EXPERIMENTAL AND MOLECULAR PATHOLOGY 34, 191-201 (1981)

Mosaicism in Female Hybrid Hares Heterozygous for Glucose-6-Phosphate Dehydrogenase (G-6-PD)’ I. General Properties of a Hybrid Hare Model with Special

Reference to Atherogenesis

K. T. LEE, W. A. THOMAS, K. JANAKIDEVI, M. KROMS, J. M. REINER, AND K. Y. BORG*

Departments of Pathology and Biochemistry, Neil Hellman Medical Research Building, Albany Medical College, Albany, New York 12208, and *National Veterinary Institute, Vppsala, Sweden

Received July 30, 1980, and in revised form October 3, 1980

Hybrid hares (Lepus europaeus male x Lepus timidus female) were fed intermittently with a cholesterol-containing diet for 3 to 13 months. Their aortas were evaluated for extent of lesions, and for glucose-dphosphate dehydrogenase (GAPD) isozyme patterns in lesions and in normal-appearing aortic media. The red cells of all animals were assayed above 50% for T (the slower-moving form of the enzyme, corresponding to type B in black women, and contributed in the cross by the L. timidus dam). The lesions and normal aortic tissues showed a similar bias toward type T, although values fell slightly below 50% in a few cases. The average percentage of T in lesions and in normal tissues from the same aorta differed significantly in some of the animals, suggesting that cells with one type of active X chromo- somes might have some selective advantage over the other in the abnormal environment of the atherosclerotic lesion. The putative selective advantage would have to be related to genes on the X chromosome other than that for G-6-PD, since deviations toward T-type G-CPD were found in some hares and toward E in others. One hare was fed for 6 months with 25-hydroxycholesterol as well as cholesterol. In this animal (and only in this one) monotypic samples were obtained from lesions, even more frequently from grossly normal regions of the aorta, and from uterus and liver. The monotypes were 100% E in liver, but 100% T in normal aorta, aortic lesions, and uterus. Skin, heart, kidney, spleen, and lung samples were all ditypic. In addition to developing aortic lesions readily on a cholesterol diet, these hybrids show marked stenotic lesions in the extramyocardial coronaries, and are therefore tentatively considered a promising model for the study of atherogenesis.

INTRODUCTION

According to Lyon’s hypothesis, one or the other of the X chromosomes in females is randomly inactivated probably early in embryonic life. If the individual is heterozygous for any gene on the X chromosome, the body will henceforth contain a mixture of two types of cells whose genotype and phenotype depend on which X chromosome was inactivated (Lyon, 1962).

The gene on the X chromosome which has been studied most extensively in this connection is that for G-6-PD. ?n this country, about one-third of the black women are G-6-PD mosaics. Proliferative lesions, neoplastic or otherwise, developing in these women sometimes consist of a single cell type and hence are monotypic. In some neoplasias, e.g., leiomyomas, this has been interpreted as indicating origin from a single cell, i.e., monoclonal origin.

Recently Benditt and Benditt (1973) and Pearson et al. (1975, 197ga, b) have shown that many advanced atherosclerotic lesions in black women who are G-6-PD heterozygotes, are monotypic, or at least have monotypic foci. They concluded that this probably means that atherosclerotic lesions are monoclonal in

I Supported by PHS Grant HL 20993.

191 0014-4800/81/020191-11$02.00/0 Copyright @ 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.

192 LEE ET AL.

origin. We have studied similar atherosclerotic lesions with monotypic foci and have concluded, from similar data to those of the Benditt and Benditt and Pearson et al., that the monotypism in this particular situation probably has causes other than monoclonism and hence does not indicate monoclonal origin of the lesions (Thomas et al., 1979).

