Xenobiotica the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Comparative metabolism of the cysteine and homocysteine conjugates of propachlor by in situ perfused kidneys of rats K. L. Davison, J. E. Bakke & G. L. Larsen To cite this article: K. L. Davison, J. E. Bakke & G. L. Larsen (1992) Comparative metabolism of the cysteine and homocysteine conjugates of propachlor by in situ perfused kidneys of rats, Xenobiotica, 22:4, 479-485 To link to this article: http://dx.doi.org/10.3109/00498259209046660
Published online: 27 Aug 2009.
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Date: 06 November 2015, At: 05:22
XENOBIOTICA,
1992, VOL. 22,
NO.
4, 479485
Comparative metabolism of the cysteine and homocysteine conjugates of propachlor by in situ perfused kidneys of rats K. L. DAVISON?, J. E. BAKKE and G. L. LARSEN Downloaded by [NUS National University of Singapore] at 05:22 06 November 2015
US Department of Agriculture, Agricultural Research Service, Biosciences Research Laboratory, Fargo, N D 58105, USA
Received 28 August 1991; accepted 3 January 1992
1 . 14C-Cysteinyl- and homocysteinylpropachlor were metabolized to their respective mercapturic acids by rat kidneys in situ. First-pass elimination of I4C in urine was 47.5%for the cysteine conjugate and 36% for the homocysteine conjugate. 2. About half of the perfused 14C-labelled material isolated from urine from kidneys perfused with homocysteinylpropachlor was unchanged homocysteinylpropachlor and about half was the corresponding mercapturic acid. However, only the corresponding mercapturic acid and the S-oxide of this mercapturic acid (3 1.4%and 1.7%of the dose) were found in urine from kidneys perfused with cysteinylpropachlor, indicating that rat kidneys more efficiently acetylated the natural substrate, the cysteine conjugate.
Introduction It is generally recognized that glutathione conjugates of xenobiotics are metabolized to cysteine conjugates by enzymes of the brush border of proximal cells of the renal tubules (Silbernagl and Heuner 1990). These cysteine conjugates are absorbed by the proximal cells and may be further metabolized within the cells by N acetyltransferase, forming mercapturic acids, or by cysteine conjugate a-lyase (C-S lyase), forming reactive thiols (Dekant et al. 1990). Cysteine conjugate 8-lyase has been observed in bovine kidney (Anderson and Schultze 1965) and purified from rat kidney (Stevens et al. 1986), with S-1,2dichlorovinylcysteine (DCVC) as substrate. T h e resultant sulphur-containing dichlorovinyl fragment is capable of binding with proteins and nucleic acids, and is nephrotoxic in a number of animal species. Subsequently, S-l,2-dichlorovinylhomocysteine (DCVHC) was more nephrotoxic than DCVC (Elfarra et al. 1986), presumably because not only was a toxic thiol formed by /I-lyase but also because some homocysteine was transformed into 2-keto-3-butenoic acid which is toxic. T h e glutathione conjugate of propachlor (2-S-glutathionyl-N-isopropylacetanilide) was metabolized to the mercapturic acid conjugate by kidneys of rats when this substrate was perfused into the kidneys in situ (Davison et al. 1990). In this report we compare the metabolism of the cysteine conjugate of propachlor with that of the homocysteine conjugate of propachlor by rat kidneys perfused in situ in an attempt to determine intrarenal differences in the processing of a natural and an unnatural substrate. Cysteinylpropachlor is an intermediate in the metabolism of the glutathione conjugate and, therefore, should be metabolized to the mercapturic acid conjugate of propachlor. On the other hand, homocysteinylpropachlor, a conjugate
t Correspondence to: K. L. Davison, USDA, ARS, Biosciences Research Laboratory, PO Box 5764 University Station, Fargo, N D 58105 USA. 0049-8254/92 $3.00
0 1992 Taylor & Francis Ltd.
480
K.L. Davison et al.
of propachlor with an unnatural amino acid, may be metabolized to the corresponding mercapturic acid or it may be metabolized to a thiol and 2-keto-3-butenoic acid, as occurred with DCVHC when it was incubated with isolated renal tubule cells (Elfarra et al. 1986). I4C-Substrate was perfused through the renal artery of one kidney and urine was collected separately from both kidneys, allowing the determination of first-pass metabolism of the substrate by difference.
