Bone marrow and renal injury associated with haloalkene cysteine conjugates in calves.

Almost 40 years ago, it was reported that cattle-feed which had been extracted with hot trichloroethylene and then fed to calves produced renal injury and a fatal aplastic anaemia. The toxic factor was subsequently identified as S-(1,2-dichlorovinyl)-L-cysteine (DCVC). These original findings have been confirmed, a single intravenous dose of DCVC at 4 mg/kg, or 0.4 mg/kg intravenously per day administered for 10 days to calves produced aplastic anaemia, and renal injury after a single dose of 4 mg/kg. The toxicity to calves of a number of other haloalkene cysteine conjugates has been examined to ascertain whether, like DCVC, they produce bone marrow and renal injury. Intravenous administration of the N-acetyl cysteine conjugate of DCVC produced renal but not bone marrow injury at a molar equivalent dose to DCVC, indicating that the calf can deacetylate the mercapturic acid and further that sufficient chemical had reached the kidney to be a substrate for the enzyme cysteine conjugate beta-lyase. However, intravenous administration of the alpha-methyl analogue of DCVC, which cannot undergo metabolism via the enzyme cysteine conjugate beta-lyase, was without toxicity at doses about five-fold higher than DCVC. These latter findings provide strong evidence that metabolism of DCVC via the enzyme beta-lyase is necessary for bone marrow and renal injury to occur. The cysteine conjugates of perchloroethylene and hexachloro-1,3-butadiene(HCBD) when given intravenously to calves at molar equivalent doses to DCVC, or above, did not produce either bone marrow or renal injury. In contrast, intravenous administration of the cysteine conjugate of tetrafluoroethylene (TFEC) produced severe renal tubular injury in calves without affecting the bone marrow. In vitro studies with these haloalkene cysteine conjugates showed, like DCVC, that they were good substrates for calf renal cysteine conjugate beta-lyase and toxic to renal cells as judged by their ability to reduce organic anion and cation transport by slices of calf renal cortex and inhibit the renal enzyme glutathione reductase. Calves were also dosed either orally or intravenously with HCBD to assess its toxicity. HCBD at higher molar equivalent doses than DCVC produced mid-zonal necrosis in the liver, renal tubular necrosis but no bone marrow injury in calves. The key findings emerging from these studies are (1) that none of the other cysteine conjugates, at molar equivalent doses to DCVC and above, produce bone marrow injury in calves, (2) TFEC produced only renal injury, suggesting that sufficient of the other conjugates had not reached the kidney for metabolism by beta-lyase to produce cytotoxicity and (3) that HCBD itself is more toxic than its cysteine or mercapturic acid conjugate, suggesting that pharmaco-kinetics and disposition are important factors in determining the toxicity of these conjugates to calves. Further studies are needed to understand the basis for the selective toxicity of DCVC to the bone marrow of calves.

Cysteine conjugate beta-lyase-catalyzed bioactivation of bromine-containing cysteine S-conjugates: stoichiometry and formation of 2,2-difluoro-3-halothiiranes.