If a suitable experimental animal were available, many of the mysteries sur- rounding monotypism in atherosclerotic lesions might be clarified. In 1965 Ohno et al. reported that a cross between two species of hare (Lepus europaeus and Lepus timidus) resulted in G-6PD mosaicism in female hybrid hares. Rabbits develop a form of atherosclerosis readily when fed a high cholesterol diet, which suggests that hybrid hares might also be susceptible.

This potential hare model had not been extensively studied either from the standpoint of G-6-PD mosaicism or susceptibility to cholesterol-diet-induced ath- erosclerosis. The goal of the current study was to obtain such hares and to begin to characterize them with particular reference to their potential usefulness in the study of atherosclerosis.

We have to date studied 52 females and 55 males considered to be progeny of L. timidus dams and L. europueus sires. We shall present the G-6-PD patterns in the blood of all of these, in the aortas of most, and in other tissues of some. We shall also present the results of feeding cholesterol alone or with one of its oxidation products to these animals for up to 13 months.

MATERIALS AND METHODS

The hybrid hares used in the current study were obtained by crossing L. europueus sires with L. timidus dams, Ohno et al. (1965) had shown that the cross could be made either way, and our choice was simply a matter of convenience. After several unsuccessful attempts to breed the hares in Albany, we contracted with a professional breeder of captive wild animals in the countryside north of Stockholm, Sweden, to breed them for us. It apparently takes considerable care and skill to get the two species to interbreed.

All hybrids that survive infancy are shipped to Albany, New York by plane. Of the total of 132 hares received, 25 died soon after arrival due to excessive stress and injury sustained during the prolonged land and air travel from the Northern part of Sweden to Albany. Most have been housed in outdoor group pens; but some of the males have been housed singly in standard rabbit cages. Thus far these animals born in captivity have adapted readily to this environment.

Blood was drawn from all hybrid hares shortly after arrival for G-6-PD analyses. These were done in duplicate by the following method. G&PD from hemolysate of erythrocytes was analysed by cellulose acetate electrophoresis using Beckmann Microzone apparatus. The chamber buffer used was that described by Sparkes er al. (1969) and modified by Benditt and Benditt (1973). Electrophoresis was carried out for 15 minutes at 2 mA. Following electrophoresis the strips were stained with a mixture containing NADP-G6P-MTT-PMS (Sparkes ef al., 1969). Stained strips were fixed in 1% formalin and scanned in a Beckman R-l 12 den- sitometer at 550 nm.

Tissue samples were extracted by repeated freeze-thawing in a Tris-EDTA- boric acid buffer containing NADP at 5 mg per 100 ml. Electrophoresis and devel- opment of enzyme reaction were carried out as described above for erythrocyte samples.

G-6-PD MOSAICISM AND ATHEROGENESIS IN HYBRID HARES 193

Serum cholesterol concentrations were determined by the method of Leffler (1959) in all hares.

Twelve of the females and seven of the males were given a diet, consisting of 2 g cholesterol, 12 ml of peanut oil, and 186 g commerical rabbit pellets per day (Table III). The cholesterol feeding was usually intermittent in order to increase the chances of survival of the hares.

All hares fed cholesterol diet for various periods of time were sacrificed and the extent of surface involvement with atherosclerotic lesions was estimated by visual inspection. Aortic samples of five of the female hares fed cholesterol and one female fed cholesterol and 25hydroxycholesterol were extensively analyzed for G-6-PD patterns. In addition, various tissues from seven untreated and two cholesterol-fed females were analyzed for G-6PD.

RESULTS

The hybrid hares thrived in captivity, and losses by death from natural causes were small except for the initial deaths after the stressful travel. They ate the cholesterol diet readily and with the intermittent feeding usually remained in good health.

In Table I are presented the data on the erythrocyte G-6-PD patterns in the 52 females and 55 male hybrid hares that were received in Albany. As expected, all of the males had only the type of L. timidus dam (type “T”). Among the females 23 exhibited only type T. Twenty-nine of the females had both types E (L. europeus) and T; however, type T concentration was greater than 50% in all of these.

