Occupational and Environmental Medicine 1994;51:735-738

Effect of bis (tributyl tin) oxide on permeability ofthe blood-brain barrier: a transient increase

Kazuo Hara, Mitsuaki Yoshizuka, Yoshiaki Doi, Sunao Fujimoto

AbstractObjectives-To study the effect of bis(tributyl tin) oxide (TBTO) on perme-ability of the blood-brain barrier.Methods-Electron microscopy and an xray microanalyser with lanthanum chlo-ride as a tracer were used, and blood tinconcentrations were determined with anatomic absorption spectrophotometer.Adult male wistar rats received0 05 ml/kg body weight ofTBTO orally.Results-A transient increase in para-cellular permeability at the blood-brainbarrier was found 2 h after the dose ofTBTO. Electron dense lanthanumdeposits penetrated tight junctions of theendothelia and permeated the subendo-thelial space. The x ray microprobe datashowed an accumulation of TBTO at thetight junctions at 2 h. Leakage of tracerdid not occur at 4 h, but oedematouschanges in the surrounding glial cellswere prominent between 4 and 8 h andhad almost returned to normal by 24 h.By atomic absorption analysis, it wasseen that blood tin concentrations rapidlyincreased at 1 h and rose to a maximumpeak at 8 h, then gradually decreased toreach zero at 24 h.Conclusions-Accumulated TBTO attight junctions could have caused thetemporary replacement of calcium ion bytin, which induces a transient increase inparacellular permeability throughout theblood-brain barrier.

(Occup Environ Med 1994;51:735-738)

Department ofAnatomy, Universityof Occupational andEnvironmentalHealth, School ofMedicine, Kitakyushu807K HaraY DoiS FujimotoDepartment ofAnatomy, KurumeUniversity School ofMedicine, Kurume830, JapanM YoshizukaCorrespondence to:Dr Kazuo Hara,Department of Anatomy,University of Occupationaland Environmental Health,School of Medicine,Kitakyushu 807, Japan.Accepted 22 June 1994

Keywords: bis (tributyl tin) oxide, blood-brain barrier,capillary permeability, atomic absorption spectropho-tometry

Tributyl tin compounds have been widelyused in the fishing industry as antifoulingagents in numerous formulations of marinepaints to prevent adhesions and growth ofbarnacles, algae, and other organisms on shiphulls, fishing nets, and cages. Recently, accu-mulation of tributyl tin, heavily toxic tomarine organisms, has been pointed out in thefood chain in crabs, oysters, salmon, andother fish. For instance, deformities such asdwarfism and kyphoscoliosis of young yellowtailed fish due to the toxic effects of tributyltin compounds were reported.' Although thetoxic effects of these compounds on mammalshave been given much attention from bio-chemical and pharmacological viewpoints,2A4

detailed ultrastructural investigations to clar-ify the aetiology of the toxicity of tributyl tinhave been limited.

Neurotoxic effects of organotin compoundshave been found in accidentally exposedhumans.56 The signs and symptoms weresevere headache, vomiting, vertigo, photopho-bia, anorexia, increased tendency to sleep,memory loss, and psychiatric disturbances.Striking interstitial oedema of the cerebralwhite matter was found in the victims, andreproduced as a specific effect of organotincompounds in experimental animals.78 Thedetailed mechanisms that cause the shift ofthe fluid into the central nervous system inorganotin intoxications remains uncertain.Our study was undertaken to determine theeffects of oral bis (tributyl tin) oxide (TBTO)on the permeability of the blood-brain barrier.We used an electron microscope and an x raymicroanalyser. Also, blood tin concentrationswere measured with an atomic absorptionspectrophotometer.

Materials and methodsANIMALSAdult male wistar UOEH rats (weight250-300 g) were housed in separate stainlesssteel cages and maintained in a 12 hour light-dark cycle. Exposure to light was six to 18hours. Animals completed an 18 hour fastbefore the dose, but were permitted freeaccess to water.

ELECTRON MICROSCOPYIn our preliminary study,9 we found that asingle dose of 0-2 ml/kg (234 mg/kg) bodyweight of TBTO given orally was the LD50

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Hara, Yoshizuka, Doi, Fujimoto

Figure 2 Lanthanum deposits (arrowheads) do not penetrate the capillary endothelium1 h after TBTO treatment. Bar represents 0 5 ,im.

