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Toxicity of triorganotin compounds: Comparative in vivo studies with a series of trialkyltin compounds and triphenyltin chloride in male rats
TOXICOLOGY
AND APPLIED
PHARMACOLOGY
s&274-286
(1985)
Toxicity of Triorganotin Compounds: Comparative in Vivo Studies with a Series of Trialkyltin Compounds and Triphenyltin Chloride in Male Rats N. J. SNOEIJ, A. A. J. VAN IERSEL, A. H. PENNINKS,
AND W. SEINEN
Department of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Sciences, Building A9. Biltstraat 172, 35 72 BP, Utrecht, The Netherlands
Received March 21, 1985: accepted July 1, 1985 Toxicity of Triorganotin Compounds: Comparative in Vivo Studies with a Series of Trialkyltin Compounds and Triphenyltin Chloride in Male Bats. SNOELJ, N. J., VAN IERSEL, A. A. J., PENNINKS, A. H., AND SEINEN, W. (1985). Toxicol. Appl. Pharmacol. 81,274-286. In 2-week feeding studies, a series of trialkyltin chlorides and triphenyltin chloride were fed to male weanhng rats at different dietary concentrations to evaluate their toxic effects, especially on the brains and the lymphoid organs, thymus and spleen. The lower trialkyltin homologs, trimethyltin chloride (TMTC) and triethyltin chloride (TETC), were neurotoxic, causing neuronal degradation and cerebral edema, respectively, at dietary concentrations of 15 ppm. The intermediate homologs, tri-npropyltin chloride (TPTC) and tri-n-butyltin chloride (TBTC), and the aromatic compound, triphenyltin chloride (TPhTC), caused a dose-related reduction of thymus weight. At a dietary concentration of 150 ppm decreases in thymus weight to 53, 39, and 81% of controls were found following treatment with TPTC, TBTC, and TPhTC, respectively. Microscopically, thymus atrophy was associated with a lymphocyte depletion in the thymic cortex. Only 16% of the total number of nucleated thymocytes could be isolated from rats fed 150 ppm TBTC. These effects were completely reversed within 2 weeks. Slight thymus atrophy was observed after feeding a relatively high dose of 150 mg tri-n-hexyltin chloride (THTC)/kg diet, whereas tri-n-octyltin chloride (TOTC) was ineffective. A dose-related decrease in spleen weight was noticed after 2 weeks feeding of TPTC, TBTC, and TPhTC. Liver weights were increased in rats fed TBTC, THTC, and TPhTC for 2 weeks. Nevertheless, no enlarged livers and normal spleen weights were found upon feeding 100 ppm TPTC or TBTC for 4 weeks, whereas thymus weight was severely decreased. Therefore, atrophy of the thymus was considered to be the predominant effect of the intermediate trialkyltins (TPTC, TBTC). From this study it is concluded that the lower trialkyltins (TMTC, TETC) are essentially neurotoxic, the intermediate trialkyltins (TPTC, TBTC) and triphenyltin are primarily immunotoxic, and the higher homologs (THTC, TOTC) are only slightly toxic or not toxic at all.
0 1985 Academic press. Inc.
Of the organotin compounds, the diorganotins and the triorganotins especially are used in a variety of technical applications (Ross, 1965). The worldwide production of the organotins was estimated in 1976 to be 27,000 tons per year and is expected to grow to about 63,000 tons in 1986 (WHO, 1980). Triorganotins are widely used as biocides (Piver, 1973). In particular, bis(tri-n-butyltin) oxide (TBTO) is used for the preservation of paper and wood and for antifouling and dis0041-008X/85
$3.00
Copyright Q 1985 by Academic Pmq Inc. AU rights of rqmduction in any form reserved.
infection of surfaces and cooling water. TBTO and tri-n-butyltin fluoride have ken proposed as molluscicides for the control of certain aquatic snails. These snails serve as the intermediate hosts for the trematode parasite that causes schistosomiasis in man (Cardarelli, 1976; Duncan, 1980). Triphenyltin compounds also are successfully used as agricultural fungicides. The mammalian toxicity of the triorganotins was reviewed by Duncan (1980) and in a 274
TOXICITY
OF TRIORGANOTINS
275
recent WHO report ( 1980). The toxicity of the trialkyltin compounds which appeared to be trialkyltin compounds is determined by the immunotoxic, additional features such as rechain length of the alkylgroup. Their acute oral versibility and stress-dependency were investoxicity to rats decreases in the following order: tigated. ethyl > methyl > n-propyl > n-butyl > nhexyl, n-octyl. Triphenyltin is as toxic to METHODS mammals as tributyltin (Barnes and Stoner, 1958; WHO, 1980). Trimethyltin (TMT) is a Compounds. The following organotin compounds were used in this study: trimethyltin chloride (TMTC), triethylpotent and persistent neurotoxicant, inducing chloride (TETC), tri-n-propyltin chloride (TPTC), trisevere tremor, hyperexcitability, and aggres- tin n-butyltin chloride (TBTC), tri-n-hexyltin chloride sion in rats and mice (Barnes and Stoner, (THTC), tri-n-octyltin chloride (TOTC), and triphenyltin 1958). Neuronal degradation and necrosis in chloride (TPhTC). These compounds were kindly provided specific areas, particularly the hippocampus, by Dr. E. J. Bulten and Dr. H. A. Meinema, Institute for are characteristic lesions (Brown et al., 1979; Applied Chemistry TNO, Utrecht, The Netherlands. Their Bouldin et al., 198 1). Intoxication of adult rats purity was more than 98%, as established by thin-layer chromatography according to Kimmel et al., (1977). with triethyltin (TET) results in interstitial Animals and diets. Specific pathogen-free Wistar rats edema in the white matter of the brain and were purchased from the Central Institute for Breeding of spinal cord (Watanabe, 1980). Neonatal ex- Laboratory Animals, TNO, Zeist, The Netherlands. The posure to TET produces reduced brain weights animals were housed in plastic cagesat room temperature and delayed myelinogenesis but no cerebral of 23 + 2°C with 50 to 60% relative humidity, and a constant 12-hr light/dark cycle. The diets were prepared 1 day edema in rats (Reiter et al., 198 1; Ruppert et before the experiment by dissolving the organotin comal., 1983). Tri-n-octyltin is considered to be pounds in 5 ml of acetone and mixing it thoroughly with nontoxic to all living species (Duncan, 1980). a commercial feed (Trouw & Co., Putten, The NetherAlthough growth reduction is a consistent lands). Homogeneous distribution of tin was confirmed by atomic absorption, using a Perkin-Elmer 400 atomic finding (Stoner, 1966; Gaines and Kimbrough, absorption spectrometer with an acetylene-air flame. Diets 1968) triphenyltin (TPhT) appears to have and tap water were constantly available. Provided the orrelatively nonspecific effects. ganotin compounds are stored dry and dark, they are conDespite the large amount of toxicologic sidered to be stable (Blunden and Chapman, 1982). In a diet containing 150 ppm TBTC even after 6 months no data, only recently have the effects of the triorganotins on the immune system been reported. degradation products were found upon chromatographing hexane extracts of the diet (97 to 108% recovery of tin). Ishaaya et al. (1976) reported lymphopenia Two-week study. Randomized groups of 10 male weanand reduction of spleen weight after feeding ling rats, weighing 35 to 50 g, were used. The organotin TBTO or TPhTC to mice. Explorative studies compounds were fed at dietary concentrations of 0, 15, pointed out that TBTC and TPhT induce thy- 50, and 150 ppm. Since TMTC and TETC caused mortality at 50 and 150 ppm, these compounds were given at mus atrophy in rats (Seinen and Penninks, doses of 0, 5, and 15 ppm. Body weight and feed con1979). Atrophy of the thymus was observed sumption were recorded twice a week. All rats that survived as a prominent feature of oral exposure to the experimental period were killed by decapitation, and TBTO or triphenyltin hydroxide (Vos et al., a necropsy was performed. Body weights and the weights 1983, 1984a, 1984b; Krajnc et al., 1984). The of thymus, spleen, liver, kidneys, adrenals, brain, and testes work of Seinen and Willems ( 1976) and Seinen were recorded. To quantify brain edema in rats fed TETC, water content was determined by drying six brains et al. (1977) has demonstrated that thymus ofbrain each group to constant weight. In addition, five spleens atrophy and subsequent immunosuppression obtained from the TBTC- and TPhTC-feeding studies were are the most pronounced effects of some of a dried and, alter digestion in nitric acid, iron content of series of dialkyltin compounds. In the present these spleens was determined by atomic absorption. A standard curve was prepared by dissolving iron sulfate in paper the toxicologic effects of a series of triorthe appropriate solvent. Occult blood was determined acganotin compounds was studied in male rats. cording to Til et al. (1972) in feces of rats fed TBTC. Special attention has been paid to their effects Thymic cell suspensions were made as described by Seinen on brain and the lymphoid organs. For those et al ( 1977).
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SNOEIJ ET AL.
Four-week study. The otganotin compounds TPTC, TBTC, and TOTC were fed to groups of six male weanling rats for a 4-week period at dietary concentrations of 0 and 100 ppm. TPTC and TBTC were given to rats weighing 35 to 40 g, and in a separate experiment TOTC was fed to rats with an initial weight of 45 to 50 g. Experimental conditions were the same as in the 2-week study. The same organs were weighed at the end of treatment. Recovery experiment. Three randomly allocated groups of 60 male rats (35 to 40 g, six rats per cage) were fed 0 or 100 ppm TPTC or 100 ppm TBTC for 4 weeks. After that period animals were returned to the stock diet to investigate the reversibility of the induced thymus atrophy. Body and thymus weight were determined in groups of six rats, killed 0, 2, 7, 9, 14, 21, 28, 35, 42, and 56 days after organotin treatment. Adrenalectomy. An additional study was performed with 12 adrenalectomized, 12 sham-adrenalectomized, and I2 nonoperated male rats (initial weight, 90 to 100 g). The adrenalectomized rats were provided physiological saline ad libitum in addition to tap water and allowed to recover during 3 days of solitary housing. After another 4 days, each group of rats was randomly divided into two groups, receiving 0 and 100 ppm of TBTC in their diet. After I3 days, the rats were killed by decapitation, and body and thymus weights were recorded. Histology. All organs weighed were fixed in 10% buffered formalin and embedded in paraplast, and 5-pm sections were cut according to routine procedures. Sections were stained with hematoxylin and eosin. In addition, thymus glands, spleens, and mesenteric lymph nodes from rats exposed to TBTC and TPhTC were embedded in glycol methacrylate, and 1-pm sections were stained with MayGriinwald-Giemsa. Statistical analysis. The data were analyzed for significance of differences by Student’s t test.
