Selective and interactive effects of D2 receptor antagonism and positive allosteric mGluR4 modulation on waiting impulsivity.

BACKGROUND: Metabotropic glutamate receptor 4 (mGluR4) and dopamine D2 receptors are specifically expressed within the indirect pathway neurons of the striato-pallidal-subthalamic pathway. This unique expression profile suggests that mGluR4 and D2 receptors may play a cooperative role in the regulation and inhibitory control of behaviour. We investigated this possibility by testing the effects of a functionally-characterised positive allosteric mGluR4 modulator, 4-((E)-styryl)-pyrimidin-2-ylamine (Cpd11), both alone and in combination with the D2 receptor antagonist eticlopride, on two distinct forms of impulsivity. METHODS: Rats were trained on the five-choice serial reaction time task (5-CSRTT) of sustained visual attention and segregated according to low, mid, and high levels of motor impulsivity (LI, MI and HI, respectively), with unscreened rats used as an additional control group. A separate group of rats was trained on a delay discounting task (DDT) to assess choice impulsivity. RESULTS: Systemic administration of Cpd11 dose-dependently increased motor impulsivity and impaired attentional accuracy on the 5-CSRTT in all groups tested. Eticlopride selectively attenuated the increase in impulsivity induced by Cpd11, but not the accompanying attentional impairment, at doses that had no significant effect on behavioural performance when administered alone. Cpd11 also decreased choice impulsivity on the DDT (i.e. increased preference for the large, delayed reward) and decreased locomotor activity. CONCLUSIONS: These findings demonstrate that mGluR4s, in conjunction with D2 receptors, affect motor- and choice-based measures of impulsivity, and therefore may be novel targets to modulate impulsive behaviour associated with a number of neuropsychiatric syndromes.

Metabolic activation of styrene by erythrocytes detected as increased sister chromatid exchanges in cultured human lymphocytes.

Styrene induces sister chromatid exchanges (SCEs) in human whole-blood lymphocyte cultures without exogenous metabolizing systems, which indicates that styrene is metabolically activated in this in vitro system. Whole-blood lymphocyte cultures from 11 male donors showed a clear increase in SCEs after a 48-hr treatment with styrene (2 mM) or with the reactive metabolite styrene 7,8-oxide (0.15 mM). Styrene (0.5 to 4 mM) induced a distinct dose-dependent increase of SCEs in whole-blood cultures (with 200 to 400 million red blood cells/ml) but only a slight effect in purified lymphocyte cultures (with 20,000 red blood cells/ml). SCE induction by styrene (2 mM) depended on the amount of red blood cells (0.02 to 2000 million/ml) added to the purified lymphocyte cultures. Cyclophosphamide, studied for comparison, clearly increased SCEs irrespective of the presence of erythrocytes. The results show that erythrocytes are essential for the activation of styrene in the lymphocyte test system. This activation probably results from the conversion of styrene into styrene 7,8-oxide by oxyhemoglobin.

Development and validation of an LC-MS/MS method for simultaneous determination of piperaquine and 97-63, the active metabolite of CDRI 97-78, in rat plasma and its application in interaction study.

Piperaquine-dihydroartemisinin combination is the latest addition to the repertoire of ACTs recommended by the World Health Organization (WHO) for treatment of falciparum malaria. Due to the increasing resistance to artemisinin derivatives, CSIR-CDRI has developed a prospective short acting, trioxane antimalarial derivative, CDRI 97-78. In the present study, a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous quantification of piperaquine (PPQ) and 97-63, the active metabolite of CDRI 97-78 found in vivo, was developed and validated in 100 µL rat plasma using halofantrine as internal standard. PPQ and 97-63 were separated using acetonitrile:methanol (50:50, v/v) and ammonium formate buffer (10 mM, pH 4.5) in the ratio of 95:5(v/v) as mobile phase under isocratic conditions at a flow rate of 0.65 mL/min on Waters Atlantis C18 (4.6 × 50 mm, 5.0 µm) column. The extraction recoveries of PPQ and 97-63 ranged from 90.58 to 105.48%, while for the internal standard, it was 94.27%. The method was accurate and precise in the linearity range 3.9-250 ng/mL for both the analytes, with a correlation coefficient (r) of ≥ 0.998. The intra- and inter-day assay precision ranged from 2.91 to 8.45% and; intra- and inter-day assay accuracy was between 92.50 and 110.20% for both the analytes. The method was successfully applied to study the effect of oral co-administration of PPQ on the pharmacokinetics of CDRI 97-78 in Sprague-dawley rats and vice versa. The co-administration of CDRI 97-78 caused significant decrease in AUC0-∞ of PPQ from 31.52 ± 2.68 to 14.84 ± 4.33 h*µg/mL. However, co-administration of PPQ did not have any significant effect on the pharmacokinetics of CDRI 97-78.

