Toxicology, 63 (1990) 73--84 Elsevier Scientific Publishers Ireland Ltd.
Inorganic arsenic methylation by rat tissue slices B. Georis, A. Cardenas*, J.P. Buchet and R. Lauwerys Unit6 de Toxicologie lndustrielle et de M6decine du Travail, Catholic University o f Louvain, Clos Chapelle-aux-Champs, 30.54. B-1200 Bruxelles (Belgium) (Received December 5th, 1989; accepted March 23th, 1990)
Summary Rat liver, kidney and lung slices methylate trivalent inorganic arsenic (Aslll) to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA); the liver has the greatest methylating capacity. Aslll enters the liver cells by a diffusion process followed by extensive binding to intracellular components which favors its extensive accumulation inside the cells. Reduced glutathione regulates Aslll metabolism through several mechanisms: facilitation of Aslll diffusion into the cells, stimulation of the first methylation reaction and increase of DMA excretion by the cells. An excess of Aslll inhibits DMA production by liver cells but this inhibition is reversible; mercuric ions inhibit both MMA and DMA production probably by decreasing inorganic arsenic (Asi) uptake and the second methylation reaction. DMA can be produced from MMA by rat liver slices and this methylation step is stimulated by GSH. In contrast to Aslli, AsV is not extensively taken up by the hepatocyte and is thus poorly methylated.
Key words: Arsenic; Methylation; Rat tissues
Introduction The biotransformation of inorganic arsenic (Asi) by mammals leads to the production of monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) which are mainly excreted in urine [1]. So far, the mechanism of Asi biotransformation has only been studied in vitro with rat liver cytosol. It has been found that the addition of GSH and of Sadenosyl-L-methionine (SAMe) as methyl group donor is required and that vitamin B-12 enhances the methylating activity. In rat liver cytosol, an excess of substrate and the addition of Hg 2" (0.1 mM) prevent the formation of DMA [21. Furthermore, the methylation of Asi is slightly inhibited by the prior enrichment of the incubation medium with MMA (feed-back inhibition) [3]. In the present study we have further investigated the mechanism of Asi methyAddress all correspondence and reprint requests to: Prof. R. Lauwerys, Industrial Toxicology and Occupational Health Unit, Clos Chapelle-aux-Champs, 30.54. B-1200 Bruxelles, Belgium *Research fellow in Toxicology of the European Science Foundation. 0300-483X/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
73
lation by using rat tissue slices which offer the advantage of preserving cell structure and may therefore be more representative of the in vivo situation than the cytosolic preparation. Materials and methods
Reagents Sodium metaarsenite (AslIl) and sodium arsenate (AsV) were obtained from Merck (Darmstadt, F.R.G.) and sodium methylarsinate (99.4070) from Carlo Erba (Milano, Italy). Vitamin B-12, DL-buthionine (S,R) sulfoximine (BSO) and GSH were purchased from Sigma Chemie Gmbh (Deisenhofen, F.R.G.). S-adenosyl-Lmethionine (SAMe), in the form of sulfuric and p-toluene suifonic acid salt, was kindly offered by Bioresearch Co (Liscate, Italy). Tetra-n-butyl ammonium hydroxyde solution (40070 in water) (TBA) and l-decanol were obtained from Aldrich-Chemie (Steinheim, F.R.G.). Other chemicals were analytical grade reagents from Merck (Darmstadt, F.R.G.).
Animals Two- or 3-month-old male Sprague--Dawley rats (150--250 g) were used. The animals were anaesthetized with pentobarbital (60 mg/kg, i.p.) and after sectioning the inferior vena cava, they were perfused with ice-cold 0.907o NaCI through a needle introduced in the left ventricle [3]. When lung preparations were used, the renal artery was first cut and the lungs were rapidly perfused in situ through the pulmonary artery [4]. In some experiments, the animals were injected i.p. with BSO dissolved in distilled isotonic water at a dose of 625 mg/kg, 3 h before sacrifice [51 in order to decrease the GSH content of the liver. Control animals were given 2 ml NaCI 0.9°7o (0.15 M) i.p.
Slice preparation and incubation The perfused organ (liver, lung or kidney) was directly transferred to a Mcllwain tissue chopper (Mickle Laboratory Engineering, Co. Ltd, UK) to prepare slices of 0.6 mm thickness [4]. About 70 mg of tissue (3 or 4 slices) were transferred to a plastic tube (30 x 70 mm) kept at 0°C and containing 3 ml of Krebs-Ringer phosphate buffer solution at pH 7.4 with glucose (11 mM), Asi and cofactors [4]. The tubes were closed and placed in a shaking water bath. Incubation was carried out at 37°C under N 2 or air. At the end of the incubation period, the slices were removed, washed in buffer, blotted on filter paper and transferred in a tube containing 1 ml TBA (1607o) which was placed in a water bath at 80°C during 2 h. The incubation medium was poured in a tube containing 50 /al concentrated HCI. The amount of Asi, MMA and DMA was measured in both the slices and medium preparation.