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Experimental Chemicals ''C-~-Cysteinylpropachlor (2-S-~ysteinyl-N-isopropyl[l-'~C]acetanilide)and ''C-~-homocysteinylpropachlor (2-S-homocysteinyl-Nisopropyl[l-'4C]acetanilide)were synthesized by a basecatalysed reaction of propachlor with the appropriate amino acid (Lamoureux and Davison 1975). Cleanup, identification and determination of radiopurity( > 99% pure by t.1.c. with two solvent systems) were conducted as described previously (Larsen and Bakke 1983). Rats Sprague-Dawley rats, 3-4 months old, weighing 35&400g, were used. The experiments were conducted in fasted (about 16 h), anaesthetized, surgically modified rats in the supine position. Dosages were administered on a per-rat basis in physiological saline (0.15 M NaCl). Anaesthesia The rats were anaesthetized with halothane (CF,CHBrCl) by using a closed-circuit anaesthesia machine with oxygen as the carrier gas. On completion of surgery, anaesthesia was maintained with sodium pentobarbital given i.m. twice or more often during the experiments. The total dose of pentobarbital was about 90mg/kg of body weight. The rats were killed at the end of the experiments by an overdose of pentobarbital given via a jugular cannula. Surgery Details of the surgical procedures have been published (Davison 1989, Davison et al. 1990). Polyethylene cannulas, 0.28 mm int. diam. x 0.61 mm overall diam., were inserted in the right jugular vein, bile duct, and both ureters. A cannula made of polyethylene tubing of similar size and equipped with 30-gauge hypodermic needle tubing bent at a 90" angle on the indwelling end was inserted into the left renal artery. Perfusions Saline was perfused at 0 1 ml/min with a syringe pump through the jugular cannula to hydrate the rats. Test substrate was dissolved in saline and perfused at 0 1 ml/min for 2 5 3 h through the cannula in the left renal artery. The total dose of cysteinylpropachlor was 1.5 pmol, specific activity 858 d.p.m./pg, and the total dose of homocysteinylpropachlor was 1.4prno1, specific activity 1111 d.p.m./pg. Assays *4C Values in bile and urine were assayed directly by conventional liquid scintillation counting methods. Lyophilized tissues were oxidized in a Packard model 306D sample oxidizer and the resultant 14C0, was assayed by liquid scintillation counting. First-pass elimination of 14Cin urine was determined by subtracting the percentage of 14Celiminated by the non-perfused kidney from that eliminated by the perfused kidney. Perfusion of only one kidney, and collection of urine from both kidneys separately, allows the determination of the contribution of the perfused kidney to the first-pass metabolism and clearance of a compound. Infused material that does not clear the perfused kidney on the first pass is transported in the general circulation and then has equal opportunity for excretion by either kidney; therefore, the first-pass clearance and metabolism by a kidney is estimated by the difference between the excretions by the two kidneys (Wideman and Braun 1982, Davison et al. 1988, Davison 1990). Isolation and identification procedures for propachlor metabolites are well documented (Larsen and Bakke 1981, 1983, Bakke and Larsen 1985), but are described briefly here. Urines from the perfused kidneys of rats given the same dose were pooled and likewise, urines from the non-perfused kidneys of these rats were pooled. The urine was applied directly to columns of Porapak Q (2 x lScm, 100-120 mesh). Non-absorbed compounds were eluted with 200ml of water, then the radioactive compounds were eluted with methanol. Methanol in the methanolic eluant was evaporated under vacuum with a rotary evaporator and the residue dissolved in water and applied to an L H 20 column (1 x 60 cm). The radioactivity was eluted with water through a radioactivity flow monitor and collected in a fraction collector. These fractions were
48 1
Metabolism of cysteine and homocysteine conjugates
lyophilized, dissolved in water and subjected to h.p.1.c. (Novapak, CIS;pumped 1 ml/min, 3 min isocratic water, 17 min linear programme 100%water to 100% methanol, 15 min isocratic methanol). The column eluant passed through a U.V. detector and a radioactivity monitor. Metabolites were identified by comparing their h.p.1.c. retention times with those of known compounds, and by interpretation of fast atom bombardment mass spectra of fractions collected from h.p.1.c. (Larsen and Ryhage 1982).
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Resu1ts Rats perfused with 14C-cysteinylpropachlor eliminated 59% of the 14C in urine from the perfused kidney, 47.5% was eliminated on first pass through the kidney (table 1). Rats perfused with ''C-homocysteinylpropachlor eliminated 49% of the 14Cin urine from the perfused kidney, 36% was eliminated on first pass through the kidney. Total elimination of 14C in urine and bile was 83% for rats perfused with cysteinylpropachlor and 79% for homocysteinylpropachlor. More 14Cwas found in the tissue of the perfused kidney than in the tissue of the non-perfused kidney. The mercapturic acids, N-acetylcysteinylpropachlor and its S-oxide (table 2), were the only compounds isolated and identified in urine and bile from rats perfused with cysteinylpropachlor (for representative mass spectra see Larsen and Ryhage 1982). Table 1 . 14CRecovery in urine, bile and tissues from rats in which the right kidney was perfused with either 14C-cysteinylpropachlor or ''C-homocysteinylpropachlor (percentage of dose). Item Urine from perfused kidney Urine from non-perfused kidney Bile Perfused kidney Non-perfused kidney Liver Gastrointestinal tract Carcass Total recovery
Cysteinylpropachlor
Homocysteinylpropachlor
58.9 k 6.9' 11.4k0.9 12.9& 0.8 1.2k0.6 0.3 k 0 . 2 5.9 F 0.7 0.7k0.1 5.0& 0.7 96.3 k4.1
488f16.9 128 k0.8 17.0 f8.6 1.4k0.7 0 7 0.4 8 9 3.7 1.2+ 1.0 9 2 f4.4 100.5k 1.4
*
'Mean k SEM; n = 3 in all cases.
Table 2.
Compounds isolated from pooled urine and bile from rats renally perfused with either I4Ccysteinylpropachlor or '4C-homocysteinylpropachlor (percentage of dose).
Where excreted and compound Urine from perfused kidney Hornocysteinylpropachlor Mercapturic acid Mercapturic acid-S-oxide Urine from non-perfused kidney Mercapturic acid Mercapturic acid-S-oxide Bile Mercapturic acid Mercapturic acid-S-oxide
Perfused with cysteinylpropachlor
Perfused with homocysteinylpropachlor
31-4 1.7
11.28 11.5 n.d.
4.8 1.5
3.2 2.2
63 26
n.d.
-
3.4
n.d. = Not detected. 'The data represent minimum values based on the amount of 14C remaining in the various fractions from which metabolites were characterized by mass spectroscopy.
