Analysis of butadiene urinary metabolites by liquid chromatography-triple quadrupole mass spectrometry.

1,3-Butadiene (BD) is a monomer produced in petrochemical production facilities and from several combustion sources. The United States Environmental Protection Agency has defined BD as a probable human carcinogen. Methods for assessing exposure and internal dose are therefore of critical interest, and one technique is the measurement of urinary metabolites. Here we describe methods for measuring two urinary metabolites, N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (referred to as MI) and an isomeric mixture of the regio- and stereoisomers (R)/(S)-N-acetyl-S-(1-(hydroxymethyl)-2-propen-yl)-L-cysteine and (R)/(S)-N-acetyl-S-(2-hydroxy-3-butenyl)-L-cysteine (referred to as MII). The method is based on isolation of the metabolites by solid-phase extraction and measurement using liquid chromatography and triple quadrupole mass spectrometry (LC-MS(3)). The LC-MS(3) allowed good selectivity with minimal sample preparation. Assay accuracy was within 10% or better, with substantial improvement in accuracy accompanying the commercial availability of deuterated internal standards for both compounds. Assay precision and linearity passed rigorous validation criteria, and precision-based limits of quantitation values were 12 and 1 ng/mL for MI and MII, respectively. Data are shown from analysis of human urine from occupationally exposed individuals and rat urine from BD exposures conducted to investigate rodent metabolic profiles. Both of these data sets clearly show that this assay can discern previously described relationships between BD exposure and the production of MI/MII.

Response of rats to low levels of sarin.

The purpose of this study was to determine whether exposure to levels of sarin causing no overt clinical signs would cause more subtle, adverse health effects that persisted after the exposure ended. Inhalation exposures of male Fischer 344 rats to 0, 0.2, or 0.4 mg/m(3) of sarin for 1 h/day for 1, 5, or 10 days under normal (25 degrees C) and heat-stressed (32 degrees C) conditions were completed and observations were made at 1 day and 1 month after the exposures. The sarin exposures had no observed effects on body weight, respiration rate, and minute volume during exposure nor in body temperature and activity during the 30-day recovery period. There was no evidence of cellular changes in brain determined by routine histopathology nor of any increase in apoptosis. Brain mRNA for interleukin (IL)-1beta, tumor necrosis factor-alpha, and IL-6 was increased in a dose-dependent manner. Autoradiographic studies demonstrated that M1 cholinergic receptor site densities were unchanged at 1 day after repeated exposures with or without heat stress. At 30 days, there was a decrease in M1 receptors in the olfactory tubercle (with and without heat), and, with heat stress, M1 sites also decreased in a dose-dependent manner in the frontal cortex, anterior olfactory nucleus, and hippocampus. M3 receptor sites were not affected by sarin exposure alone. In the presence of heat stress, there was an upregulation in binding site densities in the frontal cortex, olfactory tubercle, anterior nucleus, and striatum immediately after exposure, and these effects persisted at 30 days. Although red blood cell acetylcholinesterase (AChE) was not greatly inhibited by the 1-day exposure, there were 30 and 60% inhibitions after repeated exposures at the low and high doses, respectively. Histochemical staining for AChE demonstrated that sarin exposure alone reduced AChE in the cerebral cortex, striatum, and olfactory bulb. Sarin exposure under heat stress reduced AChE staining in the hippocampus, an area important for memory function. Thus, repeated exposures under heat-stress conditions, to levels of sarin that would not be noticed clinically, resulted in delayed development of brain alterations in cholinergic receptor subtypes that may be associated with memory loss and cognitive dysfunction.

Subclinical doses of the nerve gas sarin impair T cell responses through the autonomic nervous system.

The nerve gas sarin is a potent cholinergic agent, and exposure to high doses may cause neurotoxicity and death. Subclinical exposures to sarin have been postulated to contribute to the Gulf War syndrome; however, the biological effects of subclinical exposure are largely unknown. In this communication, evidence shows that subclinical doses (0.2 and 0.4 mg/m(3)) of sarin administered by inhalation to F344 rats for 1 h/day for 5 or 10 days inhibited the anti-sheep red blood cell antibody-forming cell response of spleen cells without affecting the distribution of lymphocyte subpopulations in the spleen. Moreover, sarin suppressed T cell responses, including the concanavalin A (Con A) and the anti-alphabeta-T cell receptor (TCR) antibody-induced T cell proliferation and the rise in the intracellular calcium following TCR ligation. These concentrations of sarin altered regional but not total brain acetylcholinesterase activity. Interestingly, serum corticosterone levels of the sarin-treated animals were dramatically lower than the control animals, indicating that sarin-induced immunosuppression did not result from the activation of the hypothalamus-pituitary-adrenal (HPA) axis. Pretreatment of animals with the ganglionic blocker chlorisondamine abrogated the inhibitory effects of sarin on spleen cell proliferation in response to Con A and anti-TCR antibodies. These results suggest that the effects of sarin on T cell responsiveness are mediated via the autonomic nervous system and are independent of the HPA axis.