Human peripheral lymphocytes as indicators of microsomal epoxide hydrolase activity in liver and lung.

In this study, we have applied an improved assay for the determination of microsomal epoxide hydrolase activity to assess enzymatic levels in human lung, liver, and blood lymphocytes. The assay is fluorescence-based and monitors the epoxide hydrolase-mediated conversion of (+/-)-benzo[a]pyrene-4,5-epoxide to (+/-)-trans-benzo[a]pyrene-4,5-dihydrodiol, using a high pressure liquid chromatography separation system. Approximately a 40-fold range in microsomal epoxide hydrolase activities was detected in blood lymphocytes collected from 70 individual donors. In 38 individuals who were sampled twice after a 3-month interval, the repeatability of an individual's lymphocyte epoxide hydrolase activity was highly correlated (r = 0.80, p < 0.02). In addition, within the same individual there appeared to be a strong correlation between lymphocyte and liver epoxide hydrolase activity (r = 0.92, p = 0.02), and some correlation between liver and lung activity (r = 0.58, p = 0.05). Activities were assessed in lymphocytes from a styrene-exposed worker population but no significant associations between blood concentrations of styrene and epoxide hydrolase activity levels were observed. Neither were any correlations detected in these workers between epoxide hydrolase activities and age, years on the job, alcohol consumption, sex, or smoking status. The results of our study suggest that blood lymphocytes are a useful sentinel cell for epoxide hydrolase activity determinations in individuals, as these measures are relatively stable over time and appear to reflect activity levels in other target organs.

Quantitative analysis of styrene monomer in polystyrene and foods including some preliminary studies of the uptake and pharmacodynamics of the monomer in rats.

A variety of food containers, drinking cups and cutlery, fabricated from polystyrene (PS) or polystyrene-related plastic, were analyzed for their styrene monomer content. Samples of yogurt, packaged in PS cups, were similarly analyzed and the leaching of styrene monomer from PS containers by some food simulants was also determined. Blood level studies with rats, dosed with styrene monomer by various routes, illustrated uptake phenomena that were dependent on the dose and route of administration and were also affected by the vehicle used to convey the styrene monomer.

DNA adducts and related biomarkers in populations exposed to environmental carcinogens.

Prevention of environmentally related cancer will be enhanced by the availability of sensitive early warning systems and by improvements in quantitative assessment of human risks. Accordingly, we have carried out a series of molecular epidemiologic studies aimed at validating a panel of biologic markers, including carcinogen-DNA and -protein adducts, sister chromatid exchange, micronucleus formation, DNA strand breaks, and DNA repair capacity. Results from three such studies illustrate the usefulness of these biomarkers in elucidating low-dose-response relationships, correlations between biomarkers, and the range of variation in biomarkers between individuals exposed to similar concentrations of carcinogens. Low-level workplace or ambient exposures to styrene, ethylene oxide, and polycyclic aromatic hydrocarbons (PAH) were associated with significant increases in both molecular dose of carcinogens (adducts) and various markers of preclinical effects. Correlations between biomarkers varied by exposure. For example, in the styrene study, sister chromatid exchange frequency was not correlated with any of the markers, in contrast to the studies of ethylene oxide and PAH. Significant molecular effects were observed not only in occupationally exposed people but also in residents of an area in Poland characterized by high levels of air pollution. For example, the mean PAH-DNA level in exposed residents (winter sample) was 30.4 adducts per 10(8) nucleotides. This level was significantly higher than that of adducts seen in summer samples from the same area (4.2/10(8), or in winter samples from residents of a rural area (11.01/10(8). Significant seasonal variation in PAH-DNA adduct formation in this group was consistent with recorded fluctuations in air pollution levels. Striking interindividual variation was observed in all three exposed populations.