RESULTS Two- Week Study Trimethyltin chloride. All rats fed 50 or 150 ppm died within a few days. Fifteen ppm TMTC caused two deaths and stopped all weight gain in the surviving animals. Relative weights of thymus and spleen were severely decreased in this group, while those of kidneys, adrenals, and testes were significantly increased. Feed intake was reduced to 60% of control, which may partly account for the severe emaciated state of these rats. Five ppm TMTC fed for 2 weeks was tolerated but some growth retardation occurred. Body weight was 8% less than control (p < 0.05). None of the weighed organs were affected.
On microscopic examination, distinct neuronal destruction was found in specific hip pocampal areas and in the pyriform cortex of rats fed 15 ppm TMTC. Many pyknotic nuclei, surrounded by a more eosinophilic cytoplasm, were seen among the granule cells of the fascia dentata (Fig. 1). In the cornu Ammonis a few necrotic nuclei were located in the dorsolateral part only (CA2, CAj,, and a part of CA, ; classification of Lorente de No, 1934). Extensive karyorrhexis was observed in the pyriform cortex and chromatolysis was noticed in certain nuclei of the brainstem (especially mesencephalic trigeminal nucleus). No signs of neuronal degeneration were noticed in the 5-ppm dosage group. Triethyltin chloride. Preliminary feeding studies with 50 or 150 ppm TETC resulted in mortality of 30 and lOO%, respectively. The seven rats that survived the 50-ppm treatment did not grow and showed edematous brains, which were increased in weight compared to normal fed controls (110%; p < 0.05). Fifteen ppm TETC caused a marked growth retardation. Body weight was 30% less than control (p < 0.001). Feed intake was decreased by approximately 30%. Relative weights of thymus and spleen were reduced, while relative adrenal weight was somewhat increased. The relative weights of liver, kidneys, and testes were not affected. In an additional study, brain water content was already increased in rats fed 10 ppm for 2 weeks (control, 79.7 f 0.2%; TETC, 80.2 f 0.3% p < 0.01). Feeding of 5 ppm for 2 weeks resulted in some reduction of growth (13% less than control; p < 0.0 1). At this dose, the recorded organ weights and water content of the brains were not affected. The other triorganotin compounds ap peared to be less toxic when given orally for 2 weeks. Doses up to 150 ppm were tolerated. Body and some of the organ weights of rats fed these compounds are presented in Table 1. Organ weights not presented in the table were not significantly changed except for those mentioned in the text. Tripropyltin chloride. The most prominent effect of TPTC was a strong and dose-related
TOXICITY
OF TRIORGANOTINS
277
FIG. 1. Fascia dentata from a rat fed 15 ppm TMTC for 2 weeks showing scattered neuronal necrosis among the granule cells. Paraplast section; H&E. X208.
decrease of the relative weights of thymus and spleen. All other organs remained unaffected at doses of 15 and 50 ppm. Treating the rats with 150 ppm caused growth retardation and altered weights of liver and brain. At this dosage, thymus weight was reduced 53% of control. Histologic examination revealed a diminishing cellularity in the thymic cortex with increasing doses. No abnormalities were noticed in liver, spleen, and brains. Tributyltin chloride. TBTC induced a doserelated and severe reduction of thymus and spleen weight. At dietary concentrations of 50 and 150 ppm relative liver weight was increased. Associated with a reduced feed intake of about 25%, body and brain weight of rats fed 150 ppm were markedly decreased, whereas relative adrenal weight was slightly increased. Thymus weight was only 39% of control weight in these animals. Microscopically, thymus atrophy was evident as judged by a numerical reduction of cortical lymphocytes, associated with a relative increase of reticulo-epithelial cells (Fig. 2). The
number of mitotic figures was also decreased. However, no signs of increased lymphocyte destruction were observed. When cell suspensions from thymus glands were made and the nucleated cell counts were determined, the severe and dose-dependent lymphocyte depletion could be accurately demonstrated (Table 2). Feeding of 50 ppm reduced the cell count to 50% of control values, while in the 150ppm group only 16% of the cells could be isolated. Despite the reduction in spleen weight, no distinct signs of lymphocyte depletion were observed. Extramedullary hematopoiesis was similar in control and treatment groups. Using atomic absorbance spectrometry, the amount of iron per spleen was decreased in a doserelated manner (Table 2). However, the iron concentration (expressed in micrograms Fe per gram dry spleen weight) was not altered (Table 2). No morphologic changes were observed in the livers. On gross examination, reddening in only a few of the mesenteric lymph nodes was noticed in each dosage group. Microscopically, a dose-related increase in the number of
278
SNOEIJ ET AL. TABLE 1 E@DY AND ORGAN
Compound
Dietary level (ppm)
WEIGHTS
OF RATS FED VARIOUS
TRIORGANOTIN
COMKNJND~
FOR
2 WEEKS’
Relative organ weights (per MY weight, g/kg) Body weight (Iid
TPTC
0 15 50 150
108 + 102+ 105+ 75 f
11 9 8 10***
TBTC
0 15 50 150
119k 1182 114+ 87 +
7 9 7 6***
THTC
0 15 50 150
108 + 106+ 106 f 98+
TOTC
0 50 150
112k 7 1172 8 117 f 10
TPhTC
0 15 50 150
120* 117k 112+ 85 -c
10 9 10 8
7 9 6* 6***
Brain weight (is) 1.57 f 1.55 f 1.55 f 1.41 k
Thymus
Spleen
Liver
0.06 0.07 0.06 0.07***
3.55 + 0.68 2.86 k 0.36** 2.35 zk 0.22*** 1.87 f 0.40***
4.16 1- 0.35 3.68 + 0.40** 3.2 1 t 0.50*** 2.69 + 0.27**’
45.0 45.6 46.9 47.5
+ f f f
3.7 2.2 2.2 2.3*
1.67 1.70 1.64 1.54
f 0.05 AC0.06 f 0.06 f 0.05***
3.76 + 0.44 3.58 + 0.30 2.46 L!I0.4 I*** 1.47 * 0.14***
3.94 3.47 3.17 2.86
f 0.68 + 0.36* f 0.23** zk 0.30***
47.1 48.1 49.4 53.2
f + f f
2.4 2.3 2.6* 4.0***
1.50 1.53 1.55 1.50
+ 0.07 f 0.10 k 0.06 -c 0.08
3.40 3.33 3.22 2.98
3.34 3.10 3.65 3.32
f * + f
47.3 48.7 49.4 51.8
f f f +
2.9 2.8 3.5 3.2**
ND ND ND 1.59 1.56 1.60 1.49
k f + +
0.06 0.09 0.05 0.06***
+ + + +
0.6 I 0.55 0.6 I 0.3 I*
0.47 0.35 0.36 0.33
3.77 f 0.44 3.78 310.26 3.72 f 0.59
3.58 It 0.49 3.46 f 0.48 3.91 + 0.48
46.8 + 2.0 48.4 f 2.6 46.5 f 2.8
3.99 3.46 3.36 3.25
3.39 3.51 2.84 2.77
48.0 48.0 47.7 51.6
+ + f f
0.31 0.39** 0.33*** 0.56***
+ 0.31 + 0.39 +- 0.33** + 0.40***
+ 2.6 -c 3.2 + 2.3 + 3.5**
OX+ SD, 10 animals per group. TPTC, tri-n-propyltin chloride; TBTC, tri-n-butyltin chloride; THTC, tri-n-hexyltin chloride; TOTC, tri-n-octyltin chloride; and TPhTC, triphenyltin chloride. * p < 0.05.
** p < 0.01. *** p < 0.001. ND, not determined.
erythrocytes, situated as rosettes around mononuclear cells, were seen throughout the medullary sinuses. No occult blood could be detected in feces of all TBTC groups. Trihexyltin chloride. Only feeding of 150 ppm THTC resulted in a small decrease in thymus weight and a slight increase in liver weight. At the 50-ppm dose, none of the organ weights differed from controls. Trioctyltin chloride. No treatment-related changes were observed in rats fed 50 or 150 ppm TOTC. Triphenyltin chloride. Decrease in body weight gain was already noticed at 50 ppm TPhTC. Reduction of relative thymus weight occurred at 15 ppm. A dose-related decrease
of spleen weight was also apparent. In the highest dose group (150 ppm), reduction of body and brain weight was observed, while livers were. enlarged. At this dose, thymus weight was reduced to 8 1% of control. On histopathologic examination, some reduction in cellularity of the thymic cortex was observed. The total amount of iron per spleen was diminished at 50 ppm (82%) and 150 ppm (70%). Iron concentration (micrograms Fe per gram dry spleen weight) was not influenced. Structure-Activity
Relationship
In Fig 3A a structure-activity relationship is given for the effect of the trialkyltins on thy-
TOXICITY
OF TRIORGANOTINS
B FIG. 2. Part of a thymus from a rat fed 0 ppm (A) or 100 ppm (B) TBTC for 4 weeks showing a decrease in number of cortical lymphocytes and a relative increase in reticula-epithelial cells. Note the absence of severe cellular destruction. Glycol methacrylate section; May-Griinwald-Giemsa. X400.
