Regional doxorubicin delivery reduces testicular toxicity

JOURNAL

OF SURGICAL

RESEARCH

43, 286-295 (1987)

Regional Doxorubicin

Delivery

Reduces Testicular

Toxicity’p2

RAPHAEL C. LUI, M.D., MARIE C. LAREGINA, D.V.M., DAVID R. HERBOLD, M.D., PH.D., JOHN A. STERN, M.D., AND FRANK E. JOHNSON,M.D.3 Department of Surgery, Maimonides Medical Center, Brooklyn, Comparative Medicine, Pathology, and Surgery St. Louis University

New York 11219, and Departments Medical Center, St. Louis, Missouri

of 63104

Submitted for publication February 24, 1986 Many anti-cancer drugs cause infertility. Regional delivery of these agents is a potential method to avoid this problem. We investigated the protective effect of normothermic testicular circulatory arrest on gonadal toxicity during doxorubicin administration in the Sprague-Dawley rat. Four groups of eight rats each were used. Animals in group 1 received no treatment. Rats in group 2 were anesthetized and received a bolus of intravenous doxorubicin (6 mg/kg). In groups 3 and 4, normothermic circulatory isolation of the left testis was induced by cross-clamping of the spermatic cord and gubernaculum immediately before doxorubicin administration. This was maintained for 15 min after doxorubicin administration in group 3 and for 45 min in group 4. Cessation and return of testicular blood flow were confirmed by Doppler. On Day 56, all rats were killed and necropsied. Testicular toxicity was evaluated qualitatively by histology and quantitatively by measurement of testicular weight, sperm count, repopulation index, and epididymal index. The results indicated that I5 min of testicular circulatory isolation mitigated testicular toxicity to a small extent and that 45 min of circulatory isolation provided moderate protection against doxorubicin-induced testicular toxicity. o 1987 Academic PWS, h.

INTRODUCTION

The acute toxic effects of many drugs used in the treatment of cancer are well known. Those organs containing self-renewing cell populations such as bone marrow and gastrointestinal tract are most commonly affected, but clinically important dysfunction is rare once chemotherapy has been discontinued. Until recently there has been little recognition of the impact of chemotherapy upon the male gonads. This has stemmed partly from the lack of any life-threatening symptoms accompanying gonadal dysfunction and partly from the paucity, until recently, of long-term survivors concerned ’ Presented at the Annual Meeting of the Association for Academic Surgery, Cincinnati, OH, November 1I-13, 1985. ’ This work was supported by American Cancer Society Institutional Grant IN-124. 3 To whom correspondence and reprint requests should be addressed at Department of Surgery, St. Louis University, 1325 South Grand Boulevard, St. Louis, MO 63104. 0022-4804187 $1.50 Copyright 0 1987 by Academic PIWS, Inc. All rights of reproduction in any form reserved.

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about their reproductive potential after the completion of chemotherapy. Many young men who have been successfully treated with chemotherapy develop azoospermia which perists long after cessation of treatment [ l-41. Virtually no satisfactory data on the frequency of return of fertility are available. Despite reports describing the adverse effects of cytotoxic agents on human male fertility, little has been done to circumvent this problem. We have recently described the quantitative relationship between doxorubicin dosage and testicular cytotoxicity in rats [5]. In the current work, we investigated the protective effect of regional drug delivery on doxorubicin-induced testicular toxicity in this model. MATERIALS

AND METHODS

Animals

Thirty - two 9 - week - old postpubertal Sprague-Dawley male rats (Sasco, St. Louis, MO) weighing 200 f 20 g were used for this

LUI

ET AL.:

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study. Animal care was in accordance with all institutional and federal guidelines. Four rats were housed per cage with free access to tap water and Purina Rodent Laboratory Chow 500 1. Five to seven days were allowed for health screening and adaptation to the new environment prior to entering this study. All rats were weighed on the day of doxorubicin administration and on the day of necropsy.