In Table II are shown the G&PD patterns in various body tissues of 9 of the bl-heterotypic females (heterotypic in blood). Type T was predominant in most but not all of these. In Table III, serum cholesterol values and extent of aortic atherosclerosis are shown for 12 female and 7 male hybrids that were fed choles- terol for 3 - 13 months.

In addition to the results presented in the tables we have addressed ourselves to the question of why some of the F, hybrid females listed in Table I show a single-enzyme phenotype. Toward resolving this, we have examined various tis-

TABLE I G-6PD Patterns in Erythrocytes of Hybrid Hares (~5. europaeus (E) x L. timidus (T)) Screened to Date

Males Females

Percentage of type T in blood

loo <55

55-59 60-69 70-79 80-89

100

Avg. 74 f 2 (excluding 100%)

No. of hares

55 0 3 5

10 11 23"

Total: 52

No&. Expressed as percentage T (Type E is 100% minus the % T). a These females are presumed to be hybrids, hence heterozygotic. They are referred to as “bl-

homotypic” (blood homotypic) to distinguish them from the overtly heterotypic cases. Possible rea- sons for the occurrence of this group are considered in the Discussion.

TABL

E II

Surv

ey o

f Va

rious

Ti

ssue

s fo

r G

-6-P

D Pa

ttern

in

Unt

reat

ed

and

Cho

lest

erol

-Fed

Fe

mal

e H

ybrid

H

ares

(L

. eu

rooa

eus

(E)

X L.

tim

idus

(T

))

Hare

no

. Bl

ood

Aorta

Sk

in

Ute

rus

Hea

rt

Unt

reat

ed

Kidn

ey

Live

r Sp

leen

Di

aphr

agm

Lu

ng

1 4 6 10

42

14

39

54(l)

75

(l)

7W

74(3

) 53

(3)

64(3

) 60

(3)

56(3

) 59

(3)

W2)

70

(l)

- 85

(2)

- 68

(l)

6’W

) W

V 52

(2)

56(2

) 56

(2)

- 56

(l)

81(l)

-

71(2

) 74

(l)

53(l)

72

(l)

69(2

) .

67(2

) -

5W)

53(2

) -

W2)

W

2)

67(2

) -

- 52

(l)

- -

75(l)

W

2)

7W)

W2)

5w

7X

2)

- W

I) -

78(2

) 7X

3 76

(2)

- 6W

70

(2)

- 84

(l)

Y/(Z

) 70

(l)

54(e

) 57

(l)

57(4

) 46

(2)

55(2

) -

572)

66

(l)

WI

12 (C

) (1

0 m

onth

s)

21 (C

OH

) (6

mon

ths)

Trea

ted

with

ch

oles

tero

l (C

) or

cho

lest

erol

an

d 25

-hyd

roxy

chol

este

rol

(CO

H)

77(l)

-

5X2)

5W

) 65

(2)

60(l)

55

(l)

- lO

O(2

) 83

(l)

86”

58(l)

w1

4 4-

W)

M(l)

O

(2)

41(2

)

- 57

(l)

- 4w

Nor

e.

Cl-6

-PD

pat

tern

ex

pres

sed

as p

erce

ntag

e of

T.

Num

ber

in p

aren

thes

es

is n

umbe

r of

sam

ples

exa

min

ed.

a D

ata

on a

ther

oscle

rotic

le

sions

in

thi

s ha

re

are

pres

ente

d in

Tab

les

V an

d VI

.