279
280
SNOEIJ ET AL. TABLE 2 ABSOLUTE WEIGHTS OF THUS AND SPLEEN, THYMIC CELL COUNT, AND SPLENICIRON CONTENT AND CONCENTRATIONOF RATS FED VARIOUS DOSESOF TBTC FOR 2 WEEKS” Thymus
Dose (ppm) 0 15 50 100 150
Spleen Cell count (X10’)
Weight (mg) 436 422 290 208 127
f + + + +
56 19 54*** 13*** 13***
111 f22 look 3 53 + 11*** 41 +- 5*** 18 f 3***
Weight (mg)
Fe content b w
425 412 357 312 231
68 + 14 62+ 13 54 f 3* 48 + 5** 41 AZ 5**
+ + + + f
71 79 8* 15** 24***
Fe concentration c Wg) 751 + 673 f 692 f 703x? 795 +
145 116 22 71 52
’ zi + SD, five animals per group. b Fe content: cg iron per spleen. ’ Fe concentration: ag iron per g dry spleen weight. * p < 0.05.
** p < 0.01. *** p < 0.001. mus weight. Being primarily neurotoxic, TMTC and TETC are excluded. TPTC is the most active homolog at low dietary concentrations (15 ppm), while TBTC causes more pronounced thymus atrophy at higher doses. Whether the effects are expressed on the basis of ppm or micromoles (Fig. 3B), the structureactivity relationship is generally the same.
V
Four- Week Study The effects of a 4-week feeding period with 100 ppm of TPTC, TBTC, or TOTC in the diet of rats are presented in Table 3. TPTC and TBTC caused reduction of thymus weight to 66 and 43% of the controls, respectively, Body weights were only slightly (TPTC) or
50 . .. l
-150
15 ppm 50 DDrn p*m
100 chain
length
(number
of C-atoms1
**
TETC
25
200 pmol
300 lrialkyltin
400
500
600
per kg feed
FIG. 3. Relationship between length of the alkyl chain of triafkyltin compounds and its effect on thymus weight determined in groups of 10 male rats fed various trialkyltin compounds for 2 weeks. (A) Dietary concentrations expressed in mg alkyltin per kg feed (ppm); (B) dietary concentrations expressed in pmol trialkyltin per kg feed. *.p < 0.05; *a, p < 0.01; ***,p < 0.01.
TOXICITY
281
OF TRIORGANOTINS TABLE 3
BODY AND ORGAN WEIGHTS OF RATS FED 0 OR 100 ppm TFTC, TBTC, OR TOTC FOR 4 WEEKS” BUY weight (9)
Brain weight (9)
Thymus
Spleen
Liver
-
171 (11)
1.63 (0.05)
3.13 (0.27)
2.55 (0.27)
52.29 (1.24)
0.14 (0.04)
TF’TC
154* (11)
(Z)
2.08*** (0.26)
2.40 (0.37)
47.95*** (1.76)
0.15 (0.03)
TBTC
142** (14)
1.58 (0.07)
1.34*** (0.32)
2.35 (0.36)
50.16 (2.53)
0.15 (0.04)
-
232 (9)
1.81 (0.06)
3.18 (0.68)
2.72 (0.20)
52.80 (1.51)
0.16 (0.02)
TOTC
228 (14)
I .74 (0.13)
3.01 (0.24)
2.58 (0.32)
52.75 (4.24)
0.16 (0.03)
Compound
Relative organ weight (g/pg body weight) Adrenals
a X; SD in parentheses. * p < 0.05.
** p < 0.01. *** p < 0.001.
moderately (TBTC) decreased. In contrast to the 2-week studies, no effects were observed on the weights of spleen and liver. The decrease in liver weight found in the lOO-ppm TPTC group is considered to be unrelated to the organotin treatment. TOTC did not influence any of the organ weights that were determined. Histologic examination of tbymus glands of rats fed TPTC or TBTC revealed a severe reduction in lymphocyte density in the cortex (Fig. 2). This feature was even more pronounced than in the 2-week feeding studies with these compounds. Histology of the spleen and liver was normal, except for slight atrophy of the periarteriolar lymphocyte sheaths (PALS) in a few spleens of rats fed TBTC. Recovery Experiment Upon feeding rats 100 ppm TPTC or TBTC for 4 weeks, body weights were significantly reduced up to 3 weeks after terminating the
treatment (Fig. 4A). The severe reduction of thymus weight induced by 100 ppm of both of the trialkyltins was followed by a fast recovery (Fig. 4B). After 1 week of feeding stock diet, thymus weights of the trialkyltin-treated rats did not differ from the controls. In the second week of rehabilitation, thymus weights were even significantly higher than the controls. This response is considered to be a compensatory reaction as is seen in many healing processes. Adrenalectomy Adrenalectomy itself caused some growth retardation, but the subsequent treatment with 100 ppm TBTC in the diet for 13 days did not affect body weight any further. Adrenalectomy also resulted in an increase in thymus weight compared to the nonoperated or sham-adrenalectomized rats. This enlargement is considered to be a consequence of decreased adrenal activity. On careful examination, no adrenal remnants were found in 10 of the 12
282
SNOEIJ ET AL.
Body weight (PI 300
200
I I our ) reeks , of reamlent
100
Stock
I
0
0 mm 100 ppm 100 ppm
TBTC TPTC
,.
4
Relativf thymus WelQht (QlkQI 3.0
6--d
diet
5
6
7
9
a
10
11
12
0 pm 100 ppm 100 PP~
TBTC TPTC
. B
h
I 1 ... I I I. . . I .*.
2.0
.* .
... 1C
I
ii:-i;;;--::; i I
Four , weeks, 01 treatment
Stock
u -
diet
I
oJ;
! 4
5
6
7 ”
6
9
10
11
12
Weeks
FIG 4. Reversibility of TPTC- and TBTC-induced growth retardation (A) and thymus atrophy (B) in rats kept on a 100 ppm of trialkyltin diet for 4 weeks and subsequently 0 to 8 weeks on stock diet (X & SD of six rats per group). *, p -z 0.05; **, p < 0.001; L**, p < 0.001.
adrenalectomized rats. As is demonstrated in Fig. 5, adrenalectomy did not abolish the TBTC-induced thymus atrophy. The relative thymus weight of these rats was decreased to the same extent as the nonoperated or shamadrenalectomized rats.
DISCUSSION From the present study it appears that the lower trialkyltins, TMTC and TETC, are essentially neurotoxic. The intermediate trialkyltins, TFTC and TBTC, together with tri-
TOXICITY
OF TRIORGANOTINS
1 l .
0 100
tIDm
TBTC
ADRENAL i
FIG. 5. Relative thymus weight of nonoperated, shamadrenalectomized, and adrenalectomized rats fed 0 or 100 ppm of TBTC for 13 days. *, p < 0.05; **, p < 0.0 1. ’ Six animals per group. b Five animals per group.
phenyltin are immunotoxic. Of the higher homologs, THTC is only slightly immunotoxic, while TOTC is not toxic at all. In 2-week oral exposure studies, TMTC was the most toxic homolog, causing deaths at dietary concentrations of 15 ppm (20%) and 50 ppm ( 100%). In contrast to acute toxicity data (Barnes and Stoner, 1958; WHO, 1980), TETC was less lethal than TMTC. Fifteen ppm of both compounds caused severe emaciation, characterized by reduction (TETC) or absence (TMTC) of growth. Feed avoidance may, at least in part, be the cause of the emaciated status. Fifteen ppm TMTC resulted in neuronal degeneration in specific areas of the central nervous system (CNS). The major pathologic features, which were described previously (Brown et al., 1979; Bouldin et al., 198 1; Chang and Dyer, 1983), consisted of pyknosis in the hippocampal formation, karyorrhexis in the pyriform cortex, and chromatolysis of certain brain stem nuclei, in particular the mesencephalic trigeminal nucleus. More detailed examination, however, revealed marked differences. Extensive neuronal destruction was seen in the fascia dentata in contrast to the observations of Bouldin et al. (198 l), who found this region to be virtually unaffected after daily oral dosing of 1 mg TMT/kg body weight given for 2 weeks.
283
Moreover, much less neuronal damage was observed in the comu Ammonis, although Brown et al. (1979) and Bouldin et al. (198 1) described this area as most severly affected upon repeated oral TMT administrations. The few necrotic cells which were found in the Ammon’s horn were mainly located in the dorsolateral areas (CA2, CA&, which are usually preserved upon TMT intoxication (Brown et al., 1979; Bouldin et ul., 198 1; Chang et al., 1983). Rat strain and dose regimen are important in the pathology of TMT poisoning (Chang et al., 1983) and may therefore account for the differences mentioned above. The observed toxicity of TETC is generally in accordance with the original findings of Magee et al. (1957). Feeding of 20 ppm triethyltin hydroxide resulted in rapid weight loss during the first 2 weeks, in which time interstitial edema of the CNS developed. Two weeks feeding of 50 ppm TETC obviously caused this type of neurotoxicity, as demonstrated by the rise in absolute brain weight. Rats fed 10 or 15 ppm showed growth retardation and increase in brain water content. No signs of neurotoxicity were observed at the 5-ppm dose of either TMTC or TETC, or with any of the other triorganotin compounds. Little attention has been paid to the effects of TMT or TET on lymphoid organs. In one study (Hioe and Jones, 1984), decreases in thymus and spleen weights were reported after ip administration of 10 mg TMT/kg. Adrenalectomy abolished these effects and therefore stress-mediated immunotoxicity was suggested. The atrophy of thymus and spleen observed in this study in the 15-ppm groups of TMTC and TETC are therefore most likely caused by stress and emaciation. The increase in adrenal weight supports this assumption. Five ppm of these compounds did not effect the lymphoid organs. The most striking effects on the lymphoid system were noticed for the intermediate trialkyltins, TF’TC and TBTC. Feeding these compounds for 2 weeks resulted in a dose-related reduction of thymus and spleen weight
284
SNOEIJ ET AL.