A nalyticai procedures Inorganic arsenic (Asi) and its metabolites, MMA and DMA, were determined
74
by flameless absorption spectrometry as described previously [1] but with the following modifications: a decanol drop was added to the sample to avoid the formation of foam during NaBH 4 addition and the U-shaped tube was warmed up at ambient temperature to improve the separation of MMA and DMA peaks from that of Asi. For non-protein thiol determination, liver slices (100 mg) were placed in a tube kept at 0°C to which were successively added 0.5 ml trichloroacetic acid (25°7o in water) and 2 ml water. After thorough mixing, the tube was centrifuged at 1200 g for 20 min. The thiol content of the supernatant was determined with Ellman reagent [6]. The results are expressed as GSH equivalent since it is known that the latter represents the major part of the low molecular weight thiols present in the liver cytosol. The leakage of LDH measured according to Korzeniewski and Callewaert [7] from tissue slices to the incubation medium was used as an index of cell membrane integrity [8]. The results are expressed in percent of total LDH activity which was determined after ultrasonication of the slices during 3 min (Vinsonic 300, Virtis Company, power setting 3) and addition of Triton X-100 up to a final concentration of 0.1%. Under the various experimental conditions selected for the study, the percentage of total LDH activity released from the slices in the incubation medium after 5 h incubation at 37°C is (mean __ S.E.) 15.4 _+ 1.1, 20.0 _+ 2.0 and 25.4 _ 3 for the kidney (n = 20), lung (n = 20) and liver (n = 16) respectively. We have also checked that LDH activity is not influenced by the reagents added to the incubation medium.
Statistical analysis The results were analysed by one- or two-way analysis of variance (differences with P < 0.05 were considered statistically significant). In case of heterogeneous variances, RANK and GLM procedures were used [9]. Comparisons between means were performed with the D U N C A N ' S test and the paired t-test. Results
The concentration of inorganic arsenic in the medium and in the liver slices was compared after several incubation times with increasing concentrations of AslII (Table I); this comparison was based upon the assumption that the density of liver slices equals 1. The AsllI uptake by the slices is proportional to its concentration in the incubation medium; the major part is taken up during the first hour and at each concentration tested, the arsenic concentration is usually more than 10 times greatei" in the slices than in the medium. Other experiments have also shown that the inorganic trivalent arsenic uptake by liver cells does not require oxygen, is temperature independent and that the cell membrane integrity is not significantly changed even at the highest arsenic concentration tested (results not shown). The comparison o f AsllI methylation by rat liver, kidney cortex and lung slices is illustrated in Fig. 1. The addition of GSH to the incubation medium has no effect on Asi methylation by lung slices but enhances the amount of MMA
75
TABLE 1 INFLUENCE OF SUBSTRATE C O N C E N T R A T I O N UPTAKE OF AslII BY RAT LIVER SLICES Incubation time (h)
AND
INCUBATION TIME
(Asi) tissue ~ M )
ON
THE
(Asi) medium (~M)
(Asi: l ~M) 1
4.1
±
0.6"
0.3
±
1.5 2 3 5
3.7 4.5 3.2 2.9
± ± ± ±
0.4 0.4 0.4 0.3
0.2 0.2 0.2 0.2
± ± ± ±
0.1 0.0 0.0 0.0 0.0
(Asi: 5/aM) 1 1.5 2 3 5
29.0 33.2 35.2 35.4 34.4
± ± ± ± ±
2.0 1.5 1.6 2.0 1.8
1.5 1.7 1.4 1.1 1.1
± ± ± ± ±
0.0 0.2 0.2 0.2 0.2
(Asi: 10 Iz M) l 1.5 2 3 5
47.9 61.1 67.1 74.3 76.2
± ± ± ± ±
1.5A b 4.0B 1.9 CB 2.8 C 0.5 C
5.3 5.3 4.7 4.2 3.6
± ± ± ± ±
0.3 0.1 0.4 0.2 0.4
• Means ± S.E. of 5 experiments. b Means with the same letter are not statistically different (Duncan test); no letter indicates no statistical difference.