195 G-6-PD MOSAICISM AND ATHEROGENESIS IN HYBRID HARES

TABLE III Data on Male and Female Hybrid Hares (L. europaeus (E) X L. hnidus (T))

Fed 1% Cholesterol for 3- 13 Months

Percentage of aortic Months surface involved by

on Initial Final atherosclerotic lesion Percentage T chol. serum serum

Hare no. Sex in blood diet chol. chol. Abd. Thorac. Arch

15 M 100 4 20 2400 -95 -95 -95 18 M 100 4 30 3000 -95 -95 -95 3 M 100 5 40 1200 -95 -40 -40

16 M 100 8 26 2588 -70 -70 -70 19 M 100 8 22 2289 -40 -40 -40 17 M 100 11 19 2424 100 100 100 22 M 100 11 29 1747 100 100 100 23 F 100 3 22 1214 100 0 10 2 F 100 4 25 2280 -5 -5 -5

13 F 100 5 20 2200 -4 -2 -40 24 F 100 6 26 1762 100 -5 -5 25 F 100 8 25 1689 -50 -25 -5 8 F 88 4 - 989 -35 0 0 9 F 77 4 - 2015 -5 0 0 7 F 61 6 - 337 -45 0 -45

21” F 83 6 - 363 -10 -2U -20 22 F 59 7 - 1338 -35 0 -35 5 F 67 12 - 1891 -60 -5 -5

26 F 79 13 - 1556 -90 -5 -5

a Also fed 25-hydroxycholesterol.

sues of 2 such animals for the distribution of the two enzyme variants. The results show that all the tissues examined follow the blood pattern of exhibiting only type T. In order to determine whether exaggerated differential activity of the two types contributes to the apparent total absence of the E type, we did a quantitative determination of the specific activities in the two pure species. These show no differences between the two variants.

In Table IV, G-6-PD profile data are presented for 143 samples from 10 large atherosclerotic lesions from the bl-heterotypic hares. Most are predominantly type T; no sample was monotypic.

In Table V, the data are presented for small lesions from bl-heterotypic hybrid hares. This includes the single hare that, after a combined cholesterol and 25 hydroxycholesterol diet, showed a single G&PD variant (monotypism) in 3 sam- ples of atherosclerotic and 33 samples from normal appearing aorta. As can be seen in Table VI, some samples from atherosclerotic lesions, and from grossly normal uterus in this animal, showed only type T, and 2 samples from the liver showed only type E.

An important morphological finding, which will be reported in detail elsewhere, is that the extramyocardial coronary arteries showed severe stenotic lesions, while the intramyocardial coronary arteries showed no such lesions. This result resembles the pattern of clinically significant coronary atherosclerosis in human subjects. This is unlike the picture found in domestic rabbits fed cholesterol nonintermittently; it may represent a true species difference, or may be due to the intermittent cholesterol feeding.

196 LEE ET AL.

TABLE IV G-6-PD Pattern (Expressed as Percentage T) in Large Atherosclerotic Lesions from

Heterozygous Female Hybrid Hares (Z,. europaeus (E) x L. hidus (T)) Fed a Hypercholesterolemic Diet for 5 to 12 Monthsa

No. of samnles Avg. percentage

Percentage T assayed -T-k SEMb-

Percentage T Lesion Hare no. in blood in aorta no. Top Bottom Top Bottom

7

9

11

5 67 63 2 3 5a

Sb

61 68 2 2 7a

7b

7c

77

55

70 f 2 9a

61 + 3 lla

llb

llc

lld

Avg. 6623

10 9

6 4

14 12

10 5

14 13

12 0

8 8

7 6

5 5

8 6

46%3 P < .002

+E 48 f 5 P < .02

+E 67 f 2

NS 53 f 3 P < .Ol

+E 53 2 3 P < .002

+E 69 2 2

NS 75 2 2 P-c.002

-+T 68 -c 2

NS

66+4 NS

54 + 4 NS

60+3

6ok2 NS

53 24 NS

72 2 2 NS

61 27 NS

56 2 2 P-c.002

+E -

69 k 1 NS

76 f 3 P < .02

+T 66*3

NS 66k2

NS

64k3

a Data on two large lesions from the hybrid fed 25-hydroxycholesterol in addition to cholesterol are presented in Table VI.

b P values represent statistical difference as compared with normal aorta. Arrows indicate the change in the direction of T to E ratio.