(Table 1). Histologic examination revealed that thymus atrophy is associated with severe lymphocyte depletion in the cortical area (Table 2). By preparing cell suspensions from thymus glands of treated rats, the loss of lymphocytes could be adequately affirmed (Table 2). Histopathologic examination of the spleens revealed no abnormalities. The decrease of iron content suggest a reduction of the red pulp. The subsequent finding that the iron concentration was not affected may be explained by a growth retardation of the whole spleen. Four-week treatment with TPTC or TBTC ( 100 ppm for both) severely affected thymus weight (Fig. 3), whereas spleens, livers, and other organs were normal (Table 3). Therefore, it is concluded that thymus atrophy induced by the intermediate trialkyltins is a selective effect. Early effects on spleen and liver are significant but transient and are considered of little toxicologic importance. A selective thymus atrophy was also noticed by Krajnc et al. (1984) and Vos et al. (1984a) upon oral treatment of rats with TBTO for 4 weeks. Spleen and liver weights were not affected up to TBTO concentrations of 80 ppm in male and 320 ppm in female rats. Erythrocyte rosettes in the sinuses of mesenteric lymph nodes were found by these authors. In this study a dose-related increase of rosettes was seen within only some of the mesenteric lymph nodes upon 2-week feeding of TBTC, but not of TPhTC. Although a test for the detection of occult blood in the feces was negative, microhemorrhages could be responsible for the presence of erythrocytes in the lymph circulation. Since no erythrophagocytosis was observed, a phagocytosis defect may underly the rosette formation. The higher trialkyltin homologs induce a slight decrease in weight of the thymus (THTC, 150 ppm) or do not affect it at all (TOTC). Although immunotoxicity was not the subject of their study, Barnes and Stoner (1958) showed a lack of toxicity of TOTC upon oral administration to rats. The effects of the aromatic triorganotin,
TPhTC, resembled those of TPTC and TBTC, but appeared to be less severe. Reductions of thymus and spleen weights were noticed at 15 and 50 ppm, respectively. Our data confirm the similar findings of Vos et al. ( 1983, 1984b) with triphenyltin hydroxide. As was described for the latter compound (Gaines and Rimbrough, 1968), TPhTC reduced weight gain at lower dietary concentrations than did the intermediate trialkyltins. A structure-activity relationship (SAR) for the intermediate and higher trialkyltin homologs with regard to thymus atrophy is presented in Fig. 3. Seinen et al. (1977) have presented a similar SAR for the dialkyltins. Dibutyltin and dioctyltin compounds have most pronounced effects on the thymus, while homologs with 12 or 18 carbon atoms per chain were inactive. As is suggested for the longchain dialkyltins, reduced intestinal uptake may render the higher trialkyltin homologs inactive. The differences in effect when 15 ppm TPTC or TBTC are compared might be related to a difference in absorption from the gut. Possible immunotoxic effects of TMTC or TETC are overshadowed by their neurotoxicity and are therefore left out of the SAR. Although TPTC- and TBTC-induced thymus atrophy is severe, the effects were completely reversible (Fig. 4). Within 1 week after the diets were changed to normal feed, thymus weight was at control values. A significant rebound was observed in the second week of recovery. Seinen et al. ( 1977) observed a similar recovery pattern of the thymus after feeding di-n-octyltin dichloride to rats. The rapid recovery suggests a short biological half-life of these organotins. Evans et al. (1979), using [14C]TBT0, indicated a rapid clearance from the body of mice when ingestion of TBTO was stopped. No exact data are available however. Although thymus atrophy can be induced by stress-related release of glucocorticosteroids (Keller et al., 1983) the effects of TBTC are not caused by stress for the following reasons. Thymus weight was as severely decreased in adrenalectomized as in sham-adrenalecto-
TOXICITY
285
OF TRIORGANOTINS
mized, TBTC-fed rats (Fig. 5). Moreover in the 2- and 4-week feeding studies relative adrenal weights were normal, except for the highest dose group of TBTC (150 ppm for 2 weeks), in which a slight weight increase was noticed. Histologically, no increased destruction of lymphocytes in the thymus was found, as is seen when glucocorticosteroid concentrations are increased. Therefore the observed depletion of thymic lymphocytes is not primarily caused by their destruction. The severe cellular destruction and decrease in cell viability noticed by Vos et al. (1984a) is possibly a secondary effect of TBTO inducing stressmediated lympholysis, since cell death was only observed at a very high dose of 320 ppm. The mechanisms by which the triorganotins induce neurotoxicity (TMTC, TETC) or immunotoxicity (TPTC, TBTC, TPhTC) are unknown. Since thymus atrophy is a sensitive parameter for immunological damage (Seinen et al., 1977; Seinen and Penninks, 1979; Vos et al., 1984a; 1984b), the mechanism of triorganotin-induced thymus atrophy is subject of further study. ACKNOWLEDGMENTS The authors thank Dr. E. J. Bulten and Dr. H. A. Meinema, Institute for Applied Chemistry, TNO, Utrecht, for the supply of organotin samples. The technical assistance of Mr. V. Rutten and Mr. F. Snel is greatly appreciated. This research was supported by the Netherlands Foundation for Technical Research (STW), Future Technical Science Branch/Division of the Netherlands Organization for the Advancement of Pure Research (ZWO).
REFERENCES BARNES, J. M., AND STONER, H. B. (1958). Toxic properties of some dialkyl and trialkyl tin salts. &it. J. Med. 15, 15-22. BLUNDEN, S. J., AND CHAPMAN, A. H. (1982). The environmental degradation of organotin compounds-A review. Environ. Technol. Lett. 3, 261-272. BOULDIN, T. W., GOINES, N. D., BAGNELL, C. R., AND KRIGMAN, M. R. (198 1). Pathogenesis of trimethyltin neuronal toxicity. Ultrastructural and cytochemical observations. Amer. J. Pathol. 104, 237-249. BROWN, A. W., ALDRIDGE, W. N., STREET, B. W., AND VERSCHOYLE, R. D. (1979). The behavioral and neuropathologic sequelae of intoxication by trimethyltin compounds in the rat. Amer. J. Pathol. 97, 59-82.
CARDARELLI, N. ( 1976). Controlled-Release Pesticide Formulations. CRC Press, Cleveland, Ohio. CHANG, L. W., WANGER, G. R., MCMILLAN, D. E., AND DYER, R. S. (1983). Species and strain comparison of acute neurotoxic effects of trimethyhin in mice and rats. Neurobehav.
Toxicol.
Teratol.
5,331-350.
CHANG, L. W., AND DYER, R. S. (1983). A time-course study of trimethyltin induced neuropathology in rats. Neurobehav.
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5,443-459.
DUNCAN, J. (1980). The toxicology of molluscicides. The organotins. Pharmacol. Ther. 10,407-429. EVANS, W. H., CARDARELLI, N. F., AND SMITH, D. J. ( 1979). Accumulation and excretion of ( l- 14C) bis(trin-butyltin)oxide in mice. J. Toxicol. Environ. Health 5, 871-877. GAINES, T. B., AND KIMBROUGH, R. D. (1968). Toxicity of fentin hydroxide to rats. Toxicol. Appl. Pharmacol. 12,397-403.
HIOE, K. M., AND JONES, J. M. (1984). Effects of trimethyltin on the immune system of rats. Toxicol. Lett. 20, 3 17-323. ISHAAYA, I., ENGEL, J. L., AND CASIDA, J. E. (1976). Dietary triorganotins affect lymphatic tissues and blood composition of mice. Pestic. Biochem. Physiol. 6,270279. KELLER, S. E., WEISS, J. M., SCHLEIFER,S. J., MILLER, N. E., AND STEIN, M. (1983). Stress-induced suppression in immunity of adrenalectomized rats. Science (Washington, D.C.) 221, 1301-1304. KIMMEL, E. C., FISH, R. H., AND CASIDA, J. E. (1977). Bioorganotin chemistry. Metabolism of organotin compounds in microsomal monooxygenase systems and in mammals. J. Agric. Food Chem. 25, l-9. KRAJNC, E. I., WESTER,P. W., LOEBER,J. G., VAN LEEUWEN, F. X. R., Vos, J. G., VAESSEN,H. A. M. G.. AND VAN DER HEIJDEN, C. A. (1984). Toxicity of bis(tri-nbutyltin)oxide in the rat. I. Short-term effects on general parameters and on the endocrine and lymphoid systems. Toxicol.
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75,363-386.
LORENTE DE N6, R. (1934). Studies on the structure of the cerebral cortex. II. Continuation of the study of the ammonic system. J. Physiol. Neurol. 46, 113-177. MAGEE, P. N., STONER,H. B., AND BARNES,J. M. (1957). The experimental production of oedema on the central nervous system of the rat by triethyltin compounds. J. Path. Bacterial. 73, 107-124. PIVER, W. T. (1973). Organotin compounds: Industrial applications and biological investigations. Env. Health Perspect.
4, 6 I-79.
REITER, L. W., HEAVNER, G. B., DEAN, K. F., AND RUPPERT, P. H. (1981). Developmental and behavioral effects of early postnatal exposure to triethyltin in rats. Neurobehav.
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Teratol.
3,285-293.
ROSS, A. (1965). Industrial applications of organotin compounds. Ann. N. Y. Acad. Sci. 125, 107-123. RUPPERT,P. H., DEAN, K. F., ANDREITER, L. W. (1983). Comparative developmental toxicity of triethyhin using
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split-litter and whole-litter dosing. J. Toxicol. Environ. Health 12, 73-87. SEINEN, W., AND WILLEMS, M. 1. (1976). Toxicity of organotin compounds. I. Atrophy ofthymus and thymusdependent lymphoid tissue in rats fed di-n-octyltindichloride. Toxicol. Appl. Pharmacol. 35, 63-75. SEINEN, W., Vos, J. G., SPANJE, I. VAN, SNOEK, M., BRANDS, R., AND HOOYKAAS, H. (1977). Toxicity of organotin compounds. II. Comparative in vivo and in vitro studies with various organotin and organolead compounds in different animal species with special emphasis on lymphocyte cytotoxicity. Toxicol. Appl. Pharmacol. 42, 197-2 12. SEINEN, W., AND PENNINKS, A. H. (1979). Immune suppression as a consequence of a selective cytotoxic activity of certain organometallic compounds on thymus and thymus-dependent lymphocytes. Ann. N. Y Acad. Sci. 320, 499-5 17. STONER, H. B. (1966). Toxicity of triphenyltin. Brit. J. Indust. Med. 23,222-229. TIL, H. P., FERON, V. J., AND DE GROOT, A. P. (1972). The toxicity of sulphur. I. Long-term feeding and multigeneration studies in rats. Food Cosm. Toxicol. 20, 291-310.
