Toxicology Letters, 30 (1986) 253-258 253

Elsevier

TOXLett. 1551

STRAIN AND

DIOCTYLTIN

SEX DIFFERENCES IN SUSCEPTIBILITY OF RATS TO

DICHLORIDE

(Organotin; thymus; immunity)

ANITA L. BOYD and JOE M. JONES*

Departments of Pathology and Microbiology/Immunology Slot 517, University of Arkansas for Medical Sciences, 4301 W. Markham, Little Rock, AR 72205-7199 (U.S.A.)

(Received November 26th, 1985)

(Revision received January 3rd, 1986)

(Accepted January 23rd, 1986)

SUMMARY

Treatment with dioctyltin dichloride (DOTC) induced increased mortality, atrophy of the thymus and

suppression of responses to concanavalin A. Brown Norway (BN) rats were more susceptible than Lewis

(LEW) to the lethal effects of DOTC and males were more susceptible than females. Increased mortality

did not correlate with changes in the histological appearance of the thymus. LEW x BN Ft hybrids

resembled the LEW parent in susceptibility to the lethal effects of DOTC and the BN parent in suscep-

tibility to changes in the thymus.

INTRODUCTION

The toxicity of alkyltin salts has been examined previously [ 1,2]. The short-chain

trialkyl tins (methyland ethyl) are significant neurotoxicants and the dialkyl com-

pounds, particularly butyl, produce lesions in the liver and bile ducts [l]. Alkyltins,

particularly dioctyltin, also affect the immune system [2]. Seinen et al. [3,4] reported

that DOTC induced atrophy of the thymus and suppression of thymus-dependent

immune responses in rats. This compound also exhibited selective toxicity for

thymocytes in vitro [3]. Others have suggested [5] that some alkyltins accumulate

in the thymus in vivo. While differences among species in susceptibility to alkyltin

*To whom reprint requests should be addressed.

Abbreviations: BN, Brown Norway, ConA, concanavalin A; DOTC, di-n-octyhin dichloride; LEW,

Lewis; PFC, plaque-forming cells; SRBC, sheep erythrocytes.

0378-4274/86/$ 03.50 0 Elsevier Science Publishers B.V. (Biomedical Division)

254

were observed in several instances [l-3], reports of sex related differences are uncommon.

METHODS

Animals and treatment BN and LEW rats, 2 to 3 months old were purchased from Charles River

Laboratories (Wilmington, MA). Fr hybrids from LEW females mated with BN males were produced on site. Approximateiy equal numbers of males and females were tested. Water and rat chow were provided ad iib.

DOTC (Crescent Chemical Co., Hauppage, NY) was dissolved in 1: 1 ethanohglycerol [3] at 2 or 4 mg/ml. Rats were injected intraperitonealiy (i.p.) with a dose of 1, 3 or 4 mg DOTC/kg body weight. Control rats were injected with equivalent volumes of solvent.

Histology Rats were weighed, exsanguinated by cardiac puncture and killed by cervical

dislocation under anesthesia. The spIeen and cervical lymph nodes were removed for assays described below. The thymus and brain were weighed and fixed in Bouin’s fixative followed by 10% buffered formalin. The tissues were embedded, sectioned and stained with hematoxylin and eosin using standard procedures and automated equipment.

Antibody production One day after injection of DOTC, each rat was immunized by an i.p. injection

of 4 ml of a 10% suspension of SRBC/kg body weight. Six days later, antibody- producing cells were detected by a plaque assay as described previously [6]. Spleen cells (2 x IO’) suspended in Hanks balanced salt solution were incubated with 0.1 ml 10% SRBC in an agarose matrix (0.6%) on glass slides (triplicate) at 37°C for 45 min. Titrated guinea pig complement (Cappel Laboratories, West Chester, PA) sufficient to wet the surface was added and incubation was continued for 2 h. The number of clear plaques was then counted.

