Testicular cytotoxicity of intravenous procarbazine in rats

Surgical

Oncology

1993; 2: 77-81

Testicular cytotoxicity F. E. JOHNSON*, Departments

Although

of *Surgery

the

previous

ally employed

by histology

repopulation

index

and kidney

histology

lar atrophy

and oligospermia testicular

at high dosages appears Oncology

index.

evaluated

atrophy,

at low

oligospermia

(500 and 700 mg kg-’

cancer treatment,

therapy

for

thousand

number

of long-term

cancer

successfully

treated

infertility,

and germinal

body weight

agents

been oligo-

long

after

completion

population

clinical

of

Surgery,

observed

This model

damage.

Surgical

on are

effects

of

small

size

their

small

blood

evaluation. method

these

human often

male

post-pubertal

male to

anti-neoplastic hinders

surgical

volume

We are currently to address

this

limits invesissue

in

Frank

E. Johnson,

University

Medical

MD,

Department

Center,

3635

of Vista

rats were

to tap water

housed

5001. Approximately

health

screening

AAALAC

(American

of Laboratory

and institutional

the performance Procarbazine LaRoche,

Association Animal

Nutley,

with

was allowed

All laboratory

Medical for

Center the

rules were

hydrochloride

for facimeet

Accredita-

Care) guidelines,

of the animal

free

Laboratory

to the new environ-

this study.

lities in the St. Louis University tion

Rodent

1 week

This

testicular

per cage

Purina

and adaptation

prior to entering

weighing

used for this study. of spontaneous

and

Chow ment

the

Sprague-Dawley

Indianapolis)

265 g were

has a low incidence Four

in

rat.

Eight-week-old

access

procarba-

cytotoxicity

METHODS

lesions.

the quantita-

between

testicular

AND

the

used

we describe

rats (Harlan-Sprague-Dawley,

of recovery

to circumvent

chemotherapy Mice

of

at Grand Blvd., PO Box 15250, St. Louis, MO 63110-

0250, USA.

were

MATERIALS

strain

their

Louis

testicu-

of procarba-

relationship

and

Sprague-Dawley

document

but

a surgical

St.

testicular

dosage

federal

Avenue

count,

lung, liver

rat, testicle.

approximately

and

Correspondence:

head

using this regimen.

are quite scanty.

reports method

available.

endocrinological tigating

have

persists

testicular

[4],

greater

who

develop

an efficient

procedures

assume

men

drugs

many

the

As the delayed

Data on the frequency

effect

USA.

these

[l-3].

is not

mg

evaluated

at day 59 for procarbazine

and qualitative

zine

chemo-

with

in this patient

evaluate

hypoplasia

LD,,

procarbazine,

increases,

agents

young

of fertility phenomena,

undergo

in the

survivors

treatment

adverse

year

anti-cancer Many

fertility

patients

each

importance.

Although

sperm

dosages

in

(O-700

was

dose-dependent

and intermediate

tive hundred

which

toxicity

on heart,

the rat [5]. In this report,

Several

spermia

weight,

to avoid drug-induced

INTRODUCTION

of

testicular

toxicity

Progressive

rat,

1993; 2: 77-81.

Keywords:

toxicities

in the

one, and have gener-

of procarbazine

body weight).

600 mg kg-’

the study of techniques

evaluated

bolus of procarbazine Testicular

by testicular Effects

St. Louis, MO, USA

we describe

later.

qualitatively.

occurred

to be approximately

will facilitate

days

and quantitatively

and epididymal were

has been

In this report,

59f2

Center,

than the intravenous

a single intravenous

necropsy

qualitatively

zine. Marked

other

regimens.

rat following with

Medical

of procarbazine

routes

multiple-dose

weight),

St. Louis University

cytotoxicity

have utilized

the Sprague-Dawley kg body

W. G. DOUBEK*, K. C. TOLMAN* AND C. G. JANNEYt

and tPatho/ogy,

testicular

models

of intravenous procarbazine in rats

and all

followed

during

experimentation. (a gift from

NJ) was dissolved

Hoffman-

in sterile

0.15

M

F. E. Johnson et al.

78

NaCl immediately wide

prior to administration.

range of dosages

tions were

prepared

were

in order to insure precision

dosage. Acute fluid overload ing the volume

Because a

used, five concentrawas avoided

of all injectates

in

by keep-

between

0.4 and

Both

testes

weighed

to

Forty rats were assigned to five treatment with eight rats in each group. Identifying ing was done after grouping.

was expressed necropsy

to estimate

groups,

evaluation albuginea

to f 1 g on the day of drug administration

(desig-

of sperm M

sodium

pentobarbital

(60

(BW), with supplemental given

as needed).

established

mg

Intravenous

while

PT 10, Brinkmann)

weight

(i.v.)