Vos, J. G., KRAJNC, E. I., BEEKHOF, P. K., AND VAN LOGTEN, M. J. (1983). Methods for testing immune effects of toxic chemicals: Evaluation of the immunotoxicity of various pesticides in the rat. In IUPAC Pesticide Chemistry. Human Welfare and the Environment (J. Miyamoto et al.. eds.), pp. 497-504; Pergamon, Oxford. Vos, J. G., DE KLERK, A., KRAJNC, E. I., KRUIZINGA, W., VAN OMMEN, B., AND ROZMG, J. (1984a). Toxicity of bis(tri-n-butyltin)oxide in the rat. II. Suppression of thymus-dependent immune responsesand of parameters of nonspecific resistance after short-term exposure. Toxicol. Appi. Pharmacol. 75, 387-408. Vos, J. G., VAN LOGTEN, M. J., KREEFTENBERG,J. G., AND KRUIZINGA, W. (1984b). Effect of triphenyltin hydroxide on the immune system of the rat. Toxicology 29, 325-336. WATANABE, I. (1980). Organotins. In Experimental and Clinical Neurotoxicology (P. S. Spencer and H. H. Schaumberg, eds.), pp. 545-557. Williams and Wilkins, Baltimore. WHO. (1980). Tin and Organotin Compounds. A Preliminary Review. Environmental Health Criteria 15. WHO, Geneva.
AND APPLIED
PHARMACOLOGY
s&274-286
(1985)
Toxicity of Triorganotin Compounds: Comparative in Vivo Studies with a Series of Trialkyltin Compounds and Triphenyltin Chloride in Male Rats N. J. SNOEIJ, A. A. J. VAN IERSEL, A. H. PENNINKS,
AND W. SEINEN
Department of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Sciences, Building A9. Biltstraat 172, 35 72 BP, Utrecht, The Netherlands
Received March 21, 1985: accepted July 1, 1985 Toxicity of Triorganotin Compounds: Comparative in Vivo Studies with a Series of Trialkyltin Compounds and Triphenyltin Chloride in Male Bats. SNOELJ, N. J., VAN IERSEL, A. A. J., PENNINKS, A. H., AND SEINEN, W. (1985). Toxicol. Appl. Pharmacol. 81,274-286. In 2-week feeding studies, a series of trialkyltin chlorides and triphenyltin chloride were fed to male weanhng rats at different dietary concentrations to evaluate their toxic effects, especially on the brains and the lymphoid organs, thymus and spleen. The lower trialkyltin homologs, trimethyltin chloride (TMTC) and triethyltin chloride (TETC), were neurotoxic, causing neuronal degradation and cerebral edema, respectively, at dietary concentrations of 15 ppm. The intermediate homologs, tri-npropyltin chloride (TPTC) and tri-n-butyltin chloride (TBTC), and the aromatic compound, triphenyltin chloride (TPhTC), caused a dose-related reduction of thymus weight. At a dietary concentration of 150 ppm decreases in thymus weight to 53, 39, and 81% of controls were found following treatment with TPTC, TBTC, and TPhTC, respectively. Microscopically, thymus atrophy was associated with a lymphocyte depletion in the thymic cortex. Only 16% of the total number of nucleated thymocytes could be isolated from rats fed 150 ppm TBTC. These effects were completely reversed within 2 weeks. Slight thymus atrophy was observed after feeding a relatively high dose of 150 mg tri-n-hexyltin chloride (THTC)/kg diet, whereas tri-n-octyltin chloride (TOTC) was ineffective. A dose-related decrease in spleen weight was noticed after 2 weeks feeding of TPTC, TBTC, and TPhTC. Liver weights were increased in rats fed TBTC, THTC, and TPhTC for 2 weeks. Nevertheless, no enlarged livers and normal spleen weights were found upon feeding 100 ppm TPTC or TBTC for 4 weeks, whereas thymus weight was severely decreased. Therefore, atrophy of the thymus was considered to be the predominant effect of the intermediate trialkyltins (TPTC, TBTC). From this study it is concluded that the lower trialkyltins (TMTC, TETC) are essentially neurotoxic, the intermediate trialkyltins (TPTC, TBTC) and triphenyltin are primarily immunotoxic, and the higher homologs (THTC, TOTC) are only slightly toxic or not toxic at all.
0 1985 Academic press. Inc.
Of the organotin compounds, the diorganotins and the triorganotins especially are used in a variety of technical applications (Ross, 1965). The worldwide production of the organotins was estimated in 1976 to be 27,000 tons per year and is expected to grow to about 63,000 tons in 1986 (WHO, 1980). Triorganotins are widely used as biocides (Piver, 1973). In particular, bis(tri-n-butyltin) oxide (TBTO) is used for the preservation of paper and wood and for antifouling and dis0041-008X/85
$3.00
Copyright Q 1985 by Academic Pmq Inc. AU rights of rqmduction in any form reserved.
infection of surfaces and cooling water. TBTO and tri-n-butyltin fluoride have ken proposed as molluscicides for the control of certain aquatic snails. These snails serve as the intermediate hosts for the trematode parasite that causes schistosomiasis in man (Cardarelli, 1976; Duncan, 1980). Triphenyltin compounds also are successfully used as agricultural fungicides. The mammalian toxicity of the triorganotins was reviewed by Duncan (1980) and in a 274
TOXICITY
OF TRIORGANOTINS
275
recent WHO report ( 1980). The toxicity of the trialkyltin compounds which appeared to be trialkyltin compounds is determined by the immunotoxic, additional features such as rechain length of the alkylgroup. Their acute oral versibility and stress-dependency were investoxicity to rats decreases in the following order: tigated. ethyl > methyl > n-propyl > n-butyl > nhexyl, n-octyl. Triphenyltin is as toxic to METHODS mammals as tributyltin (Barnes and Stoner, 1958; WHO, 1980). Trimethyltin (TMT) is a Compounds. The following organotin compounds were used in this study: trimethyltin chloride (TMTC), triethylpotent and persistent neurotoxicant, inducing chloride (TETC), tri-n-propyltin chloride (TPTC), trisevere tremor, hyperexcitability, and aggres- tin n-butyltin chloride (TBTC), tri-n-hexyltin chloride sion in rats and mice (Barnes and Stoner, (THTC), tri-n-octyltin chloride (TOTC), and triphenyltin 1958). Neuronal degradation and necrosis in chloride (TPhTC). These compounds were kindly provided specific areas, particularly the hippocampus, by Dr. E. J. Bulten and Dr. H. A. Meinema, Institute for are characteristic lesions (Brown et al., 1979; Applied Chemistry TNO, Utrecht, The Netherlands. Their Bouldin et al., 198 1). Intoxication of adult rats purity was more than 98%, as established by thin-layer chromatography according to Kimmel et al., (1977). with triethyltin (TET) results in interstitial Animals and diets. Specific pathogen-free Wistar rats edema in the white matter of the brain and were purchased from the Central Institute for Breeding of spinal cord (Watanabe, 1980). Neonatal ex- Laboratory Animals, TNO, Zeist, The Netherlands. The posure to TET produces reduced brain weights animals were housed in plastic cagesat room temperature and delayed myelinogenesis but no cerebral of 23 + 2°C with 50 to 60% relative humidity, and a constant 12-hr light/dark cycle. The diets were prepared 1 day edema in rats (Reiter et al., 198 1; Ruppert et before the experiment by dissolving the organotin comal., 1983). Tri-n-octyltin is considered to be pounds in 5 ml of acetone and mixing it thoroughly with nontoxic to all living species (Duncan, 1980). a commercial feed (Trouw & Co., Putten, The NetherAlthough growth reduction is a consistent lands). Homogeneous distribution of tin was confirmed by atomic absorption, using a Perkin-Elmer 400 atomic finding (Stoner, 1966; Gaines and Kimbrough, absorption spectrometer with an acetylene-air flame. Diets 1968) triphenyltin (TPhT) appears to have and tap water were constantly available. Provided the orrelatively nonspecific effects. ganotin compounds are stored dry and dark, they are conDespite the large amount of toxicologic sidered to be stable (Blunden and Chapman, 1982). In a diet containing 150 ppm TBTC even after 6 months no data, only recently have the effects of the triorganotins on the immune system been reported. degradation products were found upon chromatographing hexane extracts of the diet (97 to 108% recovery of tin). Ishaaya et al. (1976) reported lymphopenia Two-week study. Randomized groups of 10 male weanand reduction of spleen weight after feeding ling rats, weighing 35 to 50 g, were used. The organotin TBTO or TPhTC to mice. Explorative studies compounds were fed at dietary concentrations of 0, 15, pointed out that TBTC and TPhT induce thy- 50, and 150 ppm. Since TMTC and TETC caused mortality at 50 and 150 ppm, these compounds were given at mus atrophy in rats (Seinen and Penninks, doses of 0, 5, and 15 ppm. Body weight and feed con1979). Atrophy of the thymus was observed sumption were recorded twice a week. All rats that survived as a prominent feature of oral exposure to the experimental period were killed by decapitation, and TBTO or triphenyltin hydroxide (Vos et al., a necropsy was performed. Body weights and the weights 1983, 1984a, 1984b; Krajnc et al., 1984). The of thymus, spleen, liver, kidneys, adrenals, brain, and testes work of Seinen and Willems ( 1976) and Seinen were recorded. To quantify brain edema in rats fed TETC, water content was determined by drying six brains et al. (1977) has demonstrated that thymus ofbrain each group to constant weight. In addition, five spleens atrophy and subsequent immunosuppression obtained from the TBTC- and TPhTC-feeding studies were are the most pronounced effects of some of a dried and, alter digestion in nitric acid, iron content of series of dialkyltin compounds. In the present these spleens was determined by atomic absorption. A standard curve was prepared by dissolving iron sulfate in paper the toxicologic effects of a series of triorthe appropriate solvent. Occult blood was determined acganotin compounds was studied in male rats. cording to Til et al. (1972) in feces of rats fed TBTC. Special attention has been paid to their effects Thymic cell suspensions were made as described by Seinen on brain and the lymphoid organs. For those et al ( 1977).