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(lethal dose for 50% of the group) in malewistar rats, and an oral dose of 0-02 ml/kg(23-4 mg/kg) body weight of TBTO did notcause any ultrastructural changes in the ratvascular endothelia. A quarter of the LDso waschosen in the present study.Twenty four adult male wistar rats received

0-05 ml/kg body weight of TBTO (AldrichChemical, WI, USA, purity; 96%) orally as anemulsion in 1 ml of distilled water through astainless steel stomach tube. Another 16 con-trol rats received 1 ml of distilled water only.At 0 5, 1, 2, 4, 8, 12, 16, and 24hours afterthe dose three animals in each experimentalgroup, and two in each control group weregiven an intravenous injection of 1 ml of 2%lanthanum chloride as a tracer through thecaudal vein. Five minutes after the injection,

all animals were deeply anaesthetised and per-fused with 3-5% glutaraldehyde in 01 Mcacodylate buffer through the right commoncarotid artery. Then, each hypothalamus wasisolated, further immersed in the same fixa-tive, postfixed in 1% osmium tetroxide in thesame buffer, dehydrated by acetone, andembedded in epoxy resin. Ultrathin sectionswere stained by uranyl acetate and lead citrateand viewed with a JEM 1200EX electronmicroscope.

x RAY MICROANALYSISIn the energy dispersive x ray microanalysis,frozen sections of tissues are usually used forthe detection of cytoplasmic elements. In ourpreliminary experiment, however, the tin L-alpha peak (344 keV) was completely cov-ered by the high potassium K-alpha peak(3-31 keV) due to persistence of the intracyto-plasmic potassium ion in the frozen section.Therefore, sections fixed and embedded inresin were used.

Non-stained ultrathin sections wereanalysed for detection of accumulations ofTBTO in the hypothalamus with a JEM2000EX electron microscope equipped with aLINK QX 200J energy dispersive x ray micro-analyser.

DETERMINATION OF BLOOD TINCONCENTRATIONSTwenty four male wistar rats received thedose of TBTO orally. At 0-5, 1, 2, 4, 8, 12,16, and 24 hours after the dose three animalsfrom each group were deeply anaesthetisedwith an injection of pentobarbitone, andblood samples were collected from the leftventricle. Whole blood was diluted fourfoldwith 3N HC1 containing 10% ascorbic acid toprevent an adverse effect of iron when deter-mining tin concentrations.

Tin concentrations in the blood sampleswere determined with a Hitachi Z-8000Zeeman Atomic Absorption Spectro-photometer. To determine the blood tin con-centrations, samples were dried at 80-120 ICfor 180 s, ashed at 400-500 0C for 60 s, andatomised at 2700 °C for 10 s.

ResultsNo deaths or weight loss occurred among theexperimental animals associated with TBTOtreatment, and all treated rats seemed to be ingood condition.

Figure 1 shows data obtained from-atomicabsorption analyses of tin concentrations inthe blood samples. The blood tin concentra-tion showed a rapid increase one hour afterthe dose. This rose to the maximum peak ateight hours, then gradually decreased, andreached unmeasurable concentrations at24 hours.

By-electron microscopy, it was seen that noultrastructural changes occurred in the hypo-thalamus and that lanthanum chloride did notleak out of the true capillaries until one hourafter treatment with TBTO (fig 2). The leak-age of tracer through the tight junctions of the

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Effect of bis (tributyl tin) oxide on permeability of the blood-brain barrier

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Figure 4 Tin L-alpha peak (3 44 kel') obtainedfrom the leaky tightjunction area Icapillary 2 h after TBTO treatment.

Figure S Astrocytes (AS) surrounding the capillary show severe oedematous change8 h after TBTO treatment, whereas neural elements seem to be unaltered. Bar repress1 /Im.

endothelia first occurred at two hours afterTBTO treatment. Electron opaque depositsof lanthanum chloride permeated both sub-endothelial space and intercellular spacesamong the endfeet of the surrounding astro-cytes (fig 3). No visible ultrastructuralchanges of the cytoplasm of these endothelialcells and glial cells surrounding the capillarieswere noticed at two hours. By energy disper-sive x ray microanalysis, discriminative tin L-

alpha peaks (3 44 keV) were obtained fromthe leaky junction (fig 4). Such leakage oftracer was not found and tin peaks were notobtained in the control animals throughoutthis experiment.

