridoxine HCl, 2.2026; riboflavin, 2.2026; thiamine HCI, 2.2026; dry vitamin A palmitate, (500,008 U/g); dry vitamin D,, (500,OO U/g); dry vitamin E acetate (500 U/g); corn starch, 466.6878.
either cholesterol or phospholipid levels as well as the fatty acid composition of microsomal and mitochondrial membranes in TEPC-183 tumors.
MATERIALS AND METHODS
Female BALB/c mice (15-18 g), obtained from Dominion Labs, McLean, Virginia, were divided into three groups. One group was fed a zinc-deficient (ZD) diet, a synthetic egg white solid diet prepared according to the procedure of Luecke (14) and purchased from Teklad, Madison, Wisconsin. The composition of the diet is outlined in Table 1. An ad libitum group (AL) was fed a similar synthetic diet with the exception that zinc carbonate was added to the basal diet so that the zinc level was 100 ppm. Pair-fed animals (PF) were given the control diet in an amount equal to that of a corresponding ZD animal. After 3 weeks on the test, animals from all three groups were injected with 0.25 ml of a TEPC- 183 cell suspension (0.5 x 10’ cells/ml) subcutaneously. When the tumors reached approximately 1 cm in size, the mice were sacrificed, tumors were excised, and membrane fractions were prepared. The tumor TEPC-183 is an IgM (k)-secreting plasmacytoma obtained from Dr. Frances Havas of the Temple University School of Medicine. It is maintained by subcutaneous transfer into BALB/c mice fed normal chow (Ralston Purine, St. Louis, MO).
150 BURKE, OWENS, AND FENTON
TABLE 2 Phospholipid and Cholesterol Content of Tumor Microsomes
Group Phospholipid Cholesterol
(nmole/mg protein) (nmole/mg protein)
Cholesterol/ phospholipid (molar ratio)
ad libitum 224 + 66 71 2 28 Pair-Fed 292 2 59 62 2 11 Zinc-deficient 337 2 75 35 f 17
(P < 0.05) (P < 0.05y
Note. Values represent means k SD of five preparations. * Indicates a significant difference compared to ad libitum controls. * Indicates a significant difference compared to pair-fed controls.
0.31 f 0.07 0.22 f 0.01 0.13 + 0.04 (P < 0.05p
Tumor mitochondrial and microsomal membrane fractions were prepared ac- cording to the procedure of Koch (15). The purity of each fraction was assessed by the appropriate enzyme markers, succinate dehydrogenase (mitochondrial), and inosine diphophatase (microsomal). Isolated membrane fractions were subjected to lipid extraction according to the procedure of Folch et al. (16). The lipid extracts were analyzed for cholesterol by the procedure of Courchain et al. (17). Phospholipid levels were assessed by measuring lipid phosphorus levels according to the procedure of Bartlett (18). Analysis of phospholipid classes was conducted by utilization of a two-dimensional thin layer chromatography system using the procedure of Turner and Rouser (19). The resulting spots were identified by spraying with sulfuric acid and comparing them with the appropriate standards. The concentration of each phospholipid class was determined by measuring lipid phosphorous. Fatty acid and fatty aldehyde composition of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was determined as described by Owens et al. (20) using the BF,-methanol transesterification procedure of Morrison and Smith (21). The resulting methyl esters and dimethylacetals derived from plas- malogens were separated by gas-liquid chromatography using a Carlo Erba gas chromatograph with a 30-m capillary column packed with DB225 (J and W Scientific). Peak areas were quantitated and identified by comparison of retention times to those of standards obtained from Supelco, Inc., using a Spectra Physics SP4270 integrator.
Protein was determined by the method of Lowry et al. (22). Statistical analysis was performed on data using the Student t test.
RESULTS
Animals maintained on a zinc-deficient diet displayed altered fur texture and decreased liver/body weight ratios when compared to either PF or AL controls (data not shown). The results of the cholesterol and phospholipid analysis for microsomal membrane fractions are depicted in Table 2. A ZD diet led to or resulted in an increase in phospholipid levels when compared to AL controls. An increase was observed when compared to PF controls, but the difference was not statistically significant. On the other hand, cholesterol levels were found
ZINC DEFICIENCY AND LIPID METABOLISM 151
to decrease 50% in ZD animals. Also of interest is the fact that a sharp decline was observed in the cholesterol to phospholipid ratio. Such a large alteration of cholesterol concentration will change the lipid domain of the membrane and tend to make it more fluid. Although a slight decrease in cholesterol and an increase in phospholipid was observed in mitochondrial fractions from ZD tumors, the differences were not statistically significant (data not shown).