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Four-week study. The otganotin compounds TPTC, TBTC, and TOTC were fed to groups of six male weanling rats for a 4-week period at dietary concentrations of 0 and 100 ppm. TPTC and TBTC were given to rats weighing 35 to 40 g, and in a separate experiment TOTC was fed to rats with an initial weight of 45 to 50 g. Experimental conditions were the same as in the 2-week study. The same organs were weighed at the end of treatment. Recovery experiment. Three randomly allocated groups of 60 male rats (35 to 40 g, six rats per cage) were fed 0 or 100 ppm TPTC or 100 ppm TBTC for 4 weeks. After that period animals were returned to the stock diet to investigate the reversibility of the induced thymus atrophy. Body and thymus weight were determined in groups of six rats, killed 0, 2, 7, 9, 14, 21, 28, 35, 42, and 56 days after organotin treatment. Adrenalectomy. An additional study was performed with 12 adrenalectomized, 12 sham-adrenalectomized, and I2 nonoperated male rats (initial weight, 90 to 100 g). The adrenalectomized rats were provided physiological saline ad libitum in addition to tap water and allowed to recover during 3 days of solitary housing. After another 4 days, each group of rats was randomly divided into two groups, receiving 0 and 100 ppm of TBTC in their diet. After I3 days, the rats were killed by decapitation, and body and thymus weights were recorded. Histology. All organs weighed were fixed in 10% buffered formalin and embedded in paraplast, and 5-pm sections were cut according to routine procedures. Sections were stained with hematoxylin and eosin. In addition, thymus glands, spleens, and mesenteric lymph nodes from rats exposed to TBTC and TPhTC were embedded in glycol methacrylate, and 1-pm sections were stained with MayGriinwald-Giemsa. Statistical analysis. The data were analyzed for significance of differences by Student’s t test.
RESULTS Two- Week Study Trimethyltin chloride. All rats fed 50 or 150 ppm died within a few days. Fifteen ppm TMTC caused two deaths and stopped all weight gain in the surviving animals. Relative weights of thymus and spleen were severely decreased in this group, while those of kidneys, adrenals, and testes were significantly increased. Feed intake was reduced to 60% of control, which may partly account for the severe emaciated state of these rats. Five ppm TMTC fed for 2 weeks was tolerated but some growth retardation occurred. Body weight was 8% less than control (p < 0.05). None of the weighed organs were affected.
On microscopic examination, distinct neuronal destruction was found in specific hip pocampal areas and in the pyriform cortex of rats fed 15 ppm TMTC. Many pyknotic nuclei, surrounded by a more eosinophilic cytoplasm, were seen among the granule cells of the fascia dentata (Fig. 1). In the cornu Ammonis a few necrotic nuclei were located in the dorsolateral part only (CA2, CAj,, and a part of CA, ; classification of Lorente de No, 1934). Extensive karyorrhexis was observed in the pyriform cortex and chromatolysis was noticed in certain nuclei of the brainstem (especially mesencephalic trigeminal nucleus). No signs of neuronal degeneration were noticed in the 5-ppm dosage group. Triethyltin chloride. Preliminary feeding studies with 50 or 150 ppm TETC resulted in mortality of 30 and lOO%, respectively. The seven rats that survived the 50-ppm treatment did not grow and showed edematous brains, which were increased in weight compared to normal fed controls (110%; p < 0.05). Fifteen ppm TETC caused a marked growth retardation. Body weight was 30% less than control (p < 0.001). Feed intake was decreased by approximately 30%. Relative weights of thymus and spleen were reduced, while relative adrenal weight was somewhat increased. The relative weights of liver, kidneys, and testes were not affected. In an additional study, brain water content was already increased in rats fed 10 ppm for 2 weeks (control, 79.7 f 0.2%; TETC, 80.2 f 0.3% p < 0.01). Feeding of 5 ppm for 2 weeks resulted in some reduction of growth (13% less than control; p < 0.0 1). At this dose, the recorded organ weights and water content of the brains were not affected. The other triorganotin compounds ap peared to be less toxic when given orally for 2 weeks. Doses up to 150 ppm were tolerated. Body and some of the organ weights of rats fed these compounds are presented in Table 1. Organ weights not presented in the table were not significantly changed except for those mentioned in the text. Tripropyltin chloride. The most prominent effect of TPTC was a strong and dose-related
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OF TRIORGANOTINS
277
FIG. 1. Fascia dentata from a rat fed 15 ppm TMTC for 2 weeks showing scattered neuronal necrosis among the granule cells. Paraplast section; H&E. X208.
decrease of the relative weights of thymus and spleen. All other organs remained unaffected at doses of 15 and 50 ppm. Treating the rats with 150 ppm caused growth retardation and altered weights of liver and brain. At this dosage, thymus weight was reduced 53% of control. Histologic examination revealed a diminishing cellularity in the thymic cortex with increasing doses. No abnormalities were noticed in liver, spleen, and brains. Tributyltin chloride. TBTC induced a doserelated and severe reduction of thymus and spleen weight. At dietary concentrations of 50 and 150 ppm relative liver weight was increased. Associated with a reduced feed intake of about 25%, body and brain weight of rats fed 150 ppm were markedly decreased, whereas relative adrenal weight was slightly increased. Thymus weight was only 39% of control weight in these animals. Microscopically, thymus atrophy was evident as judged by a numerical reduction of cortical lymphocytes, associated with a relative increase of reticulo-epithelial cells (Fig. 2). The
number of mitotic figures was also decreased. However, no signs of increased lymphocyte destruction were observed. When cell suspensions from thymus glands were made and the nucleated cell counts were determined, the severe and dose-dependent lymphocyte depletion could be accurately demonstrated (Table 2). Feeding of 50 ppm reduced the cell count to 50% of control values, while in the 150ppm group only 16% of the cells could be isolated. Despite the reduction in spleen weight, no distinct signs of lymphocyte depletion were observed. Extramedullary hematopoiesis was similar in control and treatment groups. Using atomic absorbance spectrometry, the amount of iron per spleen was decreased in a doserelated manner (Table 2). However, the iron concentration (expressed in micrograms Fe per gram dry spleen weight) was not altered (Table 2). No morphologic changes were observed in the livers. On gross examination, reddening in only a few of the mesenteric lymph nodes was noticed in each dosage group. Microscopically, a dose-related increase in the number of
278
SNOEIJ ET AL. TABLE 1 E@DY AND ORGAN
Compound
Dietary level (ppm)
WEIGHTS
OF RATS FED VARIOUS
TRIORGANOTIN
COMKNJND~
FOR
2 WEEKS’
Relative organ weights (per MY weight, g/kg) Body weight (Iid
TPTC
0 15 50 150
108 + 102+ 105+ 75 f
11 9 8 10***
TBTC
0 15 50 150
119k 1182 114+ 87 +
7 9 7 6***
THTC
0 15 50 150
108 + 106+ 106 f 98+
TOTC
0 50 150
112k 7 1172 8 117 f 10
TPhTC
0 15 50 150
120* 117k 112+ 85 -c
10 9 10 8
7 9 6* 6***
Brain weight (is) 1.57 f 1.55 f 1.55 f 1.41 k
Thymus
Spleen
Liver
0.06 0.07 0.06 0.07***
3.55 + 0.68 2.86 k 0.36** 2.35 zk 0.22*** 1.87 f 0.40***
4.16 1- 0.35 3.68 + 0.40** 3.2 1 t 0.50*** 2.69 + 0.27**’
45.0 45.6 46.9 47.5
+ f f f
3.7 2.2 2.2 2.3*
1.67 1.70 1.64 1.54
f 0.05 AC0.06 f 0.06 f 0.05***
3.76 + 0.44 3.58 + 0.30 2.46 L!I0.4 I*** 1.47 * 0.14***
3.94 3.47 3.17 2.86
f 0.68 + 0.36* f 0.23** zk 0.30***
47.1 48.1 49.4 53.2
f + f f
2.4 2.3 2.6* 4.0***
1.50 1.53 1.55 1.50
+ 0.07 f 0.10 k 0.06 -c 0.08
3.40 3.33 3.22 2.98
3.34 3.10 3.65 3.32
f * + f
47.3 48.7 49.4 51.8
f f f +
2.9 2.8 3.5 3.2**
ND ND ND 1.59 1.56 1.60 1.49
k f + +
0.06 0.09 0.05 0.06***
+ + + +
0.6 I 0.55 0.6 I 0.3 I*
0.47 0.35 0.36 0.33
3.77 f 0.44 3.78 310.26 3.72 f 0.59
3.58 It 0.49 3.46 f 0.48 3.91 + 0.48
46.8 + 2.0 48.4 f 2.6 46.5 f 2.8
3.99 3.46 3.36 3.25
3.39 3.51 2.84 2.77
48.0 48.0 47.7 51.6
+ + f f
0.31 0.39** 0.33*** 0.56***
+ 0.31 + 0.39 +- 0.33** + 0.40***
+ 2.6 -c 3.2 + 2.3 + 3.5**
OX+ SD, 10 animals per group. TPTC, tri-n-propyltin chloride; TBTC, tri-n-butyltin chloride; THTC, tri-n-hexyltin chloride; TOTC, tri-n-octyltin chloride; and TPhTC, triphenyltin chloride. * p < 0.05.