Liver
Kidney
Lung
120 100 'B ¢.D
O0 ID
Inorganic arsenic methylation by rat tissue slices B. Georis, A. Cardenas*, J.P. Buchet and R. Lauwerys Unit6 de Toxicologie lndustrielle et de M6decine du Travail, Catholic University o f Louvain, Clos Chapelle-aux-Champs, 30.54. B-1200 Bruxelles (Belgium) (Received December 5th, 1989; accepted March 23th, 1990)
Summary Rat liver, kidney and lung slices methylate trivalent inorganic arsenic (Aslll) to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA); the liver has the greatest methylating capacity. Aslll enters the liver cells by a diffusion process followed by extensive binding to intracellular components which favors its extensive accumulation inside the cells. Reduced glutathione regulates Aslll metabolism through several mechanisms: facilitation of Aslll diffusion into the cells, stimulation of the first methylation reaction and increase of DMA excretion by the cells. An excess of Aslll inhibits DMA production by liver cells but this inhibition is reversible; mercuric ions inhibit both MMA and DMA production probably by decreasing inorganic arsenic (Asi) uptake and the second methylation reaction. DMA can be produced from MMA by rat liver slices and this methylation step is stimulated by GSH. In contrast to Aslli, AsV is not extensively taken up by the hepatocyte and is thus poorly methylated.
Key words: Arsenic; Methylation; Rat tissues
Introduction The biotransformation of inorganic arsenic (Asi) by mammals leads to the production of monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) which are mainly excreted in urine [1]. So far, the mechanism of Asi biotransformation has only been studied in vitro with rat liver cytosol. It has been found that the addition of GSH and of Sadenosyl-L-methionine (SAMe) as methyl group donor is required and that vitamin B-12 enhances the methylating activity. In rat liver cytosol, an excess of substrate and the addition of Hg 2" (0.1 mM) prevent the formation of DMA [21. Furthermore, the methylation of Asi is slightly inhibited by the prior enrichment of the incubation medium with MMA (feed-back inhibition) [3]. In the present study we have further investigated the mechanism of Asi methyAddress all correspondence and reprint requests to: Prof. R. Lauwerys, Industrial Toxicology and Occupational Health Unit, Clos Chapelle-aux-Champs, 30.54. B-1200 Bruxelles, Belgium *Research fellow in Toxicology of the European Science Foundation. 0300-483X/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
73
lation by using rat tissue slices which offer the advantage of preserving cell structure and may therefore be more representative of the in vivo situation than the cytosolic preparation. Materials and methods
Reagents Sodium metaarsenite (AslIl) and sodium arsenate (AsV) were obtained from Merck (Darmstadt, F.R.G.) and sodium methylarsinate (99.4070) from Carlo Erba (Milano, Italy). Vitamin B-12, DL-buthionine (S,R) sulfoximine (BSO) and GSH were purchased from Sigma Chemie Gmbh (Deisenhofen, F.R.G.). S-adenosyl-Lmethionine (SAMe), in the form of sulfuric and p-toluene suifonic acid salt, was kindly offered by Bioresearch Co (Liscate, Italy). Tetra-n-butyl ammonium hydroxyde solution (40070 in water) (TBA) and l-decanol were obtained from Aldrich-Chemie (Steinheim, F.R.G.). Other chemicals were analytical grade reagents from Merck (Darmstadt, F.R.G.).
Animals Two- or 3-month-old male Sprague--Dawley rats (150--250 g) were used. The animals were anaesthetized with pentobarbital (60 mg/kg, i.p.) and after sectioning the inferior vena cava, they were perfused with ice-cold 0.907o NaCI through a needle introduced in the left ventricle [3]. When lung preparations were used, the renal artery was first cut and the lungs were rapidly perfused in situ through the pulmonary artery [4]. In some experiments, the animals were injected i.p. with BSO dissolved in distilled isotonic water at a dose of 625 mg/kg, 3 h before sacrifice [51 in order to decrease the GSH content of the liver. Control animals were given 2 ml NaCI 0.9°7o (0.15 M) i.p.
Slice preparation and incubation The perfused organ (liver, lung or kidney) was directly transferred to a Mcllwain tissue chopper (Mickle Laboratory Engineering, Co. Ltd, UK) to prepare slices of 0.6 mm thickness [4]. About 70 mg of tissue (3 or 4 slices) were transferred to a plastic tube (30 x 70 mm) kept at 0°C and containing 3 ml of Krebs-Ringer phosphate buffer solution at pH 7.4 with glucose (11 mM), Asi and cofactors [4]. The tubes were closed and placed in a shaking water bath. Incubation was carried out at 37°C under N 2 or air. At the end of the incubation period, the slices were removed, washed in buffer, blotted on filter paper and transferred in a tube containing 1 ml TBA (1607o) which was placed in a water bath at 80°C during 2 h. The incubation medium was poured in a tube containing 50 /al concentrated HCI. The amount of Asi, MMA and DMA was measured in both the slices and medium preparation.