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each group were entered into the study each day for 4 consecutive days. Postoperatively, all rats were inspected daily for wound infection and animal mortality. All rats were killed, weighed, and necropsied on postoperative Day 56. Particular attention was paid to the presence of pleural effusion, heart size, ascites, and general appearance of the liver, kidneys, and testes. Assessment of Testicular

Toxicity

Drugs Doxorubicin (Adria Laboratory, Wilmington, DE) was stored at 4°C and dissolved in sterile 0.9% NaCl immediately prior to administration. Sodium pentobarbital (Abbott) was freshly diluted prior to each operating day. Experimental

Design

Rats were randomly assigned to four equal groups. Identifying ear notches were given after grouping. Rats in group 1 served as controls and received no treatment. Those in groups 2, 3, and 4 were anesthetized with intraperitoneal sodium pentobarbital (60 mg/kg). Supplemental doses (15 mg/kg) were given during the operative procedure as needed. Intravenous access was established by internal jugular vein cutdown and cannulation with polyethylene tubing (PE- 10). Rats in group 2 received doxorubicin at 6 mg/kg by intravenous bolus injection, but no other surgical manipulation. The left testes of rats in groups 3 and 4 were surgically exposed by sterile technique. Complete testicular circulatory arrest at room temperature was achieved by application of nontraumatic vascular clamps to the spermatic cord and gubernaculum for 15 min in group 3 rats and 45 min in group 4 rats. Doxorubicin was given by intravenous bolus at 6 mg/kg immediately after cross-clamping in group 3 and 4 rats. Cessation and return of testicular blood flow were confirmed by Doppler flow detector (Model 8 120, Parks Medical Electronics, Aloha, OR). All incisions were closed with fine polyglycolic acid sutures. Two rats from

Testicular weight. During necropsy, both testes were excised and weighed (accurate to 1 mg) after the epididymides had been removed. Sperm count. Each testis was next divided perpendicular to its long axis. The cephalic half was weighed (accurate to 1 mg) and homogenized in 2.0 ml of 0.9% NaCl using a Polytron Homogenizer (Brinkman, Model PT 10) at 80% maximum speed for 5 set, but not sonicated as described by other authors [6]. The homogenizer tip was rinsed between each use. Sperm were then manually counted using a hemocytometer. Some homogenates were further diluted with 0.9% NaCl to keep the number of sperm in each counting chamber under 600. All sperm counts were performed by the same investigator to ensure reproducibility. Histologic evaluation. The epididymis and caudad half of each testis were placed in Bouin’s solution for 3 hr. Each was then cut transversely into several 2- to 3-mm thick slices and further fixed in Bouin’s solution overnight. Fixation was continued with several changes of 70% ethanol over 24 hr, followed by immersion in 10% buffered formalin solution for 2 days before processing into standard histologic slides (hematoxylin and eosin stain). These slides were random-number coded and the testicular sections were evaluated by a pathologist who remained blind to the treatment protocol with regard to the following histologic parameters: interstitial fibrosis, edema, and inflammation; vascular endothelial and muscular hypertrophy; vasculitis; thrombosis; Leydig and

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Sertoli cell number and condition; and spermatic maturation. For each testis, the total number of seminiferous tubular sections in two testicular cross-sections was counted. Those tubules containing spermatogenic epithelium were designated repopulated tubules; tubules without spermatogenic cells were designated empty tubules. The ratio of the repopulated tubules to the total number of tubules was calculated for each testis and expressed as a percentage. This value is the repopulation index as described by Kramer

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index and maturation arrest were evaluated using the two-sample binomial test. RESULTS

Animal Morbidity and Mortality

The amount of sperm in the ductus epididymis was semiquantitatively evaluated for each testis using a numerical scale where 3+ = normal; 2+ = reduced (but greater than 30% of normal content); l+ = markedly reduced (less than 30% of normal content); and 0 = absent. This scale is referred to as the epididymal index.

There was no wound infection or animal death observed throughout the experimental period. Necropsy revealed no gross abnormality in the heart, lung, liver, or kidney of any animal. No pleural effusion or ascites was observed. Rats which had received intravenous doxorubicin (groups 2, 3, and 4) gained essentially no weight over the entire experimental period, whereas the rats in the control group (group 1) had a mean weight gain of 85%. Results are summarized in Table 1. Weights for group 1 were significantly different from those for groups 2, 3, and 4 (P < 0.01). Differences among groups 2, 3, and 4 were not significant.

Statistics

Testicular Weight

Body weight, testicular weight, sperm count, and repopulation index are expressed as means k SEM. Differences among groups in body weight, testicular weight, sperm count, and repopulation index were evaluated by analysis of variance with a Tukey test to determine factor effects. Epididymal

Both testes from all doxorubicin-treated animals were small. Results are outlined in Table 1. There was no significant difference between the weights of right vs left testicles within groups 1 and 2. In group 3 the left testes were 27% heavier than the right testes (P < 0.001). In group 4, the left testes were

et al. [7].