Since differences were observed in phospholipid levels, we were interested in determining if the relative distribution of phospholipid classes was altered in mitochondrial and microsomal membranes. In microsomal membranes, a slight decrease was observed in the phosphatidyl inositol-serine fraction from ZD animals as well as in sphingomyelin (data not shown). In mitochondrial fractions, a slight elevation was observed in the phosphatidylethanolamine fraction from ZD samples. These changes were not statisically signficant, however, and point to the conclusion that ZD diet has minimal effect on the relative distribution of phospholipid classes, in either mitochondrial or microsomal membranes.
Since reports in the literature indicate that a zinc-deficient diet alters the fatty acid composition of certain tissues (1,4,5,23,24), we analyzed the fatty acyl composition of the two predominant phospholipids, PC and PE. In the fatty acid fraction derived from mitochondrial membranes (Table 3), an elevation in the level of 18:2 was observed in ZD, PC samples compared to either AL or PF controls. A similar rise was observed in 18:2 from PE, but this was not statistically significant. However, the increase in 22:6 in this fraction from ZD mice was significant. A slight rise in PC 22:6 was also observed in ZD membranes. Microsomal PC (see Table 4) revealed a similar increase in 18:2 and 22:6 from ZD fractions and a corresponding decrease in 20:4. A decrease was also observed in PE 22:5 but this was not statistically significant. None of the ZD preparations differed significantly from either the AL or PF controls in the content of long-chain alk- I-enyl ethers. Thus although the relative distribution of phospholipid is unaltered in the ZD state, the fatty acyl composition of these fractions undergoes modifications.
DISCUSSION
The results of this study point to an effect of a zinc-deficient diet on lipid metabolism in several tumor subcellular membrane fractions. Previously, we have demonstrated that a zinc-deficient diet alters the growth of the plasmacytoma TEPC-183 in BALB/c mice (11). The incorporation of [,H]thymidine into DNA in tumor cells from ZD mice was markedly reduced. In addition, we have also found that tumor mitochondrial from ZD animals had decreased state 3 respiration and levels of cytochromes a and a3 and b compared to either PF or AL controls (25). Finally, plasmacytoma cells from ZD animals incubated with XH- or ,&- labeled leucine had decreased rates of protein secretion (26). Thus, it seems that a ZD diet has not only functional but structural effects on tumor subcellular membranes.
Although the effect of a ZD diet on the fatty acid composition of various tissues has been investigated (1,4-6,23), very few studies have examined the effect of zinc on membrane fatty acids. This point is important since a recent
TABL
E 3
Fatty
Acid
C
ompo
sitio
n of
Pho
spha
tidylc
holin
e an
d Ph
osph
atid
yleth
anol
amin
e Ph
osph
olip
ids
of T
umor
M
itoch
ondr
ial
Mem
bran
es
Fatty
ac
id c
omno
sitio
n (m
ole
%)
Phos
phat
idylc
holin
e (P
C)
Fatty
acid
ad
lib
itum
Pa
ir-fe
d Zi
nc-d
efic
ient
Phos
phat
idyle
than
olam
ine
(PE)
ad l
ibitu
m
16:0
27
.9
k 2.
1 29
.2
” 2.
6 27
.9
f 1.
3 18
:0
13.0
2
0.8
13.7
k
0.2
13.8
k
0.2
18:l
13.8
k
1.5
14.1
2
1.3
13.9
2
0.1
18:2
13
.2
k 1.
3 14
.6
-c 1
.4
16.4
f
0.7
(P
< o.
oF$
2012
1.
6 f
0.2
1.9
f 0.
3 1.
8 +
0.3
20:4
15
.2
2 1.
8 13
.4
f 3.
4 13
.6
+ 1.
1 22
:5
7.3
f 0.
4 6.
2 f
0.5
6.2
2 0.
2 22
16
2.4
k 1.
3 2.
1 f
0.7
2.8
k 0.
5
’ D
ata
expr
esse
d as
mol
e %
and
re
pres
ent
the
mea
ns
2 SD
of
five
prep
arat
ions
. *
Indi
cate
s a
sign
ifica
nt
diffe
renc
e co
mpa
red
to a
d lib
itum
an
d pa
ir-fe
d co
ntro
ls.
Pair-
fed
8.7
+ 1.