Organ distribution and nervous system binding of styrene and styrene oxide.

Ten adult male rats were injected intraperitoneally with 460 mumol of styrene oxide with radioactive label. Fifteen similar rats were injected similarly with 577 mumol of styrene. The distribution of styrene in central nervous system, blood, liver, lungs, kidneys and duodenum was studied 3, 6 and 24 h after the injection while the same studies were done with styrene oxide 3 and 6 h after the injection. The liver, brain, kidney and duodenal contents of styrene and styrene oxide were higher than that in blood, lungs and spinal cord while the macromolecule-associated styrene oxide in the central nervous system was small. The removal of injected compounds was slow between 3 and 6 h after the injection in the organ systems although lipid-soluble compounds tended to diminish in brain more rapidly than the total radioactivity.

Effects of ethanol administration on cerebral non-protein sulfhydryl content in rats exposed to styrene vapour.

Glutathione (GSH) and other non-protein sulfhydryls (NPS) are known to protect cells from oxidative stress and from potentially toxic electrophiles formed by biotransformation of xenobiotics. This study examined the effect of a simultaneous administration of styrene and ethanol on NPS content and lipid peroxidation in rat liver and brain. Hepatic cytochrome P450 and cytochrome b5 content, aniline hydroxylase and aminopyrine N-demethylase activities as well as the two major urinary metabolites of styrene, mandelic and phenylglyoxylic acids were also measured. Groups of rats given ethanol for 3 weeks in a liquid diet were exposed, starting from the second week, to 326 ppm of styrene (6 h daily, 5 days a week, for 2 weeks). In control pair-fed animals, styrene produced about 30% depletion of brain NPS and 50% depletion of hepatic NPS. Subchronic ethanol treatment did not affect hepatic NPS levels, but caused 23% depletion of brain NPS. Concomitant administration of ethanol and styrene caused a NPS depletion in brain tissue in the order of 60%. These results suggest that in the rat, simultaneous exposure to ethanol and styrene may lead to considerable depletion of brain NPS. This effect is seen when both compounds are given on a subchronic basis, a situation which better resembles possible human exposure.

Comparison of styrene-7,8-oxide adducts formed via reaction with cysteine, N-terminal valine and carboxylic acid residues in human, mouse and rat hemoglobin.

The reactive metabolite of styrene, styrene-7,8-oxide (SO), reacts with a variety of nucleophilic sites in hemoglobin (Hb) to form SO-Hb adducts. Following the in vitro incubation of SO with blood from humans, NMRI mice and Sprague-Dawley rats, the second-order reaction rate constants were determined for the reaction of SO with cysteine (through both the alpha- and beta-carbons of SO), N-terminal valine (through the beta-carbon of SO), and carboxylic acid (presumably through both the alpha- and beta-carbons of SO) residues in Hb. The rate constants for cysteine adducts vary dramatically between species [2.04, 10.7, 133 L (mol Hb)-1 h-1 (alpha binding) for humans, mice and rats, respectively] and [0.078, 2.16, 20.4 L (mol Hb)-1 h-1 (beta binding), respectively]. The considerably higher rate of reaction with cysteine in rat Hb probably reflects the presence of an additional cysteine residue at position beta 125. Although the rate constants for valine adducts (1.82, 0.80, 0.29 L (mol Hb)-1 h-1, respectively) and COOH adducts (3.55, 1.94, 2.37 L (mol Hb)-1 h-1, respectively) are much more consistent, the inter-species differences are statistically significant for the reaction of SO with the N-terminal valine of Hb. Following the i.p. administration of styrene to mice and styrene and SO to rats, the levels of adducts at each of these sites were used in conjunction with the calculated rate constants to predict the integrated blood doses of SO. While the SO doses predicted from cysteine and valine adducts were very similar, that based upon COOH-binding was significantly different, presumably due to the instability of SO-COOH adducts. This research affirms the use of both cysteine and valine adducts, but not carboxylic acid adducts, as biomarkers of exposure to styrene and SO.