TABLE BODY WEIGHT,

TESTICULAR

WEIGHT,

Testicular Chllp 1 (Control) 2 (Doxornbicin only) 3 (Doxornbicin + 15 min ischemia) 4 (Doxornbicin + 45 min &hernia)

Body

weight”

SPERM

I

COUNT,

weight*

sperm

Left

Right

AND

REFQPULATION connt

3

0.474

k 0.010

0.470

f 0.010

lOOk

5

0.217

k 0.010

0.214

f 0.010

2.99 k

0.87

lOOk

4

0.173

+ 0.014

0.220

k 0.021

0.78 +

0.23

113k

10

0.194

f 0.014

0.295

+ 0.032

1.33 +

0.50

k 20.13

242.71

ON DAY Repopulation

Left

185?

All values are means + SEM. n Expressed as percentage of initial weight. b Expressed as percentage of Day 56 body weight.

(X 106)

Right 225.64

INDEX

index

Right

+ 26.77

Left

98.16

+

0.52

3.51 AI 1.08

14.97

k

4.96

3.89 f

1.48

14.29

i-

7.1 I

?I 2.96

38.23

+ 10.85

19.47

56

98.72

k

0.36

9.72 t

12.77

57.39

k

7.40

77.77

+

9.54

LUI ET AL.: DOXORUBICIN

52% heavier than the right testes (P < 0.00 1). The right testes from groups 2, 3, and 4 weighed less than the right testes of group 1 (P < 0.0 1). No significant differences in right testicular weight were found among groups 2, 3, and 4. The left testes from groups 2, 3, and 4 weighed less than left testes from group 1 (P < O.Ol), but were not significantly different from each other.

Sperm Count Sperm counts generally reflected testicular weight and were depressed in all doxorubitin-treated animals. Results are reported in Table 1. There were no significant differences in sperm count for right vs left testes within groups 1 and 2. In group 3, sperm count from the left testis was 398% higher than the right testis sperm count (P < 0.08). In group 4, the left testicular sperm count was 1363% greater than that of the right testes (P < 0.001). The right testicular sperm

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289

counts from groups 2, 3, and 4 were lower than that of group 1 (P -C 0.0 I), but were not significantly different from each other. The left testicular sperm counts from groups 2, 3, and 4 were lower than those of group 1 (P < 0.0 1). The left testicular sperm count from group 4 was greater than the left testicular sperm counts from groups 2 and 3 (P < 0.05). There was no significant difference between the values for the left testes of groups 2 and 3.

Qualitative examination Microscopic examination of the testicular sections from group 1 animals showed normal histology and maturation of spermatogenie cells in virtually all tubules (Fig. 1). Group 2 testes had a marked reduction in diameter of the tubules (Fig. 2). Many tubules contained fibrinoid debris and over 90% were lined by Sertoli cells only. The number and morphology of Sertoli cells in

FIG. 1. Normal testis from Group 1 rat with germinal cells in various stages of maturation in all tubules. Minimal artifactual shrinkage of the extra tubular tissues is evident. (H & E, X 130)

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FIG. 2. Testis from Group 2 rat with markedly shrunken tubules, many entirely lacking germ cells and the others with diminished numbers and maturation of germ cells. (H & E, X 130)

the shrunken tubules and the number and morphology of the interstitial Leydig cells remained essentially normal. The right testes of the animals in groups 3 and 4 had essentially the same histomorphology as those of group 2. The left testes of rats from groups 3 and 4, however, had considerably more spermatogenic cells and these cells retained the ability to undergo maturation to the spermatid stage (Figs. 3 and 4). The tubular diameters, although reduced, more nearly approximated normal than those in group 2 and the right testes from groups 3 and 4. Fibrosis, interstitial inflammation, vasculitis, and thrombosis were not seen in any of the testes. There were no apparent morphologic abnormalities in the mature spermatids of any animal. Abnormalities in maturation are reported in Table 2. Repopulation Index

Approximately 500 tubules were counted for each testis. A decreased repopulation

index was seen in all doxorubicin-treated animals. Results are summarized in Table 1. No significant differences were found between right and left testes within groups 1 and 2. Within groups 3 and 4, the repopulation index was less in the right than in the left testes (P < 0.001). The repopulation index for the right testes of group 1 was greater than those of groups 2, 3, and 4 (P < O.Ol), but no differences were evident among the right testes of groups 2, 3, and 4. For the left testes, the repopulation index was greater in group 1 than in group 2 (P < O.Ol), but no significant difference was found when groups 3 and 4 were compared to group 1. The repopulation index for the left testes of group 4 was greater than that for group 2 (P < 0.05). Epididymal Index

The results of this semiquantitative evaluation are shown in Table 3. Testes receiving vascular isolation had significantly better scores than unprotected testes.