1 16
.2 +
1.
6 7.
6 f
2.8
3.7
* 0.
4
0.8
2 0.
1 15
.2 k
1.
6 17
.5 k
2.
2 7.
4 +
0.8
6.8
2 0.
6 16
.6
+ 1.
1 7.
5 2
0.4
4.1
* 0.
2
0.7
2 0.
1 17
.0
‘- 0.
8 17
.8
2 0.
2 8.
1 k
1.4
Zinc
-def
icie
nt
6.2
+ 0.
5 17
.2 +
2.
1 7.
1 +
0.0
4.7
k 1.
3
TABL
E 4
Fatty
Acid
C
ompo
sitio
n of
Pho
spha
tidylc
holin
e an
d Ph
osph
atid
yleth
anol
amin
e Ph
osph
olip
ids
of T
umor
M
icros
omal
M
embr
anes
Fatty
acid
co
mpo
sitio
n (m
ole
%)
Phos
phat
idylc
holin
e (P
C)
Phos
phat
idyle
than
olam
ine
(PE)
Fatty
acid
ad
lib
itum
16:0
29
.2
k 2.
5 18
:0
13.3
2
0.1
18:l
13.5
+
0.5
18:2
13
.8 t
0.
4
2012
1.
9 k
0.4
20:4
15
.4 +
0.
4
22~5
6.
7 2
0.9
22:6
2.
5 k
0.8
Pair-
fed
33.6
2
4.4
14.3
2
1.3
15.0
k
2.3
13.8
2
1.1
1.7
” 0.
2 12
.3
k 2.
6
4.9
T 1.
8 1.
9 2
0.3
Zinc
-def
icie
nt
29.7
f
1.8
14.2
2
1.3
15.9
2
1.2
15.9
*
1.2
(P <
0.
02)b
.C
1.7
? 0.
1 12
.9 2
0.
2 (P
<
0.02
)b
6.3
k 0.
5 3.
2 2
0.5
(P
< 0.
02)
ad l
ibitu
m
Pair-
fed
Zinc
-def
icie
nt
5.6
2 0.
9 11
.7
T 0.
6 6.
9 k
0.6
3.3
2 0.
3
7.6
+ 2.
5 7.
6 2
3.5
15.0
+
5.3
11.6
f
1.0
7.5
? 1.
2 6.
8 Ifr
0.4
4.
0 r
1.2
3.8
f 0.
5
0.9
2 0.
0 15
.2 f
0.
2 1.
2 -c
0.3
1.
0 2
0.0
13.5
2
3.4
14.7
f
0.4
20.4
2
0.9
18.8
+
4.7
12.7
f
7.6
6.3
2 1.
3 5.
1 f
1.9
4.5
k 2.
6
’ D
ata
expr
esse
d as
mol
e %
and
re
pres
ent
the
mea
ns
k SD
of
five
prep
arat
ions
. ’
Indi
cate
s a
sign
ifica
nt
diffe
renc
e co
mpa
red
to a
d lib
itum
co
ntro
ls.
’ In
dica
tes
a si
gnifi
cant
di
ffere
nce
com
pare
d to
pai
r-fed
co
ntro
ls.
E
154 BURKE, OWENS, AND FENTON
paper by Cunnane er al. (24) suggests that the effects of zinc deficiency are different when one compares the fatty acid composition of triglycerides versus phospholipid. Also, only one study has examined the relationship between zinc, cholesterol, and phospholipid in subcellular membranes. Clejan et al. (6), working with rats, found a slight decrease in microsomal phospholipid and no change in cholesterol in liver tissue isolated from ZD animals. Fatty acid analysis revealed no significant change in liver but changes in testes microsomes, particularly in the 225 fatty acid fraction. It must be pointed out, however, that this study analyzed fatty acids from total lipid extracts of microsomal membranes, not from a particular phospholipid class. With the other exception being a detailed analysis of brain lipids (27) and the effect of zinc deficiency and/or essential fatty acid deficiency on composition, no detailed studies have been conducted on the lipid composition of subcellular membranes.
Our studies reveal that a zinc-deficient diet causes an increase in phospholipid levels in microsomal membranes as well as a corresponding decrease in cholesterol levels. Thus, the cholesterol to phospholipid ratio is altered, affecting membrane fluidity. This altered cholesterol to phospholipid ratio could significantly modify the thermotropic properties of cellular membranes. On the basis of experiments with cholesterol:phospholipid mixed vesicles it has been concluded that altering the cholesterol content will induce a change in structural hydrodynamic (28) and thermotropic properties of the vesicles (29). Rates of permeation (30) and enzyme activities (31) are found to change with altered cholesterol/phospholipid ratios. The precise functional effects of this altered ratio on the microsomal membrane functions in TEPC-183 is unknown, although this may have an influence on some of the previously described data.