Between four and eight hours after TBTOtreatment, the intercellular and intracytoplas-

mic oedema of the glial cells surrounding thecapillaries was prominent, whereas the neuralelements did not seem to be affected. Thetracer did not leak out of the capillaries andno degenerative changes were found in eitherendothelial cells or pericytes (fig 5).Moreover, the accumulation of TBTO at thetight junctions of the capillaries was notdetected from four to eight hours after thedose. Oedematous changes in the surround-ing glial tissue gradually became less pro-nounced and almost recovered by 24 hoursafter TBTO treatment (fig 6).

DiscussionAs severe brain oedema was found in acuteorganotin intoxication in human cases, manyexperimental studies have been performed toclarify the aetiology of this phenomenon. Theincrease in cerebrovascular permeability tomacromolecular tracers has not beenreported.'0 Therefore, the brain oedemainduced by organotin compounds has beenthought to be a cytotoxic and not a vasogenicoedema."1The present tracer experiment clearly

showed that an oral dose ofTBTO induced atransient increase in the permeability of theblood-brain barrier of the true capillaries inthe hypothalamus. The tight junctions tem-porarily opened very early after treatmentwith TBTO and resealed rapidly. This mightbe the reason why previous investigatorscould not find the opening in tight junctions.The mechanisms that caused the opening

of the blood-brain barrier brought about byTBTO are still unclear. The present x raymicroprobe data, however, indicated thatTBTO preferentially accumulated at the leak-ing tight junctions. Although tight junctionsare known to act as the main barrier to para-cellular passage in epithelia, there do existsome structural differences in the depth andcomplexity of each tight junction. Claude andGoodenough classified so called tight junc-tions into "very tight" and "leaky tight" junc-tions.'2 Machen et al in their in vitroexperiments, reported that ionic lanthanumpermeated the tight junction of isolated rabbitgall bladder and intestinal epithelia afterimmersion in an ionic lanthanum solution forone hour." They suggested that excess incu-bation in the ionic lanthanum solution mightcause the alteration of the conformation oftight junction molecules and increase theparacellular permeability. It is therefore rea-sonable to assume that the accumulation ofTBTO at our tight junctions may have had aneffect on the configurations of the junctionalcomplex molecules and this may have led to achange from "tight" to "leaky" during alimited period.

Recently, the existence of several Ca(2+dependent cell to cell adhesion molecules invascular endothelial cells was verified. 14

Prasada Rao et al reported that organotincompounds interfered with the transport ofcalcium ions into the sarcoplasmic reticulumin in vitro cardiac muscle cells.'5 Taking these

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Hara, Yoshizuka, Doi, Fujimoto

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Figure 6 The oedematous changes of astrocytes (AS) have disappeared at 24 h afterTBTO treatment. PC = Pericyte; BM = basal lamina. Bar represents 1 im.

results into consideration, we consider at pre-

sent that accumulated TBTO at the tightjunctions caused the temporary replacementof calcium ion by tin, which induces atransient increase in paracellular ion perme-

ability throughout the blood-brain barrier.Further studies are now in progress in our lab-oratory to elucidate the interactions ofTBTOwith calcium ions at the junctional complexesbetween the endothelial cells.

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5 Alajouanine T, Derobert L, Thibfery S. Etude cliniqued'ensemble de 210 cas d'intoxication par les selsorganique d'etain. Rev Neurol (Paris) 1958;98:85-96.

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9 Yoshizuka M, Hara K, Doi Y, Mori N, Yokoyama M, OnoE, Fujimoto S. The toxic effects of bis (tributyltin) oxideon the rat thoracic aorta. Histol Histopathol 1992;7:445-9.

10 Hultstrom D, Forssen M, Pettersson A, Tengbar C, JarildM, Olsson Y. Vascular permeability in acute triethyltin-induced brain edema studied with FITC-dextrans,sodium fluorescein and horseradish peroxidase as trac-ers. Acta Neurol Scand 1984;69:255-63.

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13 Machen TE, Erlij D, Wooding FBP. Permeable junctionalcomplexes. The movement of lanthanum across rabbitgallbladder and intestine. J Cell Biol 1972;54:302- 12.

14 Heimark RL, Degner M, Schwartz SM. Identification of aCa2+-dependent cell-cell adhesion molecule in endothe-lial cells. J Cell Biol 1990; 110: 1745-56.

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transient increase.permeability of the blood-brain barrier: a Effect of bis (tributyl tin) oxide on

K Hara, M Yoshizuka, Y Doi and S Fujimoto

doi: 10.1136/oem.51.11.7351994 51: 735-738 Occup Environ Med 

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