** p < 0.01. *** p < 0.001. ND, not determined.
erythrocytes, situated as rosettes around mononuclear cells, were seen throughout the medullary sinuses. No occult blood could be detected in feces of all TBTC groups. Trihexyltin chloride. Only feeding of 150 ppm THTC resulted in a small decrease in thymus weight and a slight increase in liver weight. At the 50-ppm dose, none of the organ weights differed from controls. Trioctyltin chloride. No treatment-related changes were observed in rats fed 50 or 150 ppm TOTC. Triphenyltin chloride. Decrease in body weight gain was already noticed at 50 ppm TPhTC. Reduction of relative thymus weight occurred at 15 ppm. A dose-related decrease
of spleen weight was also apparent. In the highest dose group (150 ppm), reduction of body and brain weight was observed, while livers were. enlarged. At this dose, thymus weight was reduced to 8 1% of control. On histopathologic examination, some reduction in cellularity of the thymic cortex was observed. The total amount of iron per spleen was diminished at 50 ppm (82%) and 150 ppm (70%). Iron concentration (micrograms Fe per gram dry spleen weight) was not influenced. Structure-Activity
Relationship
In Fig 3A a structure-activity relationship is given for the effect of the trialkyltins on thy-
TOXICITY
OF TRIORGANOTINS
B FIG. 2. Part of a thymus from a rat fed 0 ppm (A) or 100 ppm (B) TBTC for 4 weeks showing a decrease in number of cortical lymphocytes and a relative increase in reticula-epithelial cells. Note the absence of severe cellular destruction. Glycol methacrylate section; May-Griinwald-Giemsa. X400.
279
280
SNOEIJ ET AL. TABLE 2 ABSOLUTE WEIGHTS OF THUS AND SPLEEN, THYMIC CELL COUNT, AND SPLENICIRON CONTENT AND CONCENTRATIONOF RATS FED VARIOUS DOSESOF TBTC FOR 2 WEEKS” Thymus
Dose (ppm) 0 15 50 100 150
Spleen Cell count (X10’)
Weight (mg) 436 422 290 208 127
f + + + +
56 19 54*** 13*** 13***
111 f22 look 3 53 + 11*** 41 +- 5*** 18 f 3***
Weight (mg)
Fe content b w
425 412 357 312 231
68 + 14 62+ 13 54 f 3* 48 + 5** 41 AZ 5**
+ + + + f
71 79 8* 15** 24***
Fe concentration c Wg) 751 + 673 f 692 f 703x? 795 +
145 116 22 71 52
’ zi + SD, five animals per group. b Fe content: cg iron per spleen. ’ Fe concentration: ag iron per g dry spleen weight. * p < 0.05.
** p < 0.01. *** p < 0.001. mus weight. Being primarily neurotoxic, TMTC and TETC are excluded. TPTC is the most active homolog at low dietary concentrations (15 ppm), while TBTC causes more pronounced thymus atrophy at higher doses. Whether the effects are expressed on the basis of ppm or micromoles (Fig. 3B), the structureactivity relationship is generally the same.
V
Four- Week Study The effects of a 4-week feeding period with 100 ppm of TPTC, TBTC, or TOTC in the diet of rats are presented in Table 3. TPTC and TBTC caused reduction of thymus weight to 66 and 43% of the controls, respectively, Body weights were only slightly (TPTC) or
50 . .. l
-150
15 ppm 50 DDrn p*m
100 chain
length
(number
of C-atoms1
**
TETC
25
200 pmol
300 lrialkyltin
400
500
600
per kg feed
FIG. 3. Relationship between length of the alkyl chain of triafkyltin compounds and its effect on thymus weight determined in groups of 10 male rats fed various trialkyltin compounds for 2 weeks. (A) Dietary concentrations expressed in mg alkyltin per kg feed (ppm); (B) dietary concentrations expressed in pmol trialkyltin per kg feed. *.p < 0.05; *a, p < 0.01; ***,p < 0.01.
TOXICITY
281
OF TRIORGANOTINS TABLE 3
BODY AND ORGAN WEIGHTS OF RATS FED 0 OR 100 ppm TFTC, TBTC, OR TOTC FOR 4 WEEKS” BUY weight (9)
Brain weight (9)
Thymus
Spleen
Liver
-
171 (11)
1.63 (0.05)
3.13 (0.27)
2.55 (0.27)
52.29 (1.24)
0.14 (0.04)
TF’TC
154* (11)
(Z)
2.08*** (0.26)
2.40 (0.37)
47.95*** (1.76)
0.15 (0.03)
TBTC
142** (14)
1.58 (0.07)
1.34*** (0.32)
2.35 (0.36)
50.16 (2.53)
0.15 (0.04)
-
232 (9)
1.81 (0.06)
3.18 (0.68)
2.72 (0.20)
52.80 (1.51)
0.16 (0.02)
TOTC
228 (14)
I .74 (0.13)
3.01 (0.24)
2.58 (0.32)
52.75 (4.24)
0.16 (0.03)
Compound
Relative organ weight (g/pg body weight) Adrenals
a X; SD in parentheses. * p < 0.05.
** p < 0.01. *** p < 0.001.
moderately (TBTC) decreased. In contrast to the 2-week studies, no effects were observed on the weights of spleen and liver. The decrease in liver weight found in the lOO-ppm TPTC group is considered to be unrelated to the organotin treatment. TOTC did not influence any of the organ weights that were determined. Histologic examination of tbymus glands of rats fed TPTC or TBTC revealed a severe reduction in lymphocyte density in the cortex (Fig. 2). This feature was even more pronounced than in the 2-week feeding studies with these compounds. Histology of the spleen and liver was normal, except for slight atrophy of the periarteriolar lymphocyte sheaths (PALS) in a few spleens of rats fed TBTC. Recovery Experiment Upon feeding rats 100 ppm TPTC or TBTC for 4 weeks, body weights were significantly reduced up to 3 weeks after terminating the
treatment (Fig. 4A). The severe reduction of thymus weight induced by 100 ppm of both of the trialkyltins was followed by a fast recovery (Fig. 4B). After 1 week of feeding stock diet, thymus weights of the trialkyltin-treated rats did not differ from the controls. In the second week of rehabilitation, thymus weights were even significantly higher than the controls. This response is considered to be a compensatory reaction as is seen in many healing processes. Adrenalectomy Adrenalectomy itself caused some growth retardation, but the subsequent treatment with 100 ppm TBTC in the diet for 13 days did not affect body weight any further. Adrenalectomy also resulted in an increase in thymus weight compared to the nonoperated or sham-adrenalectomized rats. This enlargement is considered to be a consequence of decreased adrenal activity. On careful examination, no adrenal remnants were found in 10 of the 12
282
SNOEIJ ET AL.
Body weight (PI 300
200
I I our ) reeks , of reamlent
100
Stock
I
0
0 mm 100 ppm 100 ppm
TBTC TPTC
,.
4
Relativf thymus WelQht (QlkQI 3.0
6--d
diet
5
6
7
9
a
10
11
12
0 pm 100 ppm 100 PP~
TBTC TPTC
. B
h
I 1 ... I I I. . . I .*.
2.0
.* .
... 1C
I
ii:-i;;;--::; i I
Four , weeks, 01 treatment
Stock
u -
diet
I
oJ;
! 4
5
6
7 ”
6
9
10
11
12
Weeks
FIG 4. Reversibility of TPTC- and TBTC-induced growth retardation (A) and thymus atrophy (B) in rats kept on a 100 ppm of trialkyltin diet for 4 weeks and subsequently 0 to 8 weeks on stock diet (X & SD of six rats per group). *, p -z 0.05; **, p < 0.001; L**, p < 0.001.
adrenalectomized rats. As is demonstrated in Fig. 5, adrenalectomy did not abolish the TBTC-induced thymus atrophy. The relative thymus weight of these rats was decreased to the same extent as the nonoperated or shamadrenalectomized rats.
DISCUSSION From the present study it appears that the lower trialkyltins, TMTC and TETC, are essentially neurotoxic. The intermediate trialkyltins, TFTC and TBTC, together with tri-
TOXICITY
OF TRIORGANOTINS
1 l .
0 100
tIDm
TBTC
ADRENAL i
FIG. 5. Relative thymus weight of nonoperated, shamadrenalectomized, and adrenalectomized rats fed 0 or 100 ppm of TBTC for 13 days. *, p < 0.05; **, p < 0.0 1. ’ Six animals per group. b Five animals per group.
phenyltin are immunotoxic. Of the higher homologs, THTC is only slightly immunotoxic, while TOTC is not toxic at all. In 2-week oral exposure studies, TMTC was the most toxic homolog, causing deaths at dietary concentrations of 15 ppm (20%) and 50 ppm ( 100%). In contrast to acute toxicity data (Barnes and Stoner, 1958; WHO, 1980), TETC was less lethal than TMTC. Fifteen ppm of both compounds caused severe emaciation, characterized by reduction (TETC) or absence (TMTC) of growth. Feed avoidance may, at least in part, be the cause of the emaciated status. Fifteen ppm TMTC resulted in neuronal degeneration in specific areas of the central nervous system (CNS). The major pathologic features, which were described previously (Brown et al., 1979; Bouldin et al., 198 1; Chang and Dyer, 1983), consisted of pyknosis in the hippocampal formation, karyorrhexis in the pyriform cortex, and chromatolysis of certain brain stem nuclei, in particular the mesencephalic trigeminal nucleus. More detailed examination, however, revealed marked differences. Extensive neuronal destruction was seen in the fascia dentata in contrast to the observations of Bouldin et al. (198 l), who found this region to be virtually unaffected after daily oral dosing of 1 mg TMT/kg body weight given for 2 weeks.