DISCUSSION

This study shows that it is practical to produce female hares heterozygous for G-6-PD in substantial numbers by crossing L. europaeus males with L. timidus females. Ohno et al. (1965) had shown in a very small number that it was possible with either cross. We have also shown that the hybrid hares respond to cholesterol feeding by developing aortic atherosclerotic lesions as readily as domestic rabbits. Thus, this experimental animal model appears to be potentially useful in inves- tigating the phenomenon of G-6-PD monotypism observed in many thick athero- sclerotic lesions of black women who are heterozygous for G-6-PD (Benditt and Benditt, 1973, Pearson et al. 1979, and Thomas et al. 1979).

In the one-way cross used here (male L. europueus to female L. timidus) the G-6-PD of the male offspring were always of the mother’s type, as would be expected since the male is heterogametic in these species. However, unexpect-

TABL

E V

G&P

D Pa

ttern

(E

xpre

ssed

as

Per

cent

age

T) i

n Sm

all

Athe

rosc

lero

tic

Lesio

ns

from

H

eter

ozyg

ous

Fem

ale

Hyb

rid

Har

es

(L.

euro

paeu

s (E

) x

I..

timid

us

(T))

Fed

a C

hole

ster

ol

Die

t fo

r 4

to 1

3 M

onth

s

Perc

enta

ge

T M

onth

s N

o.

of s

ampl

es a

ssay

ed

Avg.

pe

rcen

tage

T

2 SE

M”

Hare

in

on

no

. bl

ood

chol

.

9 77

4

7 61

6

5 67

12

26

79

13

21*

83

6

No.

of

W

hole

N

orm

al

Who

le

Nor

mal

le

sions

To

p Bo

ttom

th

ickn

ess

aorta

To

p Bo

ttom

th

ickn

ess

aorta

17

10

12

9 20

70

f

1 65

+ 2

66

+2

70 +

2

NS

NS

NS

10

12

13

3 18

54

f 3

53

+ 4

48

2 3

68

*2

P<.O

O2

P<.o

o2

NS

+E

+E

4 6

4 2

22

56 ?

2 53

-c5

56

+ 2

63

_’ 3

P

< .0

5 NS

NS

+E

3

8 0

7 24

75

2 2

-

69 +

1

61 +

3

P <

.02

P <

.05

+T

+T

2 di

4

di 3

di

9

di 2

4 79

82

78

86

m

ono

1 m

ono

2 m

ono

0 m

ono

33

100

loo

100

a P

valu

es r

epre

sent

st

atis

tical

di

ffere

nce

as c

ompa

red

with

no

rmal

po

rtion

of

aor

ta.

Arro

ws

indi

cate

th

e ch

ange

in

the

dire

ctio

n of

T t

o E

ratio

. *

Rec

eive

d 25

-hyd

roxy

chol

este

rol

as w

ell

as c

hole

ster

ol.

198 LEE ET AL.

TABLE VI Data on One Hybrid Hare (#21) Heterozygous for G-6-PD Who Had Some Monotypic Foci in

Atherosclerotic Lesions, in the Normal-Appearing Portion of the Aorta and in the Uterus and Liver

Percentage Type T G-6-PD in blood Months on cholesterol + 25-hydroxycholesterol Initial serum cholesterol level Average serum cholesterol level while on cholesterol Terminal serum cholesterol level Extent of atherosclerotic lesions in aorta Average % T in organs yielding only ditypic samples

(Skin, heart, kidney, spleen, lung) Large atherosclerotic lesions sampled for G-6-PD