Dosimetry of styrene 7,8-oxide in styrene- and styrene oxide-exposed mice and rats by quantification of haemoglobin adducts.

Rats (Sprague Dawley) and mice (NMRI) were administered nonlabelled or labelled styrene and styrene oxide by i.p. injection. Blood samples were collected 6 and 24 h after treatment for studies of dose-response and 6 h to 32 days after treatment for studies of adduct stability. Haemoglobin (Hb) and plasma protein adduct levels were determined by radioactivity measurements or, in the case of adducts to N-terminal valine in Hb, by the so-called N-alkyl Edman procedure. Adducts to N-terminal valine were found to be chemically stable during the life-span of the erythrocytes, whereas adducts to carboxylic acid residues showed a reduced stability. The Hb-adduct levels found after styrene oxide treatment were compatible with a linear dose-response at low doses (< or = 0.4 mmol/kg body weight). At higher doses the detoxification of styrene oxide was overloaded resulting in a higher than proportional increase in adduct levels. Saturation of detoxification of styrene oxide could also explain the non-linear dose-response relationship observed in the mouse following treatment with styrene. Styrene oxide gave 4-7 times higher adduct levels than styrene when administered to the animals at equimolar low concentration. For both compounds, the levels of adducts to N-terminal valine were 2-3 times higher in the mouse than in the rat. A comparison of Hb-adduct levels in the styrene-exposed animals with adduct levels in styrene-exposed reinforced plastics workers (Christakopoulos et al., Scand. J. Work Environ. Health, 19(4) (1993) 255-263) suggests that styrene is less effective in humans than in mice and rats.

Biomarkers in styrene-exposed boatbuilders.

14 fiberglass-reinforced plastics (FRP) boatbuilders were compared with 9 unexposed controls with respect to several chemical specific and nonspecific biomarkers measured in peripheral blood. Biomarkers included styrene-hemoglobin adducts (styrene-Hb), sister-chromatid exchanges (SCEs), micronuclei (MN), single-strand breaks (SSBs) and N-acetoxy-2-acetylaminofluorene-induced DNA binding (NA-AAF binding) as a measure of susceptibility to DNA damage. Workers' exposures averaged 11 ppm (8-h TWA; geometric mean) and ranged from 0.6 to 44 p.p.m. Mandelic acid levels were measured in end-of-shift urine samples and reflected an average styrene exposure equivalent to 15 p.p.m. There was a large though not significant difference in levels of styrene-Hb adducts among exposed workers and controls, largely the consequence of a single heavily-exposed individual with an extremely high level of adducts. Significant differences between biomarker levels in exposed workers and controls were observed with MN, SSBs and NA-AAF binding. No significant differences were seen in mean levels of SCEs nor in the incidence of cells with a high frequency of SCEs. The data suggest that exposure to levels of styrene in occupational settings near or below the current OSHA standard (50 p.p.m.) can induce damage at the cellular/molecular level. Appropriately-selected panels of biomarkers can be useful in identifying potentially harmful exposures.

Modeling the acute neurotoxicity of styrene.

Styrene is a widely used industrial solvent associated with acute neurotoxicity. To investigate the relationships between exposure, blood concentrations, and the appearance of neurotoxic effects, four healthy males were exposed to styrene concentrations of 5-200 ppm in four different exposure-time profiles. A digit recognition test and P300 event-related evoked potential were used to measure neurologic function. A physiologically based kinetic (PBK) model generated close predictions of measured styrene blood concentrations, in the range of 0.01-12 mg/L, from this and 21 previous studies. Simulated peak brain concentration, durationXaverage exposure, and peak exposure level were predictive of toxicity. Central nervous system effects were expected at a blood concentration near 2.4 mg/L. A standard of 20 ppm was expected to protect styrene-exposed workers from acute central nervous system toxicity under light work conditions.