K. L. Davison et al.
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482
About half of the I4C isolated and identified in urine from the perfused kidneys of rats given homocysteinylpropachlor was present as parent compound (table 2). T h e remaining half of the 14C was identified as N-acetylhomocysteinylpropachlor (the mercapturic acid, figure 1). T h e FAB mass spectrum of this mercapturic acid contained molecular ion adducts at m / z 353 ( M + H ) + , 375 ( M + N a ) + , 391 (M + K ) + , 397 ( M - H + 2 N a ) + and 413 ( M - H + N a + K ) + . These ions are 42a.m.u. higher than those observed for the molecular ion plus a proton or the indicated salts for synthesized authentic homocysteinylpropachlor (figure 2), the dosing material. T h e relatively high abundance of sodium and potassium salts in FAB mass spectra of compounds isolated from urine compared to spectra of synthesized compounds is thought to be related to the abundance of these salts in the urine. Ions from the salts of glycerol, the liquid support medium on the probe of the mass spectrometer, appear at m / z 115 (glycerol+Na) and 131 (glycerol+K), although ions for two glycerols+H, Na or K may appear in some spectra at mjz 185, 207 and 223, respectively. N-Acetylhomocysteinylpropachlorand its S-oxide were isolated from urine from the non-perfused kidney and from bile. T h e FAB mass spectrum of Nacetylhomocysteinylpropachlor-S-oxide(figure 3) contained molecular ion adducts at m / z 369 ( M + H ) + , 391 ( M + N a ) + and 407 ( M + K ) + , and fragment ions at m / z 223 and 207. T h e fragment ion at m / z 223 is thought to result from fragmentation of the sulphur-carbon (y-carbon of the homocysteine moiety) bond with subsequent rearrangement and loss of a proton (Larsen and Ryhage 1982). T h e fragment ion at m / z 207 is thought to result from the loss of oxygen from either the sulphoxide during rearrangement or from the fragment ion at m / z 223 or both. T h e increase in relative intensity of the ion at m / z 13 1 compared to the relative intensity of this ion in 1
100
HNCCH,
I
M+Na
N-CCH2-S-CH2CH2CHCOOH
80
II
375
0
60 131
40
M+H 353 M-H+2Na 397 20
0 100
150
200
250
300
350
400
Figure 1 . Fast atom bombardment mass spectrum of N-acetylhomocysteinylpropachlor [2-(N-acetylhomocysteiny1)-N-isopropylacetanilide]isolated from urine from the perfused kidney of rats perfused with homocysteinylpropachlor. Molecular weight= 352.
483
Metabolism of cysteine and homocysteine conjugates N 3
1oc
J
134
I
176
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2M-134 486
2M+N8 643
-IrclL 333
-I-
1.
I
100
200
300
I.
I
500
400
600
d z Figure 2.
Fast atom bombardmentmass spectrum of homocysteinylpropachlor(2-S-homocysteinyl-Nisopropylacetanilide). Molecular weight = 3 10.
115
100
0
80 131 60
M+Na
40
20
0
100
150
200
250
300
350
400
Figure 3. Fast atom bombardment mass spectrum of N-acetylhomocysteinylpropachlor-S-oxide[2(N-acetyl-homocysteinyl)-N-isopropylacetanilide-S-oxide]isolated from urine from the nonperfused kidney of rats perfused with homocysteinylpropacholor. Molecular weight = 368.
K. L. Davison et al.
484
the spectrum for N-acetylhomocysteinylpropachloris thought to come from the Nacetylalanine moiety (130 H)', which resulted from the sulphur-carbon fragmentation (Larsen and Ryhage 1982). T h e relatively low recovery of metabolites as a percentage of the dose (table 2) occurred because of the relatively small doses and the resultant small masses in bile and urine available for clean-up. Carbon-14 recoveries from the Porapak Q and L H 20 columns varied upward from 85% and 90%, respectively. Carbon-14 recoveries from the h.p.1.c. ranged from 60% to 80%. Tails of radioactive areas were cut to increase sample purity, so the data presented are minimum values, and are based on the amount of material available for mass spectroscopy. Other metabolites, if present, were not obvious.