BW

access

was

in order

extravasated.

Rats in

hematoxylin

were performed

and incisions were

closed

with fine polyglycolic

acid sutures. On each operat-

ing

each

rats

from

treatment

weeks

were

upon in rotation in order to minimize tech-

nique bias. All animals were operated 4-day

group

period,

then

inspected

and weighed

weekly.

upon within a

daily for the first 2 Those which

expired

prior to the day of necropsy were not replaced. postoperative

day

592 2, all surviving

killed by exposure to CO2 and necropsied.

On

rats were The testi-

speed

between

as described

head count was estimated

micro-

hemocytometer

tech-

(KCT) to ensure

reproducibility.

BW,

(Table 1). All cutdowns

rinsed

of the homogenate

nique [6] by a single investigator

at 100, 300, 500 and 700 mg kg-’

2 through

the tunica

maximum

tip was

using conventional

were

in groups

under sterile conditions

operated

scopically

pro-

respectively

day,

by others. Sperm

5 received

those

at 80%

was serially diluted, but not sonicated

NaCI,

0.5 ml of i.v. 0.15

after

it was homogenized

and the final volume

M

1 then received

carbazine

body

by internal jugular vein cutdown

to ensure that no procarbazine group

kg-’

doses of 15 mg kg-’

at

NaCl using a Polytron homogenizer

samples,

(i.p.)

epidi-

of procarbazine-

count:

had been removed,

in 2 ml of 0.15 (Model

head

rats were

intraperitoneal

and

of the

of body weight

the degree

for 5 s. The homogenizer

with

necropsy

The left testis was then used for

nated day 0) and on the day of necropsy. On day 0, anaesthetized

at

removal

as a percentage

atrophy.

ear notch-

All rats were weighed

excised after

dymides. The total testicular wet weight (right + left)

induced

1.2 ml.

were

f 1 mg

Samples

were

fixed

of heart, left lung, liver, and left kidney in

10%

formalin

and

and eosin for histologic

qualitatively

assessed

stained study.

by a single

with

All slides

pathologist

(CGJ). fixed

in

Bouin’s solution for 3 h, then cut perpendicular

to

The

right

testes

and

epididymes

their long axes into several 2-3-mm fixed again in Bouin’s solution

were

thick slices and

overnight.

day they were fixed in 70% ethanol

The next

saturated

LiCO, for 24 h, and finally in 10% buffered for several

days

before

processing

histologic slides using hematoxylin These

slides were

cles, heart, lungs, liver and kidneys were inspected

regard

to germinal

for gross abnormalities.

tissue,

Leydig

scored

cells,

Sertoli

into standard and eosin stain.

by the pathologist

epithelium,

vessels,

cells,

with

formalin

and

with

interstitial epididymal

Table 1. Effect of procarbazine on animal body weight, total testicular weight, sperm head count, repopulation index, epididymal index, modified Johnsen score Group

Dose

No.

Body weight

(mgkg-‘)

surviving

gain (% of

to necropsy

original BW)

date 1

0

8

46.1 k5.4

Total testicular weight Absolute

Normalized

(9)

(% BW)

3.613kO.058

0.909kO.022

Sperm

Repopulation

Epididymal

Modified

head count

index

index

Johnsen

(per testis)

29.21 k1.83

score

0.997kO.002

3kO

13.4k0.2

0.999kO.001

3+0

13.2F0.2

0.798zkO.100

2f0.33

10.8kO.7

0.624kO.093

1.33f0.21

8.5f0.2

0.356

1

8

(X106) 2

100

8*

47.1 f2.2

3.472rk0.111

0.873+0.022

27.85k2.80 (X10")

3

300

8

42.6f4.1

2.556f0.302

0.663f0.082

14.99f2.24 (X106)

4

500

6

43.5zk3.3

1.772k0.154

0.473kO.051

3.88k4.54 (X106)

5

700

1

44.6

1.302

0.350

7.8X lOa

*One animal was censored for testicular weight and sperm head count because of a congenitally absent left testicle.

79

Procarbazine toxicity structures

according

Johnsen

scoring

viously findings

is assigned

are assigned

obtainable

The number testis.