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FIG.3. Group 3 left testis with somewhat shrunken tubules. Most tubules in this area are populated with germ cells undergoing maturation to the spermatid stage. (H & E, X 130)

DISCUSSION

Many neoplastic diseases affect the younger population and chemotherapy has become an important treatment modality for these malignancies over the last several decades. With recent advances in cancer chemotherapy, the number of long-term survivors has been rising and late toxic effects of many oncolytic agents are now assuming more clinical significance. Prolonged infertility after treatment with chemotherapy in young men is a well-documented phenomenon [8, 91. The incidence of infertility appears to be dose-related [lo], though the data dealing with this topic in man are very limited. Doxorubicin and alkylating agents seem to have particularly severe effects on gonadal function. Several approaches have been used in attempts to circumvent drug-induced toxicity to spermatogenic cells. Prechemotherapy

sperm banking can be offered to those young men with potentially curable malignant diseases. However, since oligospermia and subfertile sperm quality are common in this group of patients, the applicability of this technique is limited [8, 1 I]. Rapidly dividing cell lines, such as the spermatogenic series, are generally more susceptible to cytotoxic agents than are those in a resting state. DTryp-6-LHRH, a luteinizing hormone releasing hormone analog, reversibly lowers blood levels of follicle stimulating hormone and luteinizing hormone, thus slowing or arresting sperm production in mice. This reversible “chemical castration” by D-Tryp-6LHRH has been shown to significantly reduce drug-induced testicular toxicity in this model [ 121, but a similar trial in man was unsuccessful [ 131. Chemical modification of the chemotherapeutic agent to mitigate its testicular toxicity represents an ideal alternative. Modification of the doxorubicin mole-

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FIG. 4. Left testis from Group 4 rat, still containing some sparsely populated tubules. Generally, however, the tubules contain plentiful germ cells and maturation to the spermatid stage is again evident. The tubules remain somewhat shrunken as compared to normal (Fig. 1). (H & E, X 130)

cule, for example, has been reported to allow augmentation of anti-cancer activity with diminution of cardiotoxicity [ 141. There is little evidence, however, that this will be achieved in the near future with respect to gonadal damage. Surgical procedures modifying the regional pharmacokinetics of a toxic agent offer another potential method to protect fer-

tility. Several surgical procedures involving short-term regional ischemia have been used to protect specific organ systems. Vascular isolation and perfusion of limbs with oncolytic agents is a well-established technique which minimizes systemic toxicity and allows very high regional drug levels to be achieved [ 151. Attempts to reduce drug-induced bone marrow injury by using limb

TABLE 2 NUMBEROFTESTESWITH

Group Right testes Left testes Significance

1 (Control)

018 018 NS

MATURATION

2 (Doxorubicin only)

ABNORMALITIES

3 (Doxorubicin + 15 min ischemia)

w3 w-3

NS

Note. Right vs left testis, two sample binomial test. NS, not significant. *p
w 418 *

4 (Doxorubicin + 45 min ischemia)

618 O/8 **

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TABLE 3 EPIDIDYMAL

Right

Left

Right

018 O/8 018 w

018 018 018 8/8

018 w3 O/8 018

Score 0 If 2+ 3+

ON DAY 56

3 (Doxorubicin + 15 min iscbemia)

2 (Doxorubicin only)

1 (Control) Group

INDEX

Right

Left

3/8

l/8

O/8

518 018 O/8

718

318

O/8 O/8

518 O/8

Left

4 (Doxorubicin + 45 min ischemia) Right

Left

018 w3 018 O/8

018 2/8 ($3 O/8

Statistical Analysis Right vs Left Right testes only

NS

NS

P < 0.005

Group 1 vs groups 2, 3, 4-P < 0.00 1

Group 2 vs Group

Group 3 vs Group 2-NS

3-NS

Group 2 vs Group 4-NS

Left testes only

Group 1 vs groups 2, 3, 4-P

< 0.001

3-P

< 0.005

Group 2 vs Group 4-P < 0.00 1

tourniquets [ 161 and occlusive intraaortic balloons [ 171 during drug infusion have been reported. Regional hypothermia or partial vascular occlusion of the scalp by tourniquet during drug administration to alleviate druginduced alopecia has been reported with some success [ 18, 191. In order to modify a drug’s specific toxic effect, an animal model which reproducibly demonstrates that effect is desirable. We have recently established the relationship of doxorubicin dose to testicular toxicity in the rat-an animal model large enough for convenient performance of surgical procedures [5]. In the present study, we describe the protective effect of temporary normothermic testicular circulatory isolation on doxorubitin-induced testicular toxicity. The rat is an attractive model because it can tolerate warm testicular ischemia for up to 90 min without any apparent permanent histologic changes [20]. In addition, the process of spermatogenesis in rats has been elucidated [21]