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+
Discussion T h e distribution of the 14Camong the tissues and its elimination in bile and urine was similar for the two compounds. T h e larger standard errors observed for rats given homocysteinylpropachlor were attributable, for no apparent reason, to one of the three rats given this compound. Bile was collected at hourly intervals, and urine was collected at 15 and 30min and at 30min intervals thereafter to 3 h. T h e distribution of the radioactivity among these fractions was also similar for the two compounds. Retention of 14C in kidney or other tissues was relatively small following perfusion of either the cysteinyl- or homocysteinylpropachlor. We observed greater retention of I4C when I4C-DCVC was perfused renally in rats (6% of the dose was retained in the perfused kidney) and calves (24%, Davison 1989). T h e metabolism of the two compounds differed, however. T h e metabolites observed from cysteinylpropachlor were mercapturic acids; no parent compound was found in either bile or urine. In the case of homocysteinylpropachlor, homocysteinylpropachlor itself was found in urine from the perfused kidneys, but was not found in bile or urine from the non-perfused kidneys. Also, homocysteinylmercapturic acids were found in bile and in urine from both kidneys. T h e appearance of unchanged homocysteinylpropachlor only in urine from the perfused kidneys suggests either that the proximal tubular cells were unable to reabsorb all of the homocysteinylpropachlor from the glomerular filtrate or that the unnatural substrate was a poor substrate for renal cysteine conjugate N-acetyltransferase which could enhance both thiol and 2-keto-3-butenoic acid formation. T h e finding of similar tissue residues in the groups of rats given the cysteine and homocysteine conjugates, and the finding of homocysteinylmercapturic acids but no homocysteinylpropachlor in bile or urine from the non-perfused kidneys of rats given the homocysteine conjugate, demonstrates that the tissues were capable of metabolizing homocysteinylpropachlor . Some absorption, metabolism (N-acetyltransferase) and transport of homocysteinylpropachlor obviously occurred in the renal cells because twice the quantity of mercapturic acids was eliminated in urine from the perfused kidneys than in urine from the non-perfused kidneys. However, the data do not allow determination of how much substrate entered the cells directly through the basolateral cell walls and how much entered through the tubular walls. Uptake of DCVC and DCVHC by suspensions of rat renal tubule cells was shown to be similar (Lash and Anders 1989). If Lash's observation can be extrapolated to our in situ observations, then one might conclude that the apparent slower metabolism of the homocysteinylpropachlor by
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Metabolism of cysteine and homocysteine conjugates
48 5
perfused kidneys was due to intracellular factors. DCVC is a known renotoxin, causing disruption of mitochondrial structure and function and cell death (Lash and Anders 1987). To summarize, rat kidneys perfused in situ metabolized both cysteinyl- and homocysteinylpropachlor to their respective mercapturic acids. Acetylation of cysteinylpropachlor appeared to be more efficient than acetylation of homocysteinylpropachlor. Acetylated cysteinylpropachlor was the only compound isolated from bile or urine from rats renally perfused with cysteinylpropachlor. Homocysteinylpropachlor and acetylated homocysteinylpropachlor were isolated from urine from the perfused kidneys of rats given homocysteinylpropachlor, but acetylated homocysteinylpropachlor only was isolated from bile or urine from the nonperfused kidneys of these rats. Therefore, as expected from in vitro studies (Lash and Anders 1989), the natural cysteine conjugate is the preferred substrate.
Acknowledgements We thank D. Barton, B. Jacobson, K. McDonald and V. Peterson for excellent technical and secretarial assistance. Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
References ANDERSON, P. M., and SCHULTZE, M. O., 1965, Cleavage of S-(l,2-dichlorovinyl)-~-cysteine by an enzyme of bovine origin. Archives of Biochemistry and Biophysics, 111, 593-602. BAKKE, J. E., and LARSEN, G. I,., 1985, Metabolism of 2-chloro-N-isopropylacetanilide in chickens. Chemosphere, 14, 1749-1754. DAVISON, K. L., 1989, In situ perfusion and collection techniques for studying xenobiotic metabolism in animals, in Intermediary Xenobiotic Metabolism in Animals: Methodology, Mechanisms and Significance, edited by D . H. Hutson, J. Caldwell, and G. D. Paulson (London: Taylor & Francis), pp. 315-333. DAVISON, K. L., BAKKE, J. E., and LARSEN, G. L., 1988, A kidney perfusion method for metabolism studies with chickens using propachlor as a model. Xenobiotica, 18, 323-329. DAVISON, K. L., BAKKE, J. E., and LARSEN, G. L., 1990, Metabolism of the glutathione conjugate of propachlor by in situ perfused kidneys and livers of rats. Xenobiotica, 20, 375-383. W., VAMVAKAS, S., and ANDERS, M. W., 1990, Biosynthesis, bioactivation, and mutagenicity of DEKANT, S-conjugates. Toxicology Letters, 53, 53-58. ELFARRA, A. A,, LASH,L. H., and ANDERS, M. W., 1986, Metabolic activation and detoxification of nephrotoxic cysteine and homocysteine S-conjugates. Proceedings of the National Academy of Science, 83, 2667-2671. LAMOUREUX, G. L., and DAVISON, K. L., 1975, Mercapturic acid formation in the metabolism of propachlor, CDAA, and fluorodifen in the rat. Pesticide Biochemistry and Physiology, 5,497-506. G . L., and BAKKE, J. E., 1981, Enterohepatic circulation in formation of propachlor (2-chloro-NLARSEN, isopropylacetanilide) metabolites in the rat. Xenobiotica, 11, 473480. G. L., and BAKKE, J. E., 1983, Metabolism of mercapturic acid-pathway metabolites of 2-chloroLARSEN, N-isopropylacetanilide (propachlor) by gastrointestinal bacteria. Xenobiotica, 13, 115-1 26. LARSEN, G. L., and RYHAGE, R., 1982, Fast atom bombardment mass spectra of mercapturic acidpathway metabolites of propachlor (2-chloro-N-isopropylacetanilide).Xenobiotica, 12, 855-860. M. W., 1987, Mechanism of S-(1,2-dichlorovinyl)-~-cysteine-and S-(1,2LASH,L. H., and ANDERS, dichloroviny1)-L-homocysteine induced renal mitochondrial toxicity. Molecular Pharmacology, 32, 549-556. LASH,L. H., and ANDERS, M . W., 1989, IJptake of nephrotoxic S-conjugates by isolated rat renal proximal tubular cells. Journal of Pharmacology and Experimental Therapeutics, 248, 531-537. SILBERNAGL, S., and HEUNER, A,, 1990, Renal transport and metabolism of mercapturic acids and their precursors. Toxicology Letters, 53, 45-51. STEVENS, J. L., ROBBINS, J. D., and BYRD,R. A., 1986, Purified cysteine conjugate B-lyase from rat kidney cytosol. Journal of Biological Chemistry, 261, 15529-15537. WIDEMAN, R. F., JR,and BRAUN, E. J., 1982, Ureteral urine collection from anaesthetized domestic fowl. Laboratory Animal Science, 32, 298-301.