The

of

paraffin

To

maximum

scale

1 + = markedly

to

coding

as the

dosage.

Increasing

scale than

number

index

the slides

remained

determine

factor

index

variation

groups

were

of variance effects.

In the

the two-sample

are expressed

with

generally a Tukey

case

binomial

test

of epidi-

test was used.

Histologic showed each

and

animals

Group

and seven

of the eight

died

after

soon

drug

receiving

appeared

healthy,

the rats which

and delayed

tion

arrest lined

anaesthetic

since none

of the animals any

anaesthetic rarely

None period

regimen

or thoracic

routinely

anaesthetic

which

appeared

higher

dose

congenitally

any gross

viscera,

with small

did not occur. we

procarbazine

and

of the rats which showed

were

tent with

in the lower

mortality,

encounter

BW rats

on day 0 could

due to acute

since

have

believe toxicity,

dose groups we

use

in our laboratory

this and

difficulties. survived

the entire

abnormality the exception

and

soft

study

in groups minimal

One animal

in group

left

This

2 had a

animal

was

of

Approximately were

examined

tion

index.

between

animals, tubules cells

tubules

stage,

maturaas well

disruption

numerous

Sertoli-only (Fig. Ic). There

in the epididymal sperm

group

3,

4 and

examined

con-

of sperm mixed

and

with

empty

5. There

infiltrates, tubules

or

was

vasculitis,

no or

(range = 512-1595) the repopula-

differences

were

99%. The repopulation

0.80 for group

approximately

(repopulation

4

of sper-

1 and 2 as the repopulation

to about

as

cells (Fig. 1 b). Group

per testis to determine

above

(Fig.

maturation

and appearance

No significant

uniformly

com-

containing

cells in any of the testicles.

750

groups

2 showed

disrupted

2, diminished

of Leydig

the

cross-

virtually

spermatozoa

in groups

of

populated

with

in

bron-

rest

of maturation

greater

inflammatory

abnormalities

dropped

absent

sperm

animal

incomplete

changes

debris

evidence

of the testes, in the

1 and

eosinophilic

was

in animals

by Sertoli

amount

kidney

One

tubules

exhibiting

maturation,

and

1 and

with

fully

and few mature normal

of

rats is

of the testicular

had

showed

corresponding

in abdominal

groups.

testicle.

tubules

All other

complications,

were

BW

IO4 in the

patchy

the

at the spermatid

only

a per

lungs.

seminiferous

those

5 animals

700 mg kg-’

deaths

died

the deaths suffered

500 mg kg-’

administration.

Although experienced

receiving

all

groups

normal,

among

matocytic animals

but normal

3 animals

scattered with

liver

cells at all stages

with

X

in untreated

histology,

repopulated

count

coefficient

5 had mild,

from

normal

The

drug

caused

head

heart,

examination

spermatogenic

and

Two of the eight

2, 4 and

of animals

essentially

values

increasing

abnormalities.

pneumonia,

Microscopic

pletely

of

had essentially

tubules RESULTS

sections

in groups

are

a progressive

1 to 7.8

count

a

[7].

no discernable

chiolitis

la).

head

0; results

dosage

5 rat.

as

(absolute

sperm

IO6 in group

group

of sperm

arrest,

as mean + SEM.

in

in our laboratory

sections

among

X

29.21

weight

procarbazine

surviving

procarbazine,

was

BW) with

reduction

from

4-12%

of the slides throughout

of evaluation. by analysis

testis single

reduced

and O=absent.

was used for all slides.

evaluated

progressive

epi-

expressed

on day

testicular

of The

epididymal

in total

head in the

index,

of

was

1. There

as percentage

was also

(but greater

decrease

effects

weight

in Table

included

score.

necropsy

of body

of sperm

but

repopulation

systemic at

as well

et a/. [8]. index.

weight,

and

number

epididymis

content);

number

Differences

All results

total

using a numerical

content);

to the group

of sper-

counted

by Kramer

2 + = reduced

who

signs

also

repopulation

is referred

the process

percentage summarized

the

evaluated

The investigator

evaluated

the

30% of normal

[7]. Random

was right

the analysis

weight

and Johnsen

evaluate

of the

with

body

weight

sections

was of

of

section

from

testicular

index,

tubular

in the ductus

normal

(less than

dymal

of 1. The

excluded and

body

is called

3 + = normal;

count didymal

as described

of sperm

therefore analysis

of tubules

semi-quantitatively

to

the pre-

sugges-

as a fraction

sections

This fraction

blind

of used

any abnormality

repopulation

expressed

where

have

a score of zero and normal

of seminiferous

number

matogenic

amount

we

a score

in an intact

tubular

modification

score is 14.

counted

This

the which

[7]. In this system,

tive of injury

30%

to

system

were

38% devoid

of

noted index index

3 rats. In group all of

seminiferous spermatogenic

index = 0.62), while

the majority

4

F. E. Johnson et al.

80

Figure 1. (a) Testicular from

Group

normal

tubules

each tubule.

of maturation

H&E stain,

magnification. partly

repopulated in rare tubules.