Lesion no. 1 No. of samples No. of samples, ditypic

Average % T Lesion no. 2

No. of samples No. of samples, 100% T No. of samples, ditypic

Average % T Small atherosclerotic lesions sampled for G-6-PD

No. of samples No. of samples, 100% T No. of samples, ditypic

Average % T Normal-appearing aorta from two areas

Area no. 1 No. of samples No. of samples, 100% T No. of samples, ditypic

Average % T Area no. 2

No. of samples No. of samples, 100% T No. of samples, ditypic

Average % T Uterus sampled for G-6-PD

No. of samples No. of samples 100% T No. of samples, ditypic

Average % T Liver sampled for G-6-PD

No. of samples No. of samples, 100% E

83 6

17 mg/dl 477 mg/dl 363 mg/dl

-20%

48 k 3

8 8

78 k 3

10 3 7

80 * 2

19 3

16 80 k 2

40 25 15 82 k 2

17 8 9

74 2 3

16 2

14 74 2 5

2 2

edly in the blood of half of the female hybrids we could demonstrate only the dam’s type (type T) and in the remaining half type T averaged 74% of the total as determined by densitometer.

Type T is the slower-moving band and in this respect is similar to the slower- moving B band in black women heterozygotes. Type B averages about 55% of the total in such women. With the hares we were dealing only with the F1 generation and also with only a one-way cross. With black women we are probably frequently dealing with F>, generations and with crosses in both directions. These presumed

G-6-PD MOSAICISM AND ATHEROGENESIS IN HYBRID HARES 199

conditions among the black women must be investigated in the hares before sig- nificant comparisons can be made between the two regarding ratios of fast and slow-moving G-6-PD bands.

The fact that half of the putative female hybrid hares showed only the T band in this study is puzzling. One possible explanation is that an L. timidus male was bred to some of the L. timidus females by accident. We have investigated this possibility thoroughly and it seems extremely unlikely; however, it cannot be completely ruled out.

Another possible explanation for the above is that type E in the hybrid hares was present but in quantities too small to be detected by current methods. Proba- bly quantities below 5% of the total would not be apparent. Some support for this possibility can be found in a study we are now making of skin fibroblasts from the overtly heterotypic female hares. When carried through multiple passages, one or the other type of G-6-PD occasionally completely disappear as far as detection is concerned and then reappear in a later passage (Janakidevi et al., 1981).

Another possibility is that there is some cytoplasmic component acquired from the mother that makes her type predominate in the hybrid. Reverse crosses and other types of studies need to be carried out before this possibility can be evaluated. It should also be noted that predominance of one G-6-PD is merely serving as a marker for a chromosome with many genes that may differ from its counterpart. Any combination of genes could afford a selective advantage for one set over another in a given situation and the same set of genes might afford a disadvantage in some other situation.

Our original interest in this model was derived from our hypothesis that monotypic foci in advanced atherosclerotic lesions of black women heterozygous for G-6-PD were a secondary phenomenon and not due to each lesion arising from a single cell (monoclonal origin). If the atherosclerotic lesions in the heterozygous hare arose from a single cell, we should have demonstrated monotypism with great frequency in this study, and we did not. The hare lesions are therefore not monoclonal in origin.

Pearson and his associates (1979) have asserted that this hare cross is not an acceptable model for the study of atherosclerosis. The ground offered is that the monoclonal origin of human lesions has been established, so that only a model yielding monoclonal lesions can be accepted. Since the premise of this argument is almost certainly false (Thomas et al., 1979), so is the conclusion. We shall conduct further studies before we arrive at a final decision.

The demonstration of monotypic foci in atherosclerotic lesions and elsewhere in the only hare that was fed a cholesterol oxidation product (25hydroxycholestero1, which has been demonstrated to be present in human atherosclerotic lesions (Smith and Pandya, 1973)) in addition to cholesterol is of considerable interest; but sweeping conclusions are not as yet warranted. Monotypic foci were also frequent in grossly normal parts of the aorta; and the atherosclerotic lesion with monotypic foci may have arisen from one of these.