Response to ConA Lymph node cells from each rat were washed and suspended in RPMI-1640

medium containing 10% fetal bovine serum. Triplicate cultures containing 1.25 x

10’ cells and 0.5 pg ConA or no mitogen in volumes of 0.3 ml were incubated at 37°C for 72 h [6]. 1 ,&Zi [3H]thymidine in 10 ~1 was added for the final 6 h. Cultures were harvested onto fiberglass strips and radioactivity was measured.

255

TABLE I

SURVIVAL OF BN AND LEW RATS AFTER INJECTION OF DOTC

Days and number survivingb

Straina 1

BN 218

LEW 8/8

2

O/8

S/8

3 ._.

O/8

3/8

I

O/8

l/8

9

O/8

O/8

MST’

1.25 * 0.16

4.38 + 0.99

a 4 Males and 4 females of each strain injected i.p. with DOTC (2 mg/ml) using a dose of 4 mg/kg body

weight.

b Number surviving longer than indicated day/number tested.

’ Mean survival time f SE. P<O.O5 comparing BN with LEW.

RESULTS

Survival

All BN and LEW rats injected with 4 mg DOTC/kg body weight (using a concen- tration of 2 mg DOTC/ml solvent) died within 9 days but LEW survived significant- ly longer than BN (Table I). The percentage surviving also differed significantly on days 1 and 2 but not on days 3, 7 or 9. All animals injected with an equivalent volume of solvent without DOTC survived. It was found that, when the concentra- tion of DOTC injected was increased to 4 mg/ml, both strains survived longer and this concentration was used for all studies described below.

Using the higher concentration, a difference between males and females was detected (Fig. 1). The survival rates of BN males differed significantly (P<O.OS) from that of females on all days except day 1 after injection of DOTC. LEW males differed significantly from females only on day 7. A significant difference between males of BN (17% survival at day 7) and LEW (58% survival at day 7) was also

/ BN

Days After Injection

Fig. 1. Survival of BN and LEW rats injected i.p. with DOTC. BN (12 males, 1 I females) and LEW (I2

males, 8 females) were injected with DOTC (4 mg/ml) using a dose of 3 or 4 m&kg body wt. Solid lines,

males; broken lines, females. All animals that survived 7 days were killed for other studies.

TABLE II

EFFECTS OF DOTC ON IMMUNOLOGICAL PARAMETERS OF RATS

Strain+

BN

BN

LEW

LEW

Fl

Fl

DOT@ Thymus wt (mg) ConA responsed PFC’ _ I_~~~

None 370 f 20 106818 + 16224 99 ir 22

3 mg/kg 200 -t 41’ 16847 + 4499’ 266 + 45

None 325 f 23 222024 + 39966 136 t 25

3 mgikg 190 + 19’ 20499 + 8201’ 571 i 184

None 390 + 40 Not Done 113 t 13

3 mg/kg 250 I 45 Not Done 705 I 213

a All values are averages of 4 to 12 rats f S.E. Fl, LEW x BN Fl hybrids.

b DOTC (4 mgiml) or equivalent volume of solvent injected i.p.

P<O.O5 when compared with controls injected with solvent.

d Count per minute [3H]thymidine in 1.25 x lo5 lymph node cells collected 7 days after injection of

DOTC and cultured with 0.5 fig ConA for 3 days.

e Plaque-forming cells/Z x 16 spleen cells collected 6 days after injection of SRBC and 7 days after in-

jection of DOTC.

observed. (LEW x BN) Fi hybrids (not shown) behaved like the LEW strain with 56% (5/9) of males surviving 7 days. There were no significant differences in sur- vival of females among the 3 strains. All animals (groups of 4 to 6 for each strain) injected with 1 mg DOTC/kg survived.