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Comparative metabolism of the cysteine and homocysteine conjugates of propachlor by in situ perfused kidneys of rats K. L. Davison, J. E. Bakke & G. L. Larsen To cite this article: K. L. Davison, J. E. Bakke & G. L. Larsen (1992) Comparative metabolism of the cysteine and homocysteine conjugates of propachlor by in situ perfused kidneys of rats, Xenobiotica, 22:4, 479-485 To link to this article: http://dx.doi.org/10.3109/00498259209046660
Published online: 27 Aug 2009.
Submit your article to this journal
Article views: 3
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ixen20 Download by: [NUS National University of Singapore]
Date: 06 November 2015, At: 05:22
XENOBIOTICA,
1992, VOL. 22,
NO.
4, 479485
Comparative metabolism of the cysteine and homocysteine conjugates of propachlor by in situ perfused kidneys of rats K. L. DAVISON?, J. E. BAKKE and G. L. LARSEN Downloaded by [NUS National University of Singapore] at 05:22 06 November 2015
US Department of Agriculture, Agricultural Research Service, Biosciences Research Laboratory, Fargo, N D 58105, USA
Received 28 August 1991; accepted 3 January 1992
1 . 14C-Cysteinyl- and homocysteinylpropachlor were metabolized to their respective mercapturic acids by rat kidneys in situ. First-pass elimination of I4C in urine was 47.5%for the cysteine conjugate and 36% for the homocysteine conjugate. 2. About half of the perfused 14C-labelled material isolated from urine from kidneys perfused with homocysteinylpropachlor was unchanged homocysteinylpropachlor and about half was the corresponding mercapturic acid. However, only the corresponding mercapturic acid and the S-oxide of this mercapturic acid (3 1.4%and 1.7%of the dose) were found in urine from kidneys perfused with cysteinylpropachlor, indicating that rat kidneys more efficiently acetylated the natural substrate, the cysteine conjugate.
Introduction It is generally recognized that glutathione conjugates of xenobiotics are metabolized to cysteine conjugates by enzymes of the brush border of proximal cells of the renal tubules (Silbernagl and Heuner 1990). These cysteine conjugates are absorbed by the proximal cells and may be further metabolized within the cells by N acetyltransferase, forming mercapturic acids, or by cysteine conjugate a-lyase (C-S lyase), forming reactive thiols (Dekant et al. 1990). Cysteine conjugate 8-lyase has been observed in bovine kidney (Anderson and Schultze 1965) and purified from rat kidney (Stevens et al. 1986), with S-1,2dichlorovinylcysteine (DCVC) as substrate. T h e resultant sulphur-containing dichlorovinyl fragment is capable of binding with proteins and nucleic acids, and is nephrotoxic in a number of animal species. Subsequently, S-l,2-dichlorovinylhomocysteine (DCVHC) was more nephrotoxic than DCVC (Elfarra et al. 1986), presumably because not only was a toxic thiol formed by /I-lyase but also because some homocysteine was transformed into 2-keto-3-butenoic acid which is toxic. T h e glutathione conjugate of propachlor (2-S-glutathionyl-N-isopropylacetanilide) was metabolized to the mercapturic acid conjugate by kidneys of rats when this substrate was perfused into the kidneys in situ (Davison et al. 1990). In this report we compare the metabolism of the cysteine conjugate of propachlor with that of the homocysteine conjugate of propachlor by rat kidneys perfused in situ in an attempt to determine intrarenal differences in the processing of a natural and an unnatural substrate. Cysteinylpropachlor is an intermediate in the metabolism of the glutathione conjugate and, therefore, should be metabolized to the mercapturic acid conjugate of propachlor. On the other hand, homocysteinylpropachlor, a conjugate
t Correspondence to: K. L. Davison, USDA, ARS, Biosciences Research Laboratory, PO Box 5764 University Station, Fargo, N D 58105 USA. 0049-8254/92 $3.00
0 1992 Taylor & Francis Ltd.
480
K.L. Davison et al.
of propachlor with an unnatural amino acid, may be metabolized to the corresponding mercapturic acid or it may be metabolized to a thiol and 2-keto-3-butenoic acid, as occurred with DCVHC when it was incubated with isolated renal tubule cells (Elfarra et al. 1986). I4C-Substrate was perfused through the renal artery of one kidney and urine was collected separately from both kidneys, allowing the determination of first-pass metabolism of the substrate by difference.