Group

tubules,

populated

cells. H&E stain,

(64%)

matogenic

cells and were lined by Sertoli cells only

(repopulation

in the group

index = 0.361,

5 rat had no sperindicating

stem cell killing by procarbazine from epididymal ally paralleled count,

dependent

the results obtained in groups l-2

while

other

decrease. content

dymis to another. documented 0.15

groups

showed

are

one portion

In previous

work

group,

as compared

indicating

that

procedure

approaches

induced

toxicity

hormonal regimen.

to germinal

[IO],

epithelial

in drug treatment is often viewed as

this problem

approaches

in

macokinetics

offer another potential

to modify

in

that a mechanical testicular

[9], we have

tory isolation,

and

which we have termed provides

doxorubicin-induced allows

doxorubicin-treated

anesthesia

nor the

has influenced

results

[5],

long-term

partial

germinal

retention

animals

Sprague-Dawley

of

single-dose

testicular

investigators

of fertility

in

[13], and produces

no

rat as an appropriate

intravenous

patient With

is becoming recent

percentage

an

progress of long-term

important

in

cancer

survivors

rise. Thus, late toxic effects assuming

more

mented;

cells represents

another

the to

are now

Prolonged

infertility

in young

chromosomal

treatment, has continued

of treatment

importance.

after chemotherapy

consideration.

males

is well

docu-

sterilization carbazine

of young

male

is often considered

lar injury in a number

and the formation

Pro-

as a cause of testicuprotocols

and

[I -31. on stem cell regeneration

of spermatozoa

to spermatogenic

genitor

potential

long-term

tion, infertility generally develops

hazard.

drugs in

patients.

to act as an alkylating

damage

cells. Following

injury

as a class,

implicated

of lymphoma

regimens

Male fertility depends

testicular

agents,

cancer

agent and has been implicated other treatment

procarbazine-

in limiting

The alkylating

are perhaps the most commonly cancer

epithelial

injury which should be useful for

interested

from this agent. male

circula-

of the rat

effects itself [7]. The current work estab-

lishes the model

groups.

partial protection

testis from injury

young

to pro-

approach,

in any

in the

method

[II, phar-

tect fertility. We have documented

variability

DISCUSSION of fertility

regional

of the epidi-

induced

Retention

also, but

available data suggest it is seriously inadequate

control

in the procarbazine-treated

including

germinal

and alteration

a suitable way to circumvent

difference

neither

epithelium,

of

Semen cryopreservation

with a non-treated

employed

germ

have been used to avoid drug-

manipulation

metabolism

by immature

x 100 magnification.

121. Surgical

a dose-

that animals receiving anaesthesia

parameters cutdown

sperm

summarized

no appreciable

from

NaCl have no significant

M

by repopulation

had a normal

These

Table 1. We observed the sperm

in this group. Data

index and sperm head count gener-

index. Animals head

extensive

Several

BW)

many entirely

of germ cells, and some

sparsely

of tubules

H&E (c) Testis

5 rat (700 mg kg-’

has shrunken devoid

3

shrunken

by

X 100 magnification.

from

Group

BW) shows

germ cells, with few

spermatozoa stain,

in

X 100

(b) Testis from

rat (300 mg kg-’ immature

rat showing

with spermatogenic

cells in all stages

tubules

cross-section

1 (control)

cytotoxic

from these prodrug administrarap’rdfy from lethal

Procarbazine

damage

to the differentiated

of fertility

depends

cells,

which

yield

differentiated

produce

on

repopulate

the tubules

identification

of stem

morphology,

which

to

stem

measure

involves

absence

Conversely,

cross-section

This assay

stem cell within

suffers

from

insensitive this

is

before

issue,

we

epididymal extent

injury

have

index

able technique

used

primary We The

and spermatids.

spermatogenic about

exposure

should

provide

sufficient

new

through

generation

which

supports

Spermatogenic tiated

time

should

have

which

supports

The

the

already

to

normally

stem

the differential

pathway

differentiated

sperm

repopulation already

procarbazine

to

cells, index.

testicular

is in general

within

[4], as well

given

procarbazine

from

ours,

as the results

underestimate

the

JAMA

with

as

observed

in

the findings

observed

damage.