Combined effects of solvents on the rat's auditory system: styrene and trichloroethylene.

Because exposures to toxic agents typically involve more than one substance, it is necessary to know if combined exposures pose different risks than those to single agents. Many solvents have been implicated in central nervous disorders and some of them are known to produce hearing loss, probably mediated by damage to cochlear hair cells. Hearing loss was studied by recording the brainstem auditory evoked response (BAER) in male Long Evans rats exposed 8 h/day for 5 days to mixtures of styrene (STY) and trichloroethylene (TCE). Dose groups included air or solvent pairs (STY/TCE) in the following concentrations (ppm): (0:3000), (250:2250), (500:1500), (750:750) and (1000:0). Decreased BAER amplitude, indicative of hearing loss, was correlated with blood levels of total solvent. The effects were as predicted by a linear dose-addition model, indicating neither synergistic nor antagonistic interactions at the concentrations studied.

Exposure of rabbits to styrene. Electronystagmographic findings correlated to the styrene level in blood and cerebrospinal fluid.

Objective methods for critically evaluating the toxic effect of industrial solvents are highly desirable. As many of these solvents are suspected to cause vertigo, an animal experimental model was set up for studying the effects of solvents on the vestibular systems. The vestibular function was studied by registration of involuntary eye movements--nystagmus--which are elicited via central vestibulo-oculomotor connections. During exposure to styrene a so-called positional nystagmus was demonstrated that indicated vestibular disturbances. Nystagmus is normally elicited by rotatory acceleration. During exposure to styrene the direction of this rotatory nystagmus was reversed. The incidence of the positional nystagmus correlated well with the blood level of the solvent, measured by gas chromatography. Kinetic studies also demonstrated a rapid equilibration between the level of the solvent in arterial blood and cerebrospinal fluid, and therefore suggested that estimation of the arterial level reliably indicates the level in the central nervous system.

Pharmacokinetics of inhaled styrene in rats and humans.

The pharmacokinetic profile of inhaled styrene was examined in rats exposed to levels of 80, 200, 600 or 1,200 ppm for periods of up to 24 h. At levels up to 200 ppm for 6 h, styrene was cleared from the blood according to a two-compartment linear pharmacokinetic model, but at levels of 600 ppm and above the clearance was saturated. In going from 80 to 1,200 ppm (a 15-fold increase), the area under the blood concentration/time curves (AUC) increased by a factor or 112. Fat tissue was shown to comprise the second compartment of the two-compartment pharmacokinetic model. It is suggested that saturation of styrene clearance is due mostly to saturation of the metabolic capacity for styrene. In humans exposed to 80 ppm of styrene for 6 h, styrene was cleared from the blood according to a two-compartment linear pharmacokinetic model similar to that for rats. A maximum blood concentration of 0.9 microgram/ml was reached at the end of the exposure. Most of the inhaled styrene was excreted in the urine as phenylglyoxylic and mandelic acids, and only a small amount as styrene in the expired air. Simulation of the pharmacokinetic model showed that no continued accumulation of styrene would occur during repeated, daily 8-h exposures to 80 ppm. These data reveal that the rat is a reasonable pharmacokinetic model for styrene in humans. At levels of exposure up to 200 ppm, styrene is cleared from the body very efficiently and will not continue to accumulate upon repeated exposure. But at levels of styrene sufficiently high to saturate the metabolic clearance capacity, the integrated dose (measured by the AUC) will be much greater than expected based on exposure levels alone. Therefore, the extrapolation of toxicity observed at high levels of styrene exposure to that expected at low levels may not be justified.

Biological indicators of exposure in styrene polymerization workers. Styrene in blood and adipose tissue and mandelic and phenylglyoxylic acids in urine.

