Excretion of N-acetyl-S-(1-phenyl-2-hydroxyethyl)-cysteine and N-acetyl-S-(2-phenyl-2-hydroxyethyl)-cysteine in workers exposed to styrene.
Styrene (S) has been shown to be responsible for neurotoxic effects, including behavioural changes and neuroendocrine disturbances. The initial step of S metabolism is conversion to styrene 7,8-epoxide (SO), which is present in two enantiomeric forms [(R)(+)-SO and (S)(-)-SO]; this electrophilic intermediate is considered to be directly responsible for most toxic effects of S. The major urinary metabolites derived from the biotransformation of SO in man are mandelic acid (MA) and phenylglyoxylic acid (PGA). In rats an alternative pathway has been demonstrated, which involves the conjugation of SO to glutathione (GSH), leading to the excretion of two specific mercapturic acids, N-acetyl-S-(-(1-phenyl-2-hydroxyethyl)-cysteine [M1] and N-acetyl-S-(2-phenyl-2-hydroxy-ethyl)-cysteine [M2]; a close relationship has been found between exposure to S and urinary excretion of M1 and M2 in rats. As a consequence of the chiral nature of SO, both M1 and M2 consist of two diastereoisomers (M1-'R', M1-'S', M2-'R' and M2-'S'). Early reports have shown that the conversion of S to mercapturic acids is much lower in man (below 1% of the absorbed dose) than in rats (about 10%). We propose an analytical method for the determination of urinary M1 and M2 in man, which involves a urine clean-up by a chromatographic technique with a short reversed-phase pre-column; purified samples are then deacetylated with porcine acylase and deproteinized by centrifugal ultrafiltration. A derivatization is then performed with o-phthaldialdehyde and 2-mercaptoethanol and the fluorescent derivatives are separated on a reversed-phase analytical column. The mobile phase consists of acetate buffer and methanol mixed at variable proportions, the fluorescence detector is set at 330 nm (exc.) and 440 nm (em.). M1-'S' and M1-'R' are separated (retention times = 52.8 and 73.7 min, respectively) while the diastereoisomers of M2 coelute as a single peak at 70.5 min. The detection limit is about 7 micrograms/l, the coefficients of variation are below 7% and the error percentages are less than 6%. The method was applied to 25 urine samples from workers exposed to S: significant correlations were found between mercapturic acids and MA and PGA, the best correlation being between M2 and PGA (r = 0.79). Urine samples form unexposed subjects showed no detectable amounts of the analytes. A high stereoselectivity is shown by the enzymes involved in the metabolism of S to mercapturic acids: M1-'S', which derives from (S)-SO, is excreted in much higher amounts than M1-'R', which derives from (R)-SO.
Maestri
L
L
Fondazione Salvatore Maugeri I.R.C.C.S., Pavia, Italy.
Imbriani
M
M
Ghittori
S
S
Capodaglio
E
E
Gobba
F
F
Cavalleri
A
A
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000652:urine; D000818:Animals; D001665:Binding Sites; D001711:Biotransformation; D002273:Carcinogens / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000031:analogs & derivatives / Q000652:urine; D004784:Environmental Monitoring; D004852:Epoxy Compounds / Q000652:urine; D005978:Glutathione / Q000652:urine; D006038:Glyoxylates / Q000652:urine; D006801:Humans; D008297:Male; D008333:Mandelic Acids / Q000652:urine; D008623:Mercaptoethanol / Q000737:chemistry; D016273:Occupational Exposure; D051381:Rats; D012015:Reference Standards; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D020058:Styrene; D013343:Styrenes / Q000009:adverse effects* / Q000737:chemistry / Q000652:urine; D014462:Ultrafiltration; D009764:o-Phthalaldehyde / Q000737:chemistry
D002273:Carcinogens; D004852:Epoxy Compounds; D006038:Glyoxylates; D008333:Mandelic Acids; C083581:NAPEC; D013343:Styrenes; C012482:phenylglyoxylic acid; D020058:Styrene; D008623:Mercaptoethanol; D009764:o-Phthalaldehyde; C013690:styrene oxide; D005978:Glutathione; D003545:Cysteine; C037938:mandelic acid; D000111:Acetylcysteine