1,1-Dichloroalkene-derived S-(1-chloroalkenyl)-L-cysteine conjugates, but not 1,1-difluoroalkene-derived S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates, are mutagenic in the Ames test. Recent studies have showed, however, that bromine-containing, 1,1-difluoroalkene-derived S-(2-bromo-2-halo-1,1-difluoroethyl)-L-cysteine conjugates are mutagenic [Finkelstein, M. B., et al. (1994) Chem. Res. Toxicol. 7, 157-163] and that alpha-thiolactones are formed as reactive intermediates and glyoxylate as a terminal product [Finkelstein, M. B., et al. (1995) J. Am. Chem. Soc. 117, 9590-9591]. The present studies were undertaken to examine the stoichiometry of cysteine conjugate beta-lyase-catalyzed product formation from a panel of bromine-containing and bromine-lacking cysteine S-conjugates and to search for additional metabolites. The cysteine S-conjugates were incubated with rat renal homogenates, and pyruvate:product (glyoxylate, bromide, fluoride, dihaloacetate, trihaloethene) ratios were measured. Pyruvate:glyoxylate ratios for S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine, S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine, and S-(2,2-dibromo-1,1-difluoroethyl)-L-cysteine ranged from 1:0.13 to 1:0.16. With S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine and S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine, pyruvate:bromide ratios were 1:1, but with the dibrominated conjugate S-(2,2-dibromo-1,1-difluoroethyl)-L-cysteine, the pyruvate:bromide ratio was 1:1.2. All bromine-containing cysteine S-conjugates gave less than complete conversion to fluoride. A search for additional metabolites led to the consideration of 2,2-difluoro-3-halothiiranes as putative intermediates. 2,2-Difluoro-3-halothiiranes may arise by internal displacement of bromide and cyclization of 2-bromo-2-halo-1,1-difluoroethanethiolates, which are beta-elimination products of cysteine S-conjugates. Such halogenated thiiranes may eliminate sulfur to give 1,1-difluoro-2-haloethenes. GC/MS analysis showed that trifluoroethene, 2-chloro-1,1-difluoroethene, and 2-bromo-1,1-difluoroethene were terminal products of S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine, S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine, and S-(2,2-dibromo-1,1-difluoroethyl)-L-cysteine, respectively. The bromine-lacking conjugate S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine did not yield glyoxylate or trifluoroethene as products, but the formation of chlorofluoroacetate was confirmed. The pyruvate:chlorofluoroacetate ratio was 1:0.38, indicating that other products are formed. This is the first report of the stoichiometry of the beta-lyase-catalyzed biotransformation of haloalkene-derived cysteine S-conjugates and of the formation of 2,2-difluoro-3-halothiiranes as reactive intermediates in the biotransformation of bromine-containing cysteine S-conjugates.

Metabolism of [14C]- and [35S]S-(1,2-dichlorovinyl)-L-cysteine in the male Fischer 344 rat.

The metabolic fate, tissue distribution, and elimination profile of [35S]- and [cysteine-U-14C]S-(1,2-dichlorovinyl)-L-cysteine (DCVC)--given either intravenously or intraperitoneally to male Fischer 344 rats--was investigated. Blood samples were collected periodically from 5 min to 96 hr after administration. More than 99% of the DCVC was cleared from plasma within 2.5 hr after either intravenous or intraperitoneal injection. The initial half-lives of both [35S]- and [14C]DCVC were 2.0 and 2.8 hr, respectively, and the mercapturate S-(1,2-dichlorovinyl)-N-acetyl-L-cysteine was detected in plasma within 5 min of giving DCVC. The major plasma metabolite detected after giving [35S]DCVC was inorganic sulfate, and S-(1,2-dichlorovinyl)-N-acetyl-L-cysteine and pyruvate were also detected in plasma after giving [14C]DCVC. S-(1,2-Dichlorovinyl)-N-acetyl-L-cysteine was the major urinary metabolite detected after giving [14C]DCVC, and inorganic sulfate was excreted in the urine after giving [35S]DCVC. Administration of the cysteine conjugate beta-lyase inhibitor aminooxyacetic acid led to a significant increase in the urinary excretion of radioactivity, mostly in the form of the mercapturate. The kidney contained the highest amount of radioactivity after administration of [35S]DCVC. In addition, similar amounts of radioactivity were present in brain, heart, kidney, and liver after administration of [14C]DCVC, but the 14C content of the liver was decreased in aminooxyacetic acid-treated rats. This study shows that DCVC is rapidly metabolized to inorganic sulfate and S-(1,2-dichlorovinyl)-N-acetyl-L-cysteine, which are eliminated in the urine.

Structure-mutagenicity and structure-cytotoxicity studies on bromine-containing cysteine S-conjugates and related compounds.