283
Moreover, much less neuronal damage was observed in the comu Ammonis, although Brown et al. (1979) and Bouldin et al. (198 1) described this area as most severly affected upon repeated oral TMT administrations. The few necrotic cells which were found in the Ammon’s horn were mainly located in the dorsolateral areas (CA2, CA&, which are usually preserved upon TMT intoxication (Brown et al., 1979; Bouldin et ul., 198 1; Chang et al., 1983). Rat strain and dose regimen are important in the pathology of TMT poisoning (Chang et al., 1983) and may therefore account for the differences mentioned above. The observed toxicity of TETC is generally in accordance with the original findings of Magee et al. (1957). Feeding of 20 ppm triethyltin hydroxide resulted in rapid weight loss during the first 2 weeks, in which time interstitial edema of the CNS developed. Two weeks feeding of 50 ppm TETC obviously caused this type of neurotoxicity, as demonstrated by the rise in absolute brain weight. Rats fed 10 or 15 ppm showed growth retardation and increase in brain water content. No signs of neurotoxicity were observed at the 5-ppm dose of either TMTC or TETC, or with any of the other triorganotin compounds. Little attention has been paid to the effects of TMT or TET on lymphoid organs. In one study (Hioe and Jones, 1984), decreases in thymus and spleen weights were reported after ip administration of 10 mg TMT/kg. Adrenalectomy abolished these effects and therefore stress-mediated immunotoxicity was suggested. The atrophy of thymus and spleen observed in this study in the 15-ppm groups of TMTC and TETC are therefore most likely caused by stress and emaciation. The increase in adrenal weight supports this assumption. Five ppm of these compounds did not effect the lymphoid organs. The most striking effects on the lymphoid system were noticed for the intermediate trialkyltins, TF’TC and TBTC. Feeding these compounds for 2 weeks resulted in a dose-related reduction of thymus and spleen weight
284
SNOEIJ ET AL.
(Table 1). Histologic examination revealed that thymus atrophy is associated with severe lymphocyte depletion in the cortical area (Table 2). By preparing cell suspensions from thymus glands of treated rats, the loss of lymphocytes could be adequately affirmed (Table 2). Histopathologic examination of the spleens revealed no abnormalities. The decrease of iron content suggest a reduction of the red pulp. The subsequent finding that the iron concentration was not affected may be explained by a growth retardation of the whole spleen. Four-week treatment with TPTC or TBTC ( 100 ppm for both) severely affected thymus weight (Fig. 3), whereas spleens, livers, and other organs were normal (Table 3). Therefore, it is concluded that thymus atrophy induced by the intermediate trialkyltins is a selective effect. Early effects on spleen and liver are significant but transient and are considered of little toxicologic importance. A selective thymus atrophy was also noticed by Krajnc et al. (1984) and Vos et al. (1984a) upon oral treatment of rats with TBTO for 4 weeks. Spleen and liver weights were not affected up to TBTO concentrations of 80 ppm in male and 320 ppm in female rats. Erythrocyte rosettes in the sinuses of mesenteric lymph nodes were found by these authors. In this study a dose-related increase of rosettes was seen within only some of the mesenteric lymph nodes upon 2-week feeding of TBTC, but not of TPhTC. Although a test for the detection of occult blood in the feces was negative, microhemorrhages could be responsible for the presence of erythrocytes in the lymph circulation. Since no erythrophagocytosis was observed, a phagocytosis defect may underly the rosette formation. The higher trialkyltin homologs induce a slight decrease in weight of the thymus (THTC, 150 ppm) or do not affect it at all (TOTC). Although immunotoxicity was not the subject of their study, Barnes and Stoner (1958) showed a lack of toxicity of TOTC upon oral administration to rats. The effects of the aromatic triorganotin,
TPhTC, resembled those of TPTC and TBTC, but appeared to be less severe. Reductions of thymus and spleen weights were noticed at 15 and 50 ppm, respectively. Our data confirm the similar findings of Vos et al. ( 1983, 1984b) with triphenyltin hydroxide. As was described for the latter compound (Gaines and Rimbrough, 1968), TPhTC reduced weight gain at lower dietary concentrations than did the intermediate trialkyltins. A structure-activity relationship (SAR) for the intermediate and higher trialkyltin homologs with regard to thymus atrophy is presented in Fig. 3. Seinen et al. (1977) have presented a similar SAR for the dialkyltins. Dibutyltin and dioctyltin compounds have most pronounced effects on the thymus, while homologs with 12 or 18 carbon atoms per chain were inactive. As is suggested for the longchain dialkyltins, reduced intestinal uptake may render the higher trialkyltin homologs inactive. The differences in effect when 15 ppm TPTC or TBTC are compared might be related to a difference in absorption from the gut. Possible immunotoxic effects of TMTC or TETC are overshadowed by their neurotoxicity and are therefore left out of the SAR. Although TPTC- and TBTC-induced thymus atrophy is severe, the effects were completely reversible (Fig. 4). Within 1 week after the diets were changed to normal feed, thymus weight was at control values. A significant rebound was observed in the second week of recovery. Seinen et al. ( 1977) observed a similar recovery pattern of the thymus after feeding di-n-octyltin dichloride to rats. The rapid recovery suggests a short biological half-life of these organotins. Evans et al. (1979), using [14C]TBT0, indicated a rapid clearance from the body of mice when ingestion of TBTO was stopped. No exact data are available however. Although thymus atrophy can be induced by stress-related release of glucocorticosteroids (Keller et al., 1983) the effects of TBTC are not caused by stress for the following reasons. Thymus weight was as severely decreased in adrenalectomized as in sham-adrenalecto-
TOXICITY
285
OF TRIORGANOTINS
mized, TBTC-fed rats (Fig. 5). Moreover in the 2- and 4-week feeding studies relative adrenal weights were normal, except for the highest dose group of TBTC (150 ppm for 2 weeks), in which a slight weight increase was noticed. Histologically, no increased destruction of lymphocytes in the thymus was found, as is seen when glucocorticosteroid concentrations are increased. Therefore the observed depletion of thymic lymphocytes is not primarily caused by their destruction. The severe cellular destruction and decrease in cell viability noticed by Vos et al. (1984a) is possibly a secondary effect of TBTO inducing stressmediated lympholysis, since cell death was only observed at a very high dose of 320 ppm. The mechanisms by which the triorganotins induce neurotoxicity (TMTC, TETC) or immunotoxicity (TPTC, TBTC, TPhTC) are unknown. Since thymus atrophy is a sensitive parameter for immunological damage (Seinen et al., 1977; Seinen and Penninks, 1979; Vos et al., 1984a; 1984b), the mechanism of triorganotin-induced thymus atrophy is subject of further study. ACKNOWLEDGMENTS The authors thank Dr. E. J. Bulten and Dr. H. A. Meinema, Institute for Applied Chemistry, TNO, Utrecht, for the supply of organotin samples. The technical assistance of Mr. V. Rutten and Mr. F. Snel is greatly appreciated. This research was supported by the Netherlands Foundation for Technical Research (STW), Future Technical Science Branch/Division of the Netherlands Organization for the Advancement of Pure Research (ZWO).
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CARDARELLI, N. ( 1976). Controlled-Release Pesticide Formulations. CRC Press, Cleveland, Ohio. CHANG, L. W., WANGER, G. R., MCMILLAN, D. E., AND DYER, R. S. (1983). Species and strain comparison of acute neurotoxic effects of trimethyhin in mice and rats. Neurobehav.
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split-litter and whole-litter dosing. J. Toxicol. Environ. Health 12, 73-87. SEINEN, W., AND WILLEMS, M. 1. (1976). Toxicity of organotin compounds. I. Atrophy ofthymus and thymusdependent lymphoid tissue in rats fed di-n-octyltindichloride. Toxicol. Appl. Pharmacol. 35, 63-75. SEINEN, W., Vos, J. G., SPANJE, I. VAN, SNOEK, M., BRANDS, R., AND HOOYKAAS, H. (1977). Toxicity of organotin compounds. II. Comparative in vivo and in vitro studies with various organotin and organolead compounds in different animal species with special emphasis on lymphocyte cytotoxicity. Toxicol. Appl. Pharmacol. 42, 197-2 12. SEINEN, W., AND PENNINKS, A. H. (1979). Immune suppression as a consequence of a selective cytotoxic activity of certain organometallic compounds on thymus and thymus-dependent lymphocytes. Ann. N. Y Acad. Sci. 320, 499-5 17. STONER, H. B. (1966). Toxicity of triphenyltin. Brit. J. Indust. Med. 23,222-229. TIL, H. P., FERON, V. J., AND DE GROOT, A. P. (1972). The toxicity of sulphur. I. Long-term feeding and multigeneration studies in rats. Food Cosm. Toxicol. 20, 291-310.
Vos, J. G., KRAJNC, E. I., BEEKHOF, P. K., AND VAN LOGTEN, M. J. (1983). Methods for testing immune effects of toxic chemicals: Evaluation of the immunotoxicity of various pesticides in the rat. In IUPAC Pesticide Chemistry. Human Welfare and the Environment (J. Miyamoto et al.. eds.), pp. 497-504; Pergamon, Oxford. Vos, J. G., DE KLERK, A., KRAJNC, E. I., KRUIZINGA, W., VAN OMMEN, B., AND ROZMG, J. (1984a). Toxicity of bis(tri-n-butyltin)oxide in the rat. II. Suppression of thymus-dependent immune responsesand of parameters of nonspecific resistance after short-term exposure. Toxicol. Appi. Pharmacol. 75, 387-408. Vos, J. G., VAN LOGTEN, M. J., KREEFTENBERG,J. G., AND KRUIZINGA, W. (1984b). Effect of triphenyltin hydroxide on the immune system of the rat. Toxicology 29, 325-336. WATANABE, I. (1980). Organotins. In Experimental and Clinical Neurotoxicology (P. S. Spencer and H. H. Schaumberg, eds.), pp. 545-557. Williams and Wilkins, Baltimore. WHO. (1980). Tin and Organotin Compounds. A Preliminary Review. Environmental Health Criteria 15. WHO, Geneva.