1988; 259: 2123-5.

3. Schilsky RL, Lewis B, Sherins RJ, Young RC. Gonadal

dysfunction

in patients

receiving

cheomotherapy

cancer. Ann lnt Med 1980; 93: 109-l 4. 4. Meistrich ML, Finch M, da Cunha MF, Hacker Au, WW. peutic

Damaging

drugs

effects

on mouse

of fourteen

testis

cells.

5. Lui RC, La Regina FE. Regional toxicity.

MC, Herbold

doxorubicin

for

U and

chemothera-

Cancer

Res 1982;

DR, Stern JA, Johnson

delivery

reduces

testicular

J Surg Res 1987; 43: 286-95.

6. Meistrich

ML, Hunter

after

NR, Suzuki N, Trostle

regeneration

exposure

of

mouse

to ionizing

PK. Withers

testicular

radiation.

7. Stern

JA, Lui RC, LaRegina

KC, Johnson

stem

Radiat

MC, Herbold

FE. Long-term

lar ischemia

outcome

Res

in rats

MeV fast-neutron

irradiation

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pro-

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lnt J Radiat Bioll974;

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Hughes

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in men undergoing Br J Cancer

JR, Bajorunas

13. Liebscher

of body weight

may actually

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GJ, Janney

artificial CG, La Regina

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administration.

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DR. Goldstein

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disease.

KS.

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of expression

as

25: 253-60.

DR,

J C/in Oncoll987;

produced,

DR. Tolman

following

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in the rat. J Androl

Hodgkin’s

to schedules

of atrophy

or

of gonadal

gonadal

different

degree

of childhood

JD. The relationship

and

Our method

as a percentage

survivors

cryopreservation

according

evaluate

NEJM 1987; 317: 1315-7.

and chemotherapy-induced

cyclophosphamide

59 days,

head count

atrophy

agreement

to

JJ, Myers MH, et al. Effects of treatin long-term

2. Rivkees SA, Crawford

10. Glode

differen-

administration

left the testis

suitable epithelium.

8. Kramer MF, Davis JAG, Van Der Ven TPA. Effects of 1

be longer

of stem cell survival.

our study

weight

late

of reasons.

rat

the use of the sperm

dose-related

in mice

of

be

germinal

cancer.

the

time for the surviving

were

adolescent

cells

days

which

on fertility

HR. Gradual

Fifty-nine

use of the

cells

at the

measure

of

avail-

due

even

insult.

should

injury

1978; 74: 349-62.

the

may

ment

dehydro-

damage

in

and

to a cytotoxic

cells to progress

by

for a number

cycle

48 days

The

is assay of a

only

here

to protect

period.

testicular

42: 122-31.

assay of the

lactate

testicular

on day 59+2

is seen. important

currently

spermatocytes the

is

routinely.

assayed

requires

are

functional

produced

area.

killing

tubule

Another

of

of toxins

clinically index

described

activity

it is

cell

isoenzyme, is

to a

that

(not used in this study)

which

procarbazine

a

this

is a second

germ-cell-specific

after

is the

the

the presence

doses

of procarbazine-induced

1. Byrne J, Mulvihill

repopu-

the immediate

low

of stem cell survival.

genase-X,

each

extensive

of procarbazine the study

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severe

so

when

doses during

popular

the disadvantage

because

However,

methods

result from

indicates

the higher

less weight

difficult

at a given

in sufficient

receiving slightly

epithelial

most

tubules

gained

measures

and non-repopulated

cross-sections

cells

at least a single

required

The

then

fertilization.

is rather

cross-section.

lated tubular

relatively

survival.

repopulated

of stem

used;

cells

Non-repopulated

particular

for

has led to alternative

tubular

after injury.

cells, which

animals

The model

eventually

of spermatogenic

cell

counting

seminiferous

of stem

and

spermatozoa

due to the complexities

Return

survival

spermatogenic

sufficient

Visual

spermatocytes.

adequate

81

toxicity

Surg Forum

of fertility

MC, et a/. Semen insemination

for

5: 233-8. MC, Tolman after

KC,

doxorubicin

1990; 40: 680-2.