Glutathione and cysteine S-conjugates of several haloalkenes are nephrotoxic and cytotoxic. Chloroalkene-derived S-(1-chloroalkenyl)-L-cysteine conjugates, but not fluoroalkene-derived S-(2,2-dihalo-1,1-difluorethyl)-L-cysteine conjugates, are mutagenic in the Ames test, although both types of S-conjugates are cytotoxic and nephrotoxic. Recent studies showed that bromine-containing S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates are mutagenic in the Ames test, thus challenging the generalization that S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates are not mutagenic. Hence a series of bromine-containing and bromine-lacking S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteine conjugates was prepared, and their mutagenicity was assessed in the Ames test with Salmonella typhimurium TA2638 as the test strain. In addition, several indices of cytotoxicity, including cytotoxicity in LLC-PK1 cells, induction of Ca2+ release from pig kidney mitochondria, and DNA double-strand breaks in LLC-PK1 cells, were measured. The bromine-containing S-conjugates S-(2-bromo-2-chloro-1,1-difluoroethyl)-L- cysteine (BCD-FC), S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine (BTFC), and S-(2,2-dibromo-1,1-difluoroethyl)-L-cysteine (DBDFC) were mutagenic in the Ames test, whereas S-(2-chloro-1,1,2-trifluorethyl)-L-cysteine (CTFC), S-(2,2-dichloro-1,1-difluoroethyl)-L-cysteine (DCDFC), and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFC), which lack bromine, were not. BCDFC, BTFC, CTFC, DBDFC, and TFC were cytotoxic in LLC-PK1 cells, and their cytotoxicity was blocked by the cysteine conjugate beta-lyase inhibitor (aminooxy)acetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Nephrotoxicity of the glutathione and cysteine conjugates of 2-bromo-2-chloro-1,1-difluoroethene.

The mercapturate S-(2-bromo-2-chloro-1,1-difluoroethyl)-N-acetyl-L-cysteine, which is apparently derived from the halothane degradation product 2-bromo-2-chloro-1,1-difluoroethene, is excreted in urine. S-(2-Bromo-2-chloro-1,1-difluoroethyl)glutathione (BCDFG) and S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine (BCDFC) are putative intermediates in the metabolism of 2-bromo-2-chloro- 1,1-difluoroethene and are analogs of nephrotoxic and cytotoxic S-haloalkyl glutathione and cysteine conjugates. The objective of the research was to study the nephrotoxicity and cytotoxicity of 2-bromo-2-chloro-1,1-difluoroethene-derived S-conjugates. BCDFG and BCDFC were nephrotoxic in Fischer 344 rats and caused diuresis, increases in urine glucose and protein concentrations, in blood urea nitrogen concentrations, in kidney/body weight percentages and in serum glutamate-pyruvate transaminase activities. Both S-conjugates also produced severe morphological changes in the kidneys, especially in the proximal tubules. Morphological changes indicative of hepatotoxicity were seen in some animals given BCDFG and BCDFC. Both BCDFG and BCDFC were cytotoxic to LLC-PK1 cells, as shown by lactate dehydrogenase release into the medium. The cytotoxicity of BCDFG was blocked by the gamma-glutamyltransferase inhibitor acivicin, and the cytotoxicity of both BCDFG and BCDFC was blocked by the cysteine conjugate beta-lyase inhibitor aminooxyacetic acid. Also, S-(2-bromo-2-chloro-1,1-difluoroethyl)-DL-alpha-methylcysteine, which can not be metabolized by beta-lyase, was not toxic to LLC-PK1 cells. These in vivo and in vitro data provide evidence that BCDFG and BCDFC are nephrotoxic and that their toxicity is dependent on renal bioactivation by cysteine conjugate beta-lyase.

Binding of specific ATP citrate lyase and fatty acid synthetase antibodies to heavy populations of rat liver polysomes.

The polysome fractions involved in the synthesis of the rat-liver inducible lipogenic enzymes, ATP citrate lyase and fatty acid synthetase, were identified by their binding of radioiodinated specific antibodies to enzyme. Both of these populations of specific polysomes were shown to be markedly heavier than specific polysomes involved in albumin synthesis. The quanity of antibody bound to the lipogenic enzyme-related polysomes was markedly affected by the dietary status of the animal. A dietary regimen which induced ipogenesis resulted in a tenfold increase in the hepatic activities of these enzymes found in normally fed animals. The radioactivity bound to hepatic polysomes of induced rats was likewise greater than tenfole higher, presumably reflecting an increase in the number of polysomes active in enzyme synthesis. The fasting state resulted in lower hepatic enzyme activity than normal and correspondingly less binding of ATP citrate lyase and fatty acid synthetase antibodies to the heavy polysomes of the sucrose gradient.