The frequency of monotypic foci in grossly normal portions of aorta in heterozygous black women is not known. Benditt and Bend&t (1973) found such in 2 of 59 grossly normal sites. On the basis of microscopic examination they suggested these might represent sites of very early lesions. We have occasionally found similar monotypic foci in grossly normal parts of aortas of heterozygous black women that, even on microscopic examination, failed to show a definite

200 LEE ET AL.

atherosclerotic lesion. However, even with these we have tended to conclude that an atherosclerotic lesion must be present even though we were not able to detect it. This aspect needs to be investigated further with an open mind.

The demonstration of monotypic foci in the normal-appearing uterus and liver (but not in other organs) of the same hare discussed above is also of interest. We are not aware of any studies involving extensive screening of normal organs from black women heterozygous for G-6-PD; and this needs to be done. The monotypic foci in the liver of the hare were type E in contrast to the type T found in the aorta and uterus. In the aortic atherosclerotic lesions of black women heterozygotes, monotypic foci usually tend to be of the same type, and that monotype is usually the type that predominates in the ditypic tissue. However, there are infrequent exceptions where both monotypes are found in the same aorta.

Even though monotypic foci were found in lesions in only one hare, some of the atherosclerotic lesions from other cholesterol-fed hares did show significant changes in T to E ratios as compared to normal aortic medium from the same hare. Of the four hares (not showing monotypism) studied in detail in this regard, T to E ratios moved significantly toward E in two, toward T in one, and did not change significantly in the fourth. The fact that there were significant directional changes suggests that cells with one type of X chromosome had some selective advantage over the other in this particular cellular environment. The advantage was probably not related to the G-6-PD type per se, since the movement re G-6-PD type was toward one direction in one and in the opposite in the other two.

The above results also make it seem likely that lesions (or parts of lesions) will eventually become monotypic if the hares are maintained for longer periods. Such experiments are now in progress.

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in female hybrid hares heterozygous for glucose-6-phosphate dehydrogenase (G-6PD). II. Changes in the ratios of G-6-PD types in skin tibroblast cultures carried through multiple passages. Exp. Mol. Pathol. 34, 133- 139.

LEFFLER, H. H. (1959). Estimation of cholesterol in serum. Amer. J. Clin. Pathol. 31, 310-313. LYON, M. F. (1962). Sex chromatin and gene action in the mammalian X-chromosome. Amer. J. Hum.

Genet. 14, 135-148. OHNO, S., POOLE, J., GUSTAVSSON, I. (1965). Sex-linkage of erythrocyte glucose-6phosphate dehy-

drogenase in two species of wild hares. Science. 150, 1737- 1738. Pearson, T. A., Want, A., Solez, K., and Heptinstall, R. H. (1975). Clonal characteristics of fibrous

plaques and fatty streaks from human aortas. Amer. J. Pathol. 81, 379-387. PEARSON, T. A., DILLMAN, J. M., SOLEZ, K., and HE~INSTALL, R. H. (1978a). Clonal characteristics

in layers of human atherosclerotic plaques. Amer. J. Pathol. 93(l), 93- 102. PEARSON, T. A., DILLMAN, J. M., SOLEZ, K., and HEP~INSTALL, R. H. (1978b). Clonal markers in the

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MALMIOS, H. (1979). Clonal characteristics of experimentally induced atherosclerotic lesions in the hybrid hare. Circulation 60, 166.

SMITH, L. L., and PANDYA, N. L. (1973). Sterol metabolism. Atherosclerosis. 17, 21-30. SPARKES, R., BALUDA, M. C., and TOWNSEND, D. G. (1969). Cellulose acetate electrophoresis of

human glucose&phosphate dehydrogenase. J. Lab. C/in. Med. 73, 531-534. THOMAS, W. A., JANAKIDEVI, K., REINER, J. M., and SCOTT, R. F. (1978a). Some aspects of popula-

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G-CPD MOSAICISM AND ATHEROGENESIS IN HYBRID HARES 201

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