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, i ncluding behavioural changes and neuroendocrine disturbances. The init ial step of S metabolism is conversion to styrene 7,8-epoxide (SO), wh ich is present in two enantiomeric forms [(R)(+)-SO and (S)(-)-SO]; th is electrophilic intermediate is considered to be directly responsible for most toxic effects of S. The major urinary metabolites derived fr om the biotransformation of SO in man are mandelic acid (MA) and pheny lglyoxylic acid (PGA). In rats an alternative pathway has been demonst rated, which involves the conjugation of SO to glutathione (GSH), lead ing to the excretion of two specific mercapturic acids, N-acetyl-S-(l- phenyl-2-hydroxyethyl)-cysteine (M1) and N-acetyl-S-(2-phenyl-2-hydrox y-ethyl)-cysteine [M2]; a close relationship has been found between ex posure to S and urinary excretion of M1 and M2 in rats. As a consequen ce of the chiral nature of SO, both M1 and M2 consist of two diastereo isomers (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 (be low 1% of the absorbed dose) than in rats (about 10%). We propose an a nalytical method for the determination of urinary M1 and M2 in man, wh ich involves a urine clean-up by a chromatographic technique with a sh ort 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-merc aptoethanol and the fluorescent derivatives are separated on a reverse d-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 separ ated (retention times = 52.8 and 73.7 min, respectively) while the dia stereoisomers of M2 coelute as a single peak at 70.5 min. The detectio n limit is about 7 mu g/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 w ere found between mercapturic acids and MA and PGA, the best correlati on being between M2 and PGA (r= 0.79). Urine samples from unexposed su bjects showed no detectable amounts of the analytes. A high stereosele ctivity is shown by the enzymes involved in the metabolism of S to mer capturic acids: M1-'S', which derives from (S)-SO, is excreted in much higher amounts than M1-'R', which derives from (R)-SO. (C) 1997 Elsev ier Science B.V.