The concentrations of mandelic and phenylglyoxylic acids, urinary metabolites of styrene, and styrene in blood were determined for 491 styrene polymerization workers. Styrene in subcutaneous fat was determined for 25 workers. The levels of styrene exposure were estimated to be less than 10 ppm, and urinary metabolite and blood styrene concentrations indicated that significant recent exposure (within 4 h) had occurred among workers in areas of styrene polymerization and styrene monomer production. Styrene analysis of subcutaneous fat suggested that the monomer may have been present for as long as 3 d after exposure, a time when urinary metabolites and blood styrene were almost invariably undetectable. All three biological parameters were correlated with recency of exposure and estimated intensity of exposure within job categories.

Urinary styrene in the biological monitoring of styrene exposure.

The urinary excretion of styrene represents a promising indicator of exposure to this solvent. Nevertheless extensive research under field conditions is scant. In this investigation 214 styrene-exposed workers from 10 fiberglass-reinforced plastics factories were studied. Environmental monitoring was performed by personal passive sampling. Blood styrene and the urinary excretion of styrene and its main metabolites, mandelic acid (MA) and phenylglyoxylic acid (PGA), were measured. The correlation coefficient between the time-weighted average of environmental styrene and the mean urinary excretion of styrene was 0.88 (0.91 after logarithmic transformation), compared with the 0.82 and 0.78 of the end-of-shift MA and PGA values, respectively. A high correlation (0.86) was also found between styrene in the blood and urine. The results, obtained under field conditions with a large group of exposed workers, confirm the usefulness of the urinary excretion of styrene as an exposure index for the biological monitoring of styrene exposure.

Health status of styrene-polystyrene polymerization workers.

Styrene monomer is a greatly used chemical, chiefly in the production of polystyrene. A cross-sectional health survey of 493 production workers was undertaken at the oldest and largest monomer production, polymerization, and extrusion facility in the United States. Relative exposure durations and levels were obtained from occupational histories and corroborated by spot air sampling, blood and fat styrene concentrations and levels of urinary mandelic and phenylglyoxylic acids. Statistically significant differences between the prevalence of abnormalities in high and low exposure groups were found for the following: history of acute prenarcotic symptoms, history of acute lower respiratory symptoms, peroneal nerve conduction velocities, relative lymphocytosis, and elevated gamma glutamyl transpetsidase. The following showed no distinct pattern in prevalence when analyzed by exposure group: chest radiographic changes; indices or restrictive, obstructive and small airway dysfunction; other hepatic and hematological parameters; carcinoembryonic antigen level; sputum cytopathology; radial nerve conduction velocities; and ophthalmological findings. Clinically significant abnormalities were rare.

Human blood samples as indicators of occupational exposure to persistent chlorinated hydrocarbons.

The value of using human blood as an indicator of occupational exposure to persistent organochlorine compounds is demonstrated. Blood samples from a total of 35 persons divided into three different groups, with and without exposure to chlorinated hydrocarbons in the work atmosphere, have been investigated by gas chromatography using electron capture detection. It is shown that the group of workers with an occupational exposure to pentachlorobenzene, hexachlorobenzene, heptachlorostyrene and octachlorostyrene had a higher level of these chlorinated hydrocarbons in their blood samples than did the other groups. On the average, the concentration of hexachlorobenzene is about 20 times higher in blood samples from the occupationally exposed workers than from the control group. The level of hexachlorobenzene in blood samples of the control groups is low compared to recent studies of blood samples from the general population in other industrialized countries. Furthermore, the average value obtained for the exposed workers is of the same magnitude as the general population in these industrialized countries.

Solvent uptake in relation to physical activity.

The influence of lung ventilation and free fatty acids in serum on the internal exposure of organic solvents were investigated. 68 healthy male subjects were exposed 1 h at rest and during exercise to styrene, toluene or xylene. Uptake is influenced by the solvent concentration in inspired air, retention index and pulmonary ventilation. Internal exposure is characterized by venous blood concentrations and metabolites in urine. An increase in free fatty acids could be observed dependent on the internal exposure, preventing increases in the content of free fatty acids is a problem of susceptibility and medical care of exposed workers. Biological limits for the control of internal exposure are suggested.