Whether the decreased cholesterol content of the microsomal membrane fraction is due to decreased synthesis of cholesterol, decreased uptake from the circulation or decreased transport within the tumor cell is unknown at the present time. We have found that total serum cholesterol does not differ significantly among PF, AL, or ZD animals which have been injected with TEPC-183 (13). In addition, preliminary experiments suggest that the activity of one key rate limiting enzyme in cholesterol biosynthesis, HMG CoA reductase, is inhibited 30% in tumor microsomes isolated from ZD animals (Burke, Owens, Fenton, unpublished ob- servations). Since studies conducted have implicated a lowering of cholesterol levels with a slowing of tumor growth (32), we are at present examining the effect of a ZD diet on several aspects of cholesterol metabolism.
The changes in fatty acid composition reported here agree in part with the results of Cunnane ef al. (23), that is, a ZD diet causes an elevation in the levels of the 18:2 fatty acid fraction. Evaluation of the observed differences in our study are difficult to compare with other groups due to the fact that our analysis was conducted on isolated membranes. That the elevation in levels of 18:2 in several phospholipid fractions may be due to decreased activity of one or more of the desaturase enzymes (23) is presently under investigation.
The possibility that the altered lipid composition in TEPC-183 subcellular membranes reported here may account for the decreased rate of tumor growth observed in ZD animals is open to question. Bartoli and Galeotti (33) found that
ZINC DEFICIENCY AND LIPID METABOLISM 155
microsomes and mitochondria from Morris hepatoma and Ehrlich ascites tumor cells exhibit lower rates of lipid peroxidation than corresponding fractions of rat liver. Interestingly, the peroxidative capacity appeared to be inversely proportional to the growth rate of the tumor, with slower growing tumors having a high rate of lipid peroxidation. We have demonstrated that the rate of lipid peroxidation in vitro is increased 6 to lO-fold in tumor mitochondrial and microsomal membrane fractions from ZD animals (11). In addition, Reitz et al. (34) have reported that certain hepatomas have alterations in phospholipid levels compared to normal livers, primarily an increase in polyenoic fatty acids. Hartz et al. (35) found differences in fatty acyl composition between hepatomas of varying growth rate. The faster the growth rate, the larger the sum of the contents of 16:1, 18: 1, and 18:2 and the smaller the sum of the contents of all polyunsaturated fatty acids with four or more double bonds per molecule. Thus, it is apparent that the growth rate of tumors may be influenced by the lipid composition of various cellular membranes and that zinc deficiency alters lipid metabolism in the tumor cell. Further work is required to assess the role of this trace element in membrane structure and function. In addition, the effect of the alteration in choles- terol/phospholipid ratio on the physical-chemical properties of membranes may effect transport mechanisms which may modify the potential effectiveness of certain chemotherapeutic agents.
SUMMARY
A zinc-deficient diet caused an increase in microsomal membrane phospholipid levels compared to ad libitum controls. Cholesterol levels were found to be decreased 50% compared to either pair-fed or ad libitum controls, resulting in a sharp decline in the cholesterol/phospholipid ratio. No differences were observed in the distribution of phospholipid classes among all three groups, either in mitochondrial or microsomal membrane fractions. Fatty acid analysis of PC and PE revealed a rise in the 18:2 fraction from zinc-deficient mitochondrial and microsomal membrane fractions. Mitochondrial PE and PC from zinc-deficient animals revealed a rise in the 22:6 fatty acid fraction while microsomal PC also revealed a corresponding decrease in 20:4. None of the zinc-deficient preparations differed significantly from either ad libitum or pair-fed controls in the content of long-chain alk-1-enyl ethers. The results of this study point to an effect of a zinc-deficient diet on lipid metabolism in tumor subcellular membranes which may account for the decreased rate of tumor growth observed in zinc-deficient animals.
ACKNOWLEDGMENTS
The authors thank Ms. Beth Bakley for excellent technical assistance and Ms. Lenora Duppins for skillful typing of the manuscript. This investigation was supported by Grant CA32256 awarded by the National Cancer Institute, Department of Health and Human Services.
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