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Experimental Chemicals ''C-~-Cysteinylpropachlor (2-S-~ysteinyl-N-isopropyl[l-'~C]acetanilide)and ''C-~-homocysteinylpropachlor (2-S-homocysteinyl-Nisopropyl[l-'4C]acetanilide)were synthesized by a basecatalysed reaction of propachlor with the appropriate amino acid (Lamoureux and Davison 1975). Cleanup, identification and determination of radiopurity( > 99% pure by t.1.c. with two solvent systems) were conducted as described previously (Larsen and Bakke 1983). Rats Sprague-Dawley rats, 3-4 months old, weighing 35&400g, were used. The experiments were conducted in fasted (about 16 h), anaesthetized, surgically modified rats in the supine position. Dosages were administered on a per-rat basis in physiological saline (0.15 M NaCl). Anaesthesia The rats were anaesthetized with halothane (CF,CHBrCl) by using a closed-circuit anaesthesia machine with oxygen as the carrier gas. On completion of surgery, anaesthesia was maintained with sodium pentobarbital given i.m. twice or more often during the experiments. The total dose of pentobarbital was about 90mg/kg of body weight. The rats were killed at the end of the experiments by an overdose of pentobarbital given via a jugular cannula. Surgery Details of the surgical procedures have been published (Davison 1989, Davison et al. 1990). Polyethylene cannulas, 0.28 mm int. diam. x 0.61 mm overall diam., were inserted in the right jugular vein, bile duct, and both ureters. A cannula made of polyethylene tubing of similar size and equipped with 30-gauge hypodermic needle tubing bent at a 90" angle on the indwelling end was inserted into the left renal artery. Perfusions Saline was perfused at 0 1 ml/min with a syringe pump through the jugular cannula to hydrate the rats. Test substrate was dissolved in saline and perfused at 0 1 ml/min for 2 5 3 h through the cannula in the left renal artery. The total dose of cysteinylpropachlor was 1.5 pmol, specific activity 858 d.p.m./pg, and the total dose of homocysteinylpropachlor was 1.4prno1, specific activity 1111 d.p.m./pg. Assays *4C Values in bile and urine were assayed directly by conventional liquid scintillation counting methods. Lyophilized tissues were oxidized in a Packard model 306D sample oxidizer and the resultant 14C0, was assayed by liquid scintillation counting. First-pass elimination of 14Cin urine was determined by subtracting the percentage of 14Celiminated by the non-perfused kidney from that eliminated by the perfused kidney. Perfusion of only one kidney, and collection of urine from both kidneys separately, allows the determination of the contribution of the perfused kidney to the first-pass metabolism and clearance of a compound. Infused material that does not clear the perfused kidney on the first pass is transported in the general circulation and then has equal opportunity for excretion by either kidney; therefore, the first-pass clearance and metabolism by a kidney is estimated by the difference between the excretions by the two kidneys (Wideman and Braun 1982, Davison et al. 1988, Davison 1990). Isolation and identification procedures for propachlor metabolites are well documented (Larsen and Bakke 1981, 1983, Bakke and Larsen 1985), but are described briefly here. Urines from the perfused kidneys of rats given the same dose were pooled and likewise, urines from the non-perfused kidneys of these rats were pooled. The urine was applied directly to columns of Porapak Q (2 x lScm, 100-120 mesh). Non-absorbed compounds were eluted with 200ml of water, then the radioactive compounds were eluted with methanol. Methanol in the methanolic eluant was evaporated under vacuum with a rotary evaporator and the residue dissolved in water and applied to an L H 20 column (1 x 60 cm). The radioactivity was eluted with water through a radioactivity flow monitor and collected in a fraction collector. These fractions were
48 1
Metabolism of cysteine and homocysteine conjugates
lyophilized, dissolved in water and subjected to h.p.1.c. (Novapak, CIS;pumped 1 ml/min, 3 min isocratic water, 17 min linear programme 100%water to 100% methanol, 15 min isocratic methanol). The column eluant passed through a U.V. detector and a radioactivity monitor. Metabolites were identified by comparing their h.p.1.c. retention times with those of known compounds, and by interpretation of fast atom bombardment mass spectra of fractions collected from h.p.1.c. (Larsen and Ryhage 1982).
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Resu1ts Rats perfused with 14C-cysteinylpropachlor eliminated 59% of the 14C in urine from the perfused kidney, 47.5% was eliminated on first pass through the kidney (table 1). Rats perfused with ''C-homocysteinylpropachlor eliminated 49% of the 14Cin urine from the perfused kidney, 36% was eliminated on first pass through the kidney. Total elimination of 14C in urine and bile was 83% for rats perfused with cysteinylpropachlor and 79% for homocysteinylpropachlor. More 14Cwas found in the tissue of the perfused kidney than in the tissue of the non-perfused kidney. The mercapturic acids, N-acetylcysteinylpropachlor and its S-oxide (table 2), were the only compounds isolated and identified in urine and bile from rats perfused with cysteinylpropachlor (for representative mass spectra see Larsen and Ryhage 1982). Table 1 . 14CRecovery in urine, bile and tissues from rats in which the right kidney was perfused with either 14C-cysteinylpropachlor or ''C-homocysteinylpropachlor (percentage of dose). Item Urine from perfused kidney Urine from non-perfused kidney Bile Perfused kidney Non-perfused kidney Liver Gastrointestinal tract Carcass Total recovery
Cysteinylpropachlor
Homocysteinylpropachlor
58.9 k 6.9' 11.4k0.9 12.9& 0.8 1.2k0.6 0.3 k 0 . 2 5.9 F 0.7 0.7k0.1 5.0& 0.7 96.3 k4.1
488f16.9 128 k0.8 17.0 f8.6 1.4k0.7 0 7 0.4 8 9 3.7 1.2+ 1.0 9 2 f4.4 100.5k 1.4
*
'Mean k SEM; n = 3 in all cases.
Table 2.
Compounds isolated from pooled urine and bile from rats renally perfused with either I4Ccysteinylpropachlor or '4C-homocysteinylpropachlor (percentage of dose).
Where excreted and compound Urine from perfused kidney Hornocysteinylpropachlor Mercapturic acid Mercapturic acid-S-oxide Urine from non-perfused kidney Mercapturic acid Mercapturic acid-S-oxide Bile Mercapturic acid Mercapturic acid-S-oxide
Perfused with cysteinylpropachlor
Perfused with homocysteinylpropachlor
31-4 1.7
11.28 11.5 n.d.
4.8 1.5
3.2 2.2
63 26
n.d.
-
3.4
n.d. = Not detected. 'The data represent minimum values based on the amount of 14C remaining in the various fractions from which metabolites were characterized by mass spectroscopy.