~mrnuno~ogi&u~ parameters No significant changes in body weights or spleen weights were detected in any of

the 3 strains when rats injected with DOTC were compared to those injected with solvent alone. The thymic weights and responses to ConA were decreased significantly when BN and LEW rats treated with DOTC were compared to controls (Table II). In contrast, the antibody responses of the 3 strains were increased although the increase was not statistically significant. Responses of rats injected with 1 mg DOTC/kg body weight were comparable to those of controls. No signifi- cant differences between males and females were detected for any of these parameters.

TABLE 111

ALTERATIONS IN HISTOLOGY OF THYMUS IN RATS TREATED WITH DOTC. STRAIN, SEX,

AND DEGREE OF ALTERATIONb

Treatment BN LEW Fl ._ .._~~

M F M F M F

Solvent 0 0 0 0 0 0

DOTC” 0 3+ 4+ 4+ 0 3-t

a Thymus collected 7 days after i.p. injection of 3 mg DOTC/kg wt.

b M, male; F, female, 0, no change compared to controls; 4+, extensive change.

251

Histopathology The thymus glands of the animals indicated in Table II were evaluated

histologically (Table III). There was a correlation between decreased thymic weights and alterations detected histologically. BN rats and Fi hybrids were similar and significant changes were detected only in females treated with DOTC. With LEW rats, significant changes were detected in both males and females. Tissues from rats treated with solvent alone were all normal.

The major change observed in affected tissues was a marked depletion and thin- ning of the cortex. The junction between cortex and medulla was also somewhat obscured. With LEW rats, a significant increase in fibrous connective tissue was also observed in the thymus. Because some organotins are potent neurotoxins [ 1,2,7], the hippocampal formations of brains from the animals were also examined; all were normal. Other areas of the brain were not examined.

DISCUSSION

Rats were reported [3] to be more susceptible to DOTC than were guinea pigs, mice or quail. Chang et al. [7] found Long Evans rats to be more sensitive than Sprague-Dawley rats to the neurotoxic effects of trimethyltin. Most studies, however, have not reported a difference in susceptibility between males and females [1,3,4]. In other instances [8,9] only one sex was tested and a difference could not have been detected. The present studies suggest that it may be of value to test both sexes of more than one strain for the evaluation of a toxic agent.

Three manifestations of toxicity were detected in our rats treated with DOTC (decreased survival, thymus atrophy, decreased response to ConA). The 2 strains tested and the 2 sexes differed in susceptibility to at least 2 of these manifestations. BN and LEW rats differ in a variety of genetically determined traits including coat color, enzyme polymorphisms, histocompatibility alleles and immune responses [lo-121, but whether the susceptibility to DOTC is related to any known difference was not examined. LEW x BN Fi hybrids resembled the LEW parent in suscep- tibility to the lethal effects of DOTC but resembled the BN parent susceptibility to changes in the thymus detected histologically. Interestingly, there was not a concor- dance between mortality and thymus atrophy; a result that was similar to that observed using trimethyltin [6]. As expected, there was a correlation between atrophy of the thymus indicated by decreased weight and alterations detected histologically.

Mortality was greatest for males with all 3 strains tested but BN males were more susceptible than LEW or LEW x BN Fl males. This suggests a possible role for gonadally derived hormones as well as a genetic influence. The effects of castration or administration of estrogens, androgens or other hormones were not examined but could be informative. Previous studies have shown that stress related hormones may be important for the effects of trimethyltin [6] but not for the effects of DOTC [3].

258

A decreased response to Con A by lymphocytes from rats treated with DOTC has been reported [2] and was observed in the present study. In contrast to others [4,8], we did not observe a decrease in the antibody response to SRBC. This may be related to the different routes of administration and times of testing used in the in- vestigations. At the time interval used, we observed a small increase in the antibody response. Others have found [13] that mice treated with dibutyltin or tributyltin were more resistant to growth of mammary tumors even though the animals ex- hibited atrophy of the thymus glands.

ACKNOWLEDGEMENTS

The authors are grateful to Dr. Louis Chang for advice on interpretation of histological sections. Supported by UAMS GSRF.

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