A nalyticai procedures Inorganic arsenic (Asi) and its metabolites, MMA and DMA, were determined
74
by flameless absorption spectrometry as described previously [1] but with the following modifications: a decanol drop was added to the sample to avoid the formation of foam during NaBH 4 addition and the U-shaped tube was warmed up at ambient temperature to improve the separation of MMA and DMA peaks from that of Asi. For non-protein thiol determination, liver slices (100 mg) were placed in a tube kept at 0°C to which were successively added 0.5 ml trichloroacetic acid (25°7o in water) and 2 ml water. After thorough mixing, the tube was centrifuged at 1200 g for 20 min. The thiol content of the supernatant was determined with Ellman reagent [6]. The results are expressed as GSH equivalent since it is known that the latter represents the major part of the low molecular weight thiols present in the liver cytosol. The leakage of LDH measured according to Korzeniewski and Callewaert [7] from tissue slices to the incubation medium was used as an index of cell membrane integrity [8]. The results are expressed in percent of total LDH activity which was determined after ultrasonication of the slices during 3 min (Vinsonic 300, Virtis Company, power setting 3) and addition of Triton X-100 up to a final concentration of 0.1%. Under the various experimental conditions selected for the study, the percentage of total LDH activity released from the slices in the incubation medium after 5 h incubation at 37°C is (mean __ S.E.) 15.4 _+ 1.1, 20.0 _+ 2.0 and 25.4 _ 3 for the kidney (n = 20), lung (n = 20) and liver (n = 16) respectively. We have also checked that LDH activity is not influenced by the reagents added to the incubation medium.
Statistical analysis The results were analysed by one- or two-way analysis of variance (differences with P < 0.05 were considered statistically significant). In case of heterogeneous variances, RANK and GLM procedures were used [9]. Comparisons between means were performed with the D U N C A N ' S test and the paired t-test. Results
The concentration of inorganic arsenic in the medium and in the liver slices was compared after several incubation times with increasing concentrations of AslII (Table I); this comparison was based upon the assumption that the density of liver slices equals 1. The AsllI uptake by the slices is proportional to its concentration in the incubation medium; the major part is taken up during the first hour and at each concentration tested, the arsenic concentration is usually more than 10 times greatei" in the slices than in the medium. Other experiments have also shown that the inorganic trivalent arsenic uptake by liver cells does not require oxygen, is temperature independent and that the cell membrane integrity is not significantly changed even at the highest arsenic concentration tested (results not shown). The comparison o f AsllI methylation by rat liver, kidney cortex and lung slices is illustrated in Fig. 1. The addition of GSH to the incubation medium has no effect on Asi methylation by lung slices but enhances the amount of MMA
75
TABLE 1 INFLUENCE OF SUBSTRATE C O N C E N T R A T I O N UPTAKE OF AslII BY RAT LIVER SLICES Incubation time (h)
AND
INCUBATION TIME
(Asi) tissue ~ M )
ON
THE
(Asi) medium (~M)
(Asi: l ~M) 1
4.1
±
0.6"
0.3
±
1.5 2 3 5
3.7 4.5 3.2 2.9
± ± ± ±
0.4 0.4 0.4 0.3
0.2 0.2 0.2 0.2
± ± ± ±
0.1 0.0 0.0 0.0 0.0
(Asi: 5/aM) 1 1.5 2 3 5
29.0 33.2 35.2 35.4 34.4
± ± ± ± ±
2.0 1.5 1.6 2.0 1.8
1.5 1.7 1.4 1.1 1.1
± ± ± ± ±
0.0 0.2 0.2 0.2 0.2
(Asi: 10 Iz M) l 1.5 2 3 5
47.9 61.1 67.1 74.3 76.2
± ± ± ± ±
1.5A b 4.0B 1.9 CB 2.8 C 0.5 C
5.3 5.3 4.7 4.2 3.6
± ± ± ± ±
0.3 0.1 0.4 0.2 0.4
• Means ± S.E. of 5 experiments. b Means with the same letter are not statistically different (Duncan test); no letter indicates no statistical difference.
Liver
Kidney
Lung
120 100 'B ¢.D
O0 ID