< 0.005

Group 3 vs Group 4-NS

Group 4 vs Group Group 4 vs Group 3-NS

Group 3 vs Group 2-P

0.001

2-NS

Group 3 vs Group 4-NS

Group 2 vs Group

P <

Group 4 vs Group 2-P < 0.00 1 Group 4 vs Group 3-NS

and provides a way of monitoring the cytotoxic effects of doxorubicin on the testes. Doxorubicin is a common component of many multiagent regimens for the treatment of various tumors. When administered as a single intravenous bolus in man, its plasma concentration decays as a double exponential function of time, with a plasma half-life of 4 to 7 min during the first phase [22, 231. Similar pharmacokinetics have been observed in rats [23]. If the testicular circulation can be isolated from the systemic circulation beginning just before drug administration and continuing for several half-lives of the drug, at the time of testicular reperfusion the plasma concentration of the drug will be a small fraction of the peak concentration. This should result in much less testicular cytotoxicity since a threshold effect exists [5]; below a certain dose, testicular damage is minimal. Other lines of evidence also suggest that peak concentration of doxorubicin is the most important pharmacokinetic character-

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istic in determining cytotoxicity [24]. This easily accessible, the clinical adaptation of a study was carried out to test the validity of technique similar to that outlined here may this hyothesis. represent a method of fertility preservation Male fertility depends on the continuous for young men about to receive chemotherself-renewal of stem cells and their differen- apy. Since nearly all chemotherapy regimens tiation into mature spermatocytes in the se- consist of multiple drug administration miniferous tubules. Following cytotoxic in- cycles, a closed technique would seem prefersults, immediate infertility results from lethal able to the open technique described here. damage to the differentiated spermatocytes. Further studies appear warranted to investiSubsequent return of fertility depends on re- gate the mechanisms of ischemic damage in generation of adequate numbers of stem cells this model in order to extend the tolerable and their maturation into spermatozoa. The period of circulatory isolation. Additional direct morphologic identification of stem areas of interest include examination of the cells is difficult and laborious due to their intratesticular pharmacokinetic changes in paucity and varied morphology. An indirect drug metabolism related to circulatory arrest way of measuring stem cell survival after cy- and studies of other drugs known to damage totoxic treatment involves counting repopuspermatogenic epithelium. Since the semilated (spermatogenic cell-containing) tubules niferous tubules, which make up 90% of and empty tubules at a given interval-S testicular mass in most mammals, are avasweeks in this study. The percentage of re- cular, their high demand for energy and populated tubules reflects the number of nutrient substrates must be met by diffusurviving stem cells. We chose to assay the sion-related mechanisms. This, in turn, is testicular toxicity of doxorubicin on Day 56 dependent on capillary permeability and because the spermatogenic cycle in rats nor- subsequent production of interstitial fluid. mally requires 48 days [2 1] and may be even The impact of testicular circulatory arrest longer after exposure to cytotoxic treatments and anti-cancer agents on these processes is [25]. Fifty-six days should provide sufficient unknown. A potential risk of testicular circutime for the surviving stem cells to regenerate latory arrest is the development of sympaand differentiate into spermatogonia to re- thetic orchiopathy, which has been described populate the tubules. Direct sperm count on following testicular detorsion [26]. This is Day 56 therefore indicates not only the pres- presumed to be autoimmune in origin and ence of surviving stem cells, but also their related to disruption of the blood-testis barability to divide and produce spermatozoa. rier by loss of tight junctions between Sertoli Doxorubicin treatment clearly caused sig- cells, but was not encountered in our experinificant systemic and testicular toxicity in mental preparation. Finally, since fertility this experiment, confirming the adequacy of depends on many factors in addition to the dose used. Complete testicular circulasperm production, mating studies appear estory isolation did provide protection against sential. doxorubicin-induced testicular toxicity. Forty-five minutes of testicular circulatory ACKNOWLEDGMENT arrest gave more protection than 15 min, and The authors express their appreciation to Adria Labomore recent work indicates that this is a reratory (Wilmington, DE) for their generous donation of producible phenomenon (data not shown). It doxorubicin. has been reported that human testes can tolerate up to several hours of warm ischemia REFERENCES from testicular torsion without significant long-term functional impairment [26]. Since 1. Chapman, R. M., Sutcliffe, S. B., Rees, L. H., Edthe testicle and spermatic cord in man are wards, C. R. W., and Malpas, J. S. Cyclical combi-

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