Toxicology Letters, 30 (1986) 35-39 Elsevier

35

TOXLett. 1523

DOSE-DEPENDENT BRAIN TIN CONCENTRATION IN RATS GIVEN

STANNOUS CHLORIDE IN DRINKING WATER

(Rat; stannous chloride; brain tin; acetylcholinesterase; blood tin)

H. SAVOLAINEN and SINIKKA VALKONEN

Department of Industrial Hygiene and Toxicology, Institute of Occupational Health, SF-00290 Helsinki, Finland

(Received September 13th, 1985)

(Revision received November 19th. 1985)

(Accepted November 21st. 1985)

SUMMARY

Male Wistar rats were given either 100 mg SnCls ‘2H20 per litre (0.44 mM), 250 mg/l (1.11 mM) or

500 mg/l (2.22 mM) in their drinking water for 1-18 weeks. Tin detected by a novel atomic absorption

spectrophotometric method accumulated in the cerebrum at the highest dose level (2.22 mM) throughout

the experiment. In brain, tin concentrations above the 1.11.mM dose were only found after 15 and 18

weeks. Tin did not increase in the brain at the 0.44-mM dose level. Blood tin increased promptly after

one week at the highest dose (2.22 mM) without further accumulation. Blood tin at the 0.44 mM dose

level did not differ from controls. Tin exposure caused a dose-dependent increase in the cerebral and

muscle acetylcholinesterase activity at the two higher doses.

INTRODUCTION

206 000 metric tons of tin mined over the world in 1975 and an additional 222 000 metric tons were recycled into the production process. This makes inorganic tin a common metal in the everyday environment. However, inorganic tin salts are rather innocuous by the common experience [l]. This might result from the fact that only about 1% or less of the dose is absorbed in the gastrointestinal tract [2]. Another aspect in the experimental study of tin toxicity is that its analysis at the relevant con- centrations in biological material is not easy [3]. When tin is injected in rabbits parenterally it affects the distribution of various other metal ions [3]. The same authors have shown that tin ions also inhibit erythrocyte Saminolevulinic acid dehydratase [4]. Therefore, it appears that inorganic tin could have biochemical ef- fects once a sufficient dose has been absorbed. This might be a real possibility as

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

36

higher peroral doses of stannous chloride cause degeneration of gut mucosa [5] allowing increased absorption of tin ions. With this in mind, we gave rats stannous chloride in their drinking water at three concentration levels and developed a method for the trace analysis of tin ions in the nervous system. The brain burden was compared with the effect on acetylcholinesterase activity known to be activated by various divalent cations [6].

MATERIALS AND METHODS

93-month-old male Wistar rats (365 f 17 g, + S.D.) were divided in three groups and were given drinking water containing either 100 mg SnC12.2HzO/l (0.44 mM), 250 mgIl(l.11 mM), or 500 mg/l (2.22 mM) for 1-18 weeks. 30 rats (368 f 25 g, + S.D.) served as controls and were maintained on tap water. The consumption of water in each 5-rat cage was measured daily. 5 rats in each group were killed by decapitation after 1, 4, 8, 12, 15 or 18 weeks of exposure. The cerebral hemispheres, 1 g of right gluteal muscle and a blood sample in a heparinized tube were taken at autopsy.

Tin in the right cerebral hemispheres and in the blood samples was analyzed as follows. The brain samples were weighed and lyophilized; 150 mg of dry material or 1 ml blood were digested in 1 ml of a nitric, sulphuric and perchloric acid mixture (3: 1: 1, per vol.) for 3 h while the temperature was gradually increased to 275°C. The clear residue was washed with 10 ml of deionized water into the reaction vessel of the metal hydride-generating system (Perkin-Elmer MHS-20). Stannane (SnH4) was generated by adding 2.5 ml NaBH4 (E. Merck, F.R.G., lot No. 6371) in 1.0% (w/v) sodium hydroxide. The generated hydride was purged with argon for 20 s into the Perkin-Elmer 400 atomic absorption spectrophotometer cell. The cell temperature was 900°C for 15 s, and after the analysis it was purged with argon for 30 s. An electrode-less discharge lamp was operated at 286.3 nm. Commercial tin standards (Titrisol, E. Merck, F.R.G.) were used, and they were analyzed in the same manner as the actual samples.

Lyophilized urine with a known tin content (Nyegaard Diagnostica, Oslo, Nor- way) was analyzed for quality control. Special care was taken to avoid contamina- tion. All glassware was rinsed overnight in a detergent solution before the analysis. The same NaOH and NaBH4 lots were used at all times, and the quartz cell of the spectrophotometer was decontaminated by purging it twice with hydrofluoric acid vapor after 20 analyses. With these precautions, the intraseries variation in the ex- traction and analysis was 15.7% at 5 ng tin and 11.9% at 10 ng tin per sample, respectively (N = 12). The results deviated from known controls f 10% at 5 ng tin and +_ 4% at 10 ng tin content, respectively. The detection limit was 0.6 ng cor- responding to 5 pmol/g wet weight.