K. L. Davison et al.
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482
About half of the I4C isolated and identified in urine from the perfused kidneys of rats given homocysteinylpropachlor was present as parent compound (table 2). T h e remaining half of the 14C was identified as N-acetylhomocysteinylpropachlor (the mercapturic acid, figure 1). T h e FAB mass spectrum of this mercapturic acid contained molecular ion adducts at m / z 353 ( M + H ) + , 375 ( M + N a ) + , 391 (M + K ) + , 397 ( M - H + 2 N a ) + and 413 ( M - H + N a + K ) + . These ions are 42a.m.u. higher than those observed for the molecular ion plus a proton or the indicated salts for synthesized authentic homocysteinylpropachlor (figure 2), the dosing material. T h e relatively high abundance of sodium and potassium salts in FAB mass spectra of compounds isolated from urine compared to spectra of synthesized compounds is thought to be related to the abundance of these salts in the urine. Ions from the salts of glycerol, the liquid support medium on the probe of the mass spectrometer, appear at m / z 115 (glycerol+Na) and 131 (glycerol+K), although ions for two glycerols+H, Na or K may appear in some spectra at mjz 185, 207 and 223, respectively. N-Acetylhomocysteinylpropachlorand its S-oxide were isolated from urine from the non-perfused kidney and from bile. T h e FAB mass spectrum of Nacetylhomocysteinylpropachlor-S-oxide(figure 3) contained molecular ion adducts at m / z 369 ( M + H ) + , 391 ( M + N a ) + and 407 ( M + K ) + , and fragment ions at m / z 223 and 207. T h e fragment ion at m / z 223 is thought to result from fragmentation of the sulphur-carbon (y-carbon of the homocysteine moiety) bond with subsequent rearrangement and loss of a proton (Larsen and Ryhage 1982). T h e fragment ion at m / z 207 is thought to result from the loss of oxygen from either the sulphoxide during rearrangement or from the fragment ion at m / z 223 or both. T h e increase in relative intensity of the ion at m / z 13 1 compared to the relative intensity of this ion in 1
100
HNCCH,
I
M+Na
N-CCH2-S-CH2CH2CHCOOH
80
II
375
0
60 131
40
M+H 353 M-H+2Na 397 20
0 100
150
200
250
300
350
400
Figure 1 . Fast atom bombardment mass spectrum of N-acetylhomocysteinylpropachlor [2-(N-acetylhomocysteiny1)-N-isopropylacetanilide]isolated from urine from the perfused kidney of rats perfused with homocysteinylpropachlor. Molecular weight= 352.
483
Metabolism of cysteine and homocysteine conjugates N 3
1oc
J
134
I
176
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2M-134 486
2M+N8 643
-IrclL 333
-I-
1.
I
100
200
300
I.
I
500
400
600
d z Figure 2.
Fast atom bombardmentmass spectrum of homocysteinylpropachlor(2-S-homocysteinyl-Nisopropylacetanilide). Molecular weight = 3 10.
115
100
0
80 131 60
M+Na
40
20
0
100
150
200
250
300
350
400
Figure 3. Fast atom bombardment mass spectrum of N-acetylhomocysteinylpropachlor-S-oxide[2(N-acetyl-homocysteinyl)-N-isopropylacetanilide-S-oxide]isolated from urine from the nonperfused kidney of rats perfused with homocysteinylpropacholor. Molecular weight = 368.
K. L. Davison et al.
484
the spectrum for N-acetylhomocysteinylpropachloris thought to come from the Nacetylalanine moiety (130 H)', which resulted from the sulphur-carbon fragmentation (Larsen and Ryhage 1982). T h e relatively low recovery of metabolites as a percentage of the dose (table 2) occurred because of the relatively small doses and the resultant small masses in bile and urine available for clean-up. Carbon-14 recoveries from the Porapak Q and L H 20 columns varied upward from 85% and 90%, respectively. Carbon-14 recoveries from the h.p.1.c. ranged from 60% to 80%. Tails of radioactive areas were cut to increase sample purity, so the data presented are minimum values, and are based on the amount of material available for mass spectroscopy. Other metabolites, if present, were not obvious.
