Chem. Res. Toxicol. 1995,8, 917-923
917
The Sulfoxidation of the Hexachlorobutadiene Metabolite N-Acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-~-cysteine Is Catalyzed by Human Cytochrome P450 3A Enzymes Michael Werner,$ Zuyu Guo,~Gerhard Birner,$ Wolfgang Dekant,*>$and F. Peter Guengerichs Institut fir Toxikologie der Universitat Wurzburg, Versbacherstrasse 9, 97078 Wurzburg, FRG, and Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 Received January 30, 1995@ The sulfoxidation of the mercapturic acid N-acetyl-S-(l,2,3,4,4-pentachlorobuta-1,3-dienyl)L-cysteine (N-Ac-PCBC),a urinary metabolite of the renal toxin hexachlorobutadiene (HCBD), was studied in human liver microsomes and with purified cDNA expressed human liver cytochrome P450 (P450) enzymes. N-Acetyl-S-(l,2,3,4,4-pentachlorobuta-l,3-dienyl)-~-cysteine sulfoxide (N-Ac-PCBC SO) is a major urinary metabolite of HCBD in male rats; only liver microsomes from male rats catalyze the sulfoxidation of N-Ac-PCBC. Our results presented here show that human liver microsomes from both male and female donors are capable of oxidizing N-Ac-PCBC t o the corresponding sulfoxide diastereomers. The correlation of N-AcPCBC sulfoxidation with the rates of oxidation of P450 enzyme specific substrates suggests that only P450 3A enzymes oxidize N-Ac-PCBC. Moreover, only gestodene and troleandomycin, two selective inhibitors for P450 of the 3A family, significantly reduced the rates of N-AcPCBC sulfoxidation. No reduction in sulfoxidation rates was observed with inhibitors for other P450 enzymes, i.e., diethyldithiocarbamate, 4-methylpyrazole, 7,8-benzoflavone, or sulfaphenazole. Incubations of N-Ac-PCBC with purified and reconstituted recombinant P450s 1A2, 2E1,3A4, and 3A5 resulted in sulfoxide formation only with P450s 3A4 and 3A5. In summary, these results indicate that P450 from the 3A family may sulfoxidize N-Ac-PCBC. Since these P450 enzymes account for a major fraction of the P450 in human liver and are also present in human kidney, the sulfoxidation reaction may also be expected to occur in humans exposed to HCBD.
Introduction Hexachlorobuta-1,3-diene(HCBD)' is a byproduct in the synthesis of trichloro- and perchloroethene and persists in the environment because of its chemical inertness. HCBD is a selective nephrotoxin in rats and mice and a potent nephrocarcinogen damaging selectively the pars recta of the proximal tubules in the rat (1-5). Its organotropic toxicity is based upon a multistep bioactivation mechanism involving hepatic and renal enzymes (6,7) and is initiated by the conjugation of HCBD with glutathione (GSH) by GSH S-transferases to form S-(1,2,3,4,4-pentachlorobuta1,3-dienyl)-~-glutathione (PCBG) (8, 9). Because the activities of GSH S-transferases are much higher in the liver than in the kidney of rats, this conjugation is presumed to take place predominantly in the liver. After elimination into the
* Address correspondence to this author at the Institut fur Toxikologie, Universitat Wurzburg, Versbacherstrasse 9,97078 Wurzburg, FRG. Universitat Wurzburg. e Vanderbilt University School of Medicine. Abstract published in Advance ACS Abstracts, July 1, 1995. Abbreviations: P450, cytochrome P450; HPLC, high-performance liquid chromatography; HCBD, hexachlorobuta-1,3-diene;PCBC, S-(1,2,3,4,4-pentachlorobuta-l,3-dienyl)-~-cysteine; N-Ac-PCBC,N-acetylS-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-~-cysteine; PCBG, S-(1,2,3,4,4pentachlorobuta-1,3-dienyl)-~-glutathione; GSH, reduced glutathione; FMO, flavin-containing monooxygenase; PLP-mix, 1:l:1 mixture (w/ w) of dilauroylphosphatidylcholine/dioleoylphosphatidylcholine/phosphatidylserine. @
bile and intestinal reabsorption, PCBG is subsequently translocated to the kidney and catabolized by y-glutamyltranspeptidases and dipeptidases to the cysteine conjugate S-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-~-cysteine (PCBC). PCBC may be acetylated by the action of renal and hepatic N-acetyltransferases to the mercapturic acid N-acetyl&( 1,2,3,4,4-pentachlorobuta-l,3-dienyl)-~-cysteine (N-Ac-PCBC). Both PCBC and N-Ac-PCBC are accumulated in the kidney by the organic anion transport system. While N-Ac-PCBC may be eliminated with the urine, PCBC may also be cleaved by renal cysteine conjugate P-lyase to a reactive thioketene (10, 11)whose interaction with cellular macromolecules is thought to be responsible for the nephrotoxic effects observed (Figure 1). Previous studies in male and female rats revealed pronounced sex differences in the metabolism of HCBD as judged from the urinary metabolites identified after oral administration of [l4C1HCBD(12).Only in male rats N-acetyl-S-(1,2,3,4,4-pentachlorobuta-l,3-dienyl)-~-cysteine sulfoxide (N-Ac-PCBC SO) was found. The formation of this vinyl alkyl sulfoxide can principally be mediated by the action of cytochrome P450 (P450) monooxygenases or the flavin-containing monooxygenases (FMOs). Generally, nucleophilic sulfur atoms may be oxidized by the FMOs (13-27) and also by cytochromes P450, whereas nonnucleophilic sulfur as is the case with N-Ac-PCBC is oxidized mainly by cytochromes P450 (1820). S-Oxygenations mediated by the FMOs can be
0893-228d95/2708-0917$09.00/00 1995 American Chemical Society
918 Chem. Res. Toxicol., Vol. 8, No. 7, 1995
Hz, l H , -NHCOCH3). I3C-NMR (100 MHz, ds-DMSOj: b 22.50 (q, -COCH3); 46.70 (d, -CH); 53.15 (t, -CH2); 122.58 ( s , C-4); 126.42 (s, C-2); 126.6 (s, C-3); 142.37 (s, '2-1); 169.54 (s, -COCH3); 171.02 ( s , -COOHj. Thermospray MS: m / z 402 (M + H, loo), 424 (M Na, 42). Electrospray MS: m l r 402 (M H, 1001. General Methods. Human liver microsomes were prepared from organ donors as described (23, 24). Liver samples were provided by Tennessee Donor Services, Nashville, TN. The samples utilized in most incubations were HL 98, 100, 107, 108, 109, 110, 112, 115, 118, 123, 129, 130, and 134 (the abbreviation "HL" denotes "human liver"). Protein concentrations were determined by the bicinchoninic acid procedure (Pierce Chemical Co., Rockford, IL). P450 concentrations were measured a s described (25).
CI
CI Cl+Cl
CI
CI
Werner et al.
+
t
CI
accummulation in the kidney
hH3@
&I
1
acylases
CI
CI
kHCOCH3
R-Lyase
::