The left cerebral hemispheres and the muscle specimens were homogenized in 10 ~01s. of 0.1 M phosphate buffer at pH 7.2 for the assay of acetylcholinesterase ac-

37

tivity [7]. To verify the direct effect of SN2+ ions on acetylcholinesterase, samples of control cerebral homogenate were incubated for 30 min at 37°C in 0.1 M phosphate buffer (pH 7.2) with 10V2, 10d3, 10m4 or lo-’ M stannous chloride and the enzyme activity was determined [7].

All results were statistically evaiuated with the variance analysis, and tin ac- cumulation in brain was mathematicaIly simulated with functions found by the least-squares method.

RESULTS AND DISCUSSION

The weight gain of the exposed rats did not differ from controls (not shown). The bIood tin content of contro1 rats varied from 5 to 9 pmob’g. At the 0.44 mM dose, it was 7-15 pmoI/g throughout the experiment, and at the 1.11 mM and 2.22 mM doses 5-22 pmol/g and 16-60 pmol/g, respectively. However, after the first week of exposure no further increase in the blood concentrations occurred.

Brain tin of control rats varied from 5-10 pmol/g wet weight. At the 0.44-mM dose, it was 7-19 pmol/g. This difference was statistical by not significance. After 15 weeks of exposure, brain tin at the 1.11 mM dose was 19 -t 8 pmol/g wet weight (+ S.D., N = 5), and 3 weeks later it was 38 + 8 pmoI/g. At the highest dose level brain tin content increased exponentially throughout the experiment (Fig. 1).

Brain acetylcholinesterase activity was continuously enhanced by exposure to the

pmol/g

12 15 18 weeks

1 1 I 1 1 1 cumulative intake 0.039 3.5 7.7 ‘*.* 14.7 ‘7.7(mmol/kg bodywt)

Fig. 1. Increase in brain tin content after peroral intakeof Sn” (2.22 mM in drinking water). Cumulative

ingested dose has been calculated from daily drinking rates. The exponential function shown simulates

accumulation better than a straight fine. Each point represents the mean of 5 rats + S.D.

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TABLE 1

BRAIN AND MUSCLE ACETYLCHOLINEST~RAS~ ACTlVlTY IN PERORAL STANNOUS CHLORIDE EXPOSED RATS

The enzyme activity (nmol/min x mg protein) is the mean of five rats ( f SD.). aDiffers from control at *P <0.05, **P <O.Ol, and ***P <O.OOl.

‘Dose’ Time (mmol/l drinking (weeks)

-. water)

I 18

Brain” Muscle Brain” Muscle ~~

0 157 + 21 12 I 0.2 166 * 22 15 + 0.8 0.44 167t 6 12 +- 0.5 I66 f 11 16 * 0.3 1.11 172 z+z 10 13 * 0.6 190 * 12** 19 + 0.9*** 2.22 181 k ll* 13 + 1.1 200 F lo*** 17 r 0.8**

highest dose (Table I). It was also increased at the 1.1 I-mM dose while brain tin con- centration reached 38 pnmol/g. Tin concentration corresponded roughly to that found at the 2.22-mM dose after 8 weeks (Fig. 1). Muscle acetylcholinesterase show- ed a similar trend. Compared to controls (140 + 18 nmol/min x mg protein, + S.D., N = 5) IO-* M stannous chloride activated acetylcholinesterase (4.50 + 40 nmoI/min x mg protein + SD., N = 5); at 10Y3 M the activity was 250 + 30, at 10e4M 200 rt 20 and at IO-‘M 135 rt I9 nmollmin x mg protein.

The fact that tin ions are effectively prevented from entering the circulation [2] is proven by the blood tin concentrations. The actual tin content of food or water is obviously more important than the cumulative dose. Tin is effectively excreted by the kidneys [2] and it may even increase its own elimination as tin ions are toxic to the kidney tubules [8]. These factors, as well as biliary elimination, may account for the virtual steady-state concentrations of tin in the blood even at the highest dose.

Low-M, solutes may traverse the blood-brain barrier by passive diffusion [9]. Tin may follow suit while the postulated ‘polarity’ of the barrier may prevent rapid elimination from the brain [IO] leading to accumulation. An analogous increase has been shown for brain lead at low peroral exposure [ll]. The fact that control rats had some tin in their brain and blood indicates that the commercial rat food or the tap water used contains tin ions.

Tin ions activated acetylchoIinesterase as did calcium, magnesium and divalent manganese ions [6]. This might be due to an effect on the deacylation phase of the enzyme-substrate reaction [6], although the tin concentration used is very high com- pard to that found in vivo. But, our findings in brain and in muscle are mutually supportive.

In conclusion, despite the fact that mucosal barrier mechanisms effectively pre- vent tin absorption they can be overcome by very high tin doses. Neurochemical ef- fects, however, may occur only with extreme brain tin burdens and are therefore probably not relevant.

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