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+
Discussion T h e distribution of the 14Camong the tissues and its elimination in bile and urine was similar for the two compounds. T h e larger standard errors observed for rats given homocysteinylpropachlor were attributable, for no apparent reason, to one of the three rats given this compound. Bile was collected at hourly intervals, and urine was collected at 15 and 30min and at 30min intervals thereafter to 3 h. T h e distribution of the radioactivity among these fractions was also similar for the two compounds. Retention of 14C in kidney or other tissues was relatively small following perfusion of either the cysteinyl- or homocysteinylpropachlor. We observed greater retention of I4C when I4C-DCVC was perfused renally in rats (6% of the dose was retained in the perfused kidney) and calves (24%, Davison 1989). T h e metabolism of the two compounds differed, however. T h e metabolites observed from cysteinylpropachlor were mercapturic acids; no parent compound was found in either bile or urine. In the case of homocysteinylpropachlor, homocysteinylpropachlor itself was found in urine from the perfused kidneys, but was not found in bile or urine from the non-perfused kidneys. Also, homocysteinylmercapturic acids were found in bile and in urine from both kidneys. T h e appearance of unchanged homocysteinylpropachlor only in urine from the perfused kidneys suggests either that the proximal tubular cells were unable to reabsorb all of the homocysteinylpropachlor from the glomerular filtrate or that the unnatural substrate was a poor substrate for renal cysteine conjugate N-acetyltransferase which could enhance both thiol and 2-keto-3-butenoic acid formation. T h e finding of similar tissue residues in the groups of rats given the cysteine and homocysteine conjugates, and the finding of homocysteinylmercapturic acids but no homocysteinylpropachlor in bile or urine from the non-perfused kidneys of rats given the homocysteine conjugate, demonstrates that the tissues were capable of metabolizing homocysteinylpropachlor . Some absorption, metabolism (N-acetyltransferase) and transport of homocysteinylpropachlor obviously occurred in the renal cells because twice the quantity of mercapturic acids was eliminated in urine from the perfused kidneys than in urine from the non-perfused kidneys. However, the data do not allow determination of how much substrate entered the cells directly through the basolateral cell walls and how much entered through the tubular walls. Uptake of DCVC and DCVHC by suspensions of rat renal tubule cells was shown to be similar (Lash and Anders 1989). If Lash's observation can be extrapolated to our in situ observations, then one might conclude that the apparent slower metabolism of the homocysteinylpropachlor by
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Metabolism of cysteine and homocysteine conjugates
48 5
perfused kidneys was due to intracellular factors. DCVC is a known renotoxin, causing disruption of mitochondrial structure and function and cell death (Lash and Anders 1987). To summarize, rat kidneys perfused in situ metabolized both cysteinyl- and homocysteinylpropachlor to their respective mercapturic acids. Acetylation of cysteinylpropachlor appeared to be more efficient than acetylation of homocysteinylpropachlor. Acetylated cysteinylpropachlor was the only compound isolated from bile or urine from rats renally perfused with cysteinylpropachlor. Homocysteinylpropachlor and acetylated homocysteinylpropachlor were isolated from urine from the perfused kidneys of rats given homocysteinylpropachlor, but acetylated homocysteinylpropachlor only was isolated from bile or urine from the nonperfused kidneys of these rats. Therefore, as expected from in vitro studies (Lash and Anders 1989), the natural cysteine conjugate is the preferred substrate.
Acknowledgements We thank D. Barton, B. Jacobson, K. McDonald and V. Peterson for excellent technical and secretarial assistance. Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
References ANDERSON, P. M., and SCHULTZE, M. O., 1965, Cleavage of S-(l,2-dichlorovinyl)-~-cysteine by an enzyme of bovine origin. Archives of Biochemistry and Biophysics, 111, 593-602. BAKKE, J. E., and LARSEN, G. I,., 1985, Metabolism of 2-chloro-N-isopropylacetanilide in chickens. Chemosphere, 14, 1749-1754. DAVISON, K. L., 1989, In situ perfusion and collection techniques for studying xenobiotic metabolism in animals, in Intermediary Xenobiotic Metabolism in Animals: Methodology, Mechanisms and Significance, edited by D . H. Hutson, J. Caldwell, and G. D. Paulson (London: Taylor & Francis), pp. 315-333. DAVISON, K. L., BAKKE, J. E., and LARSEN, G. L., 1988, A kidney perfusion method for metabolism studies with chickens using propachlor as a model. Xenobiotica, 18, 323-329. DAVISON, K. L., BAKKE, J. E., and LARSEN, G. L., 1990, Metabolism of the glutathione conjugate of propachlor by in situ perfused kidneys and livers of rats. Xenobiotica, 20, 375-383. W., VAMVAKAS, S., and ANDERS, M. W., 1990, Biosynthesis, bioactivation, and mutagenicity of DEKANT, S-conjugates. Toxicology Letters, 53, 53-58. ELFARRA, A. A,, LASH,L. H., and ANDERS, M. W., 1986, Metabolic activation and detoxification of nephrotoxic cysteine and homocysteine S-conjugates. Proceedings of the National Academy of Science, 83, 2667-2671. LAMOUREUX, G. L., and DAVISON, K. L., 1975, Mercapturic acid formation in the metabolism of propachlor, CDAA, and fluorodifen in the rat. Pesticide Biochemistry and Physiology, 5,497-506. G . L., and BAKKE, J. E., 1981, Enterohepatic circulation in formation of propachlor (2-chloro-NLARSEN, isopropylacetanilide) metabolites in the rat. Xenobiotica, 11, 473480. G. L., and BAKKE, J. E., 1983, Metabolism of mercapturic acid-pathway metabolites of 2-chloroLARSEN, N-isopropylacetanilide (propachlor) by gastrointestinal bacteria. Xenobiotica, 13, 115-1 26. LARSEN, G. L., and RYHAGE, R., 1982, Fast atom bombardment mass spectra of mercapturic acidpathway metabolites of propachlor (2-chloro-N-isopropylacetanilide).Xenobiotica, 12, 855-860. M. W., 1987, Mechanism of S-(1,2-dichlorovinyl)-~-cysteine-and S-(1,2LASH,L. H., and ANDERS, dichloroviny1)-L-homocysteine induced renal mitochondrial toxicity. Molecular Pharmacology, 32, 549-556. LASH,L. H., and ANDERS, M . W., 1989, IJptake of nephrotoxic S-conjugates by isolated rat renal proximal tubular cells. Journal of Pharmacology and Experimental Therapeutics, 248, 531-537. SILBERNAGL, S., and HEUNER, A,, 1990, Renal transport and metabolism of mercapturic acids and their precursors. Toxicology Letters, 53, 45-51. STEVENS, J. L., ROBBINS, J. D., and BYRD,R. A., 1986, Purified cysteine conjugate B-lyase from rat kidney cytosol. Journal of Biological Chemistry, 261, 15529-15537. WIDEMAN, R. F., JR,and BRAUN, E. J., 1982, Ureteral urine collection from anaesthetized domestic fowl. Laboratory Animal Science, 32, 298-301.
