Synthesis of the fully phosphorylated GPI anchor pseudohexasaccharide of Toxoplasma gondii.

Retrosynthesis of the fully phosphorylated glycosylphosphatidyl inositol (GPI) anchor pseudohexasaccharide 1a led to building blocks 2-6, of which 5 and 6 are known. The formation of pseudodisaccharide building block 2 is based on readily available building block 7, which gave, via derivative 11 and its glycosylation with known donor 12, the desired compound 2. Building block 3, with the required access to all hydroxy groups being permitted, was prepared from mannose in five steps. From a readily available precursor, building block 4 was obtained, which on reaction with 3 gave disaccharide 23. The synthesis of the decisive pseudohexasaccharide intermediate 32 was based on the reaction of 23 with 5, then with 6, and finally with 2. To obtain high stereoselectivity and good yields in the glycosylation reactions, anchimeric assistance was employed. To enable regioselective attachment of the two different phosphorus esters, the 6f-O-silyl group of 32 was first removed and the aminoethyl phosphate residue was attached. Then the MPM group was oxidatively removed, and the second phosphate residue was introduced. Unprotected 1a was then liberated in two steps: treatment with sodium methanolate removed the acetyl protecting groups, and finally, catalytic hydrogenation afforded the desired target molecule, which could be fully structurally assigned.

Results of biomonitoring analyses in Biomonitoring Laboratory, Helsinki, Finland in 1997.

In 1997 a total of 4848 results of 47 different analytes from blood or urine specimens, were performed in the Finnish Institute of Occupational Health, Biomonitoring Laboratory, Helsinki, Finland. The results of these service analyses were registered in a database with additional information concerning the worker and the work place. The biomonitoring register, containing one or more results of about 30,000 workers, enables the follow-up of chemical exposure on individual or working group levels. In general, the levels of chemicals or their metabolites in biological specimens have been slowly but continuously declined in Finland during the last decade. In 1997 the decrease in the levels of heavy metals was particularly important. The most problematic organic solvent in Finland is styrene. Styrene exposures have remained in unacceptable levels in work places and still in 1997 more than a third of the workers analysed had very high concentrations of styrene metabolites in their urine. In most major analyte groups studied, there were workers whose exposure level exceeded the Finnish biomonitoring action level (BAL), and in about half of the specimens the level exceeded the upper reference limits (URL), of the non-exposed persons.

Exposure to methyl tert-butyl ether and tert-amyl methyl ether from gasoline during tank lorry loading and its measurement using biological monitoring.

OBJECTIVE AND METHODS: The exposure of Finnish tank lorry drivers to methyl tert-butyl ether (MTBE) and tert-amyl methyl ether (TAME) during loading of gasoline was studied using biological and breathing-zone sampling. During the field measurements in October 1994 and August 1995 the gasolines (95, 98, 99 RON) contained MTBE to 5.2-11.8% and TAME to 0-6%. RESULTS: The geometric mean (GM) breathing-zone concentration of MTBE was 4.3 mg/m3 (n = 15) in October and 6.4 mg/m3 (n = 20) in August. The GM concentration of TAME, measured only in August, was 0.98 mg/m3. The mean loading/sampling times were 37 and 35 min, the mean wind speeds were 0.8 and 0.6 m/s, and the mean air temperatures were -4.9 degrees and + 14.1 degrees C, respectively. Blood samples collected on average at 20 min after gasoline loading/exposure showed an MTBE concentration of 143 nmol/l (GM, n = 14) in October and 213 nmol/l (GM, n = 20) in August. Pearson's coefficient of correlation (r) between the MTBE breathing-zone concentrations and MTBE in blood was 0.86 (P = 0.0001) in October and 0.81 (P = 0.00001) in August. No correlation was found between MTBE in air and the metabolite tert-butanol (TBA) in blood. MTBE, but not TBA, in urine samples collected on average at 2.5 h after exposure showed a correlation with MTBE in air. The concentrations of TAME and its metabolite tert-amyl alcohol were below the quantitation limits ( < 7 and < 100 nmol/l, respectively) in most blood and urine samples. CONCLUSIONS: The breathing-zone measurements showed low levels of exposure to the two oxygenates, the concentrations being well below the current hygienic standards for MTBE (250-360 mg/m3 for 15 min and 90-180 mg/m3 for 8 h). The linear correlations obtained for MTBE suggest that MTBE in blood or urine can be adopted as a valid biological exposure index.

Exposure of gasoline road-tanker drivers to methyl tert-butyl ether and methyl tert-amyl ether.

Organic oxygenates, namely, methyl tert-butyl ether (MTBE) and methyl tert-amyl ether (MTAE), are added to gasoline to reduce carbon monoxide in exhausts and to enhance the octane number. The aim of this study was to investigate road-tanker drivers' exposure to oxygenate vapors during road-tanker loading and unloading as well as to evaluate the measurements of these ethers and their metabolites in the urine as a means of assessing the uptake of the ethers. A total of 11 drivers in different parts of Finland were trained to monitor their exposure with personal samplers, to report their working conditions, and to collect their whole-day urine samples. Charcoal tubes of the air samples were analyzed for MTBE, MTAE, benzene, toluene, and aliphatic hydrocarbons. For biological monitoring purposes the two main oxygenates, tertiary ethers MTBE and MTAE, as well as their main metabolites, tertiary alcohols tert-butanol (TBA) and tert-amyl alcohol (TAA), were determined in urine specimens. On average the drivers were exposed to vapors for short periods (21 +/- 14 min) three times during a work shift. The mean concentrations of MTBE and MTAE (mean +/- SD) were 8.1 +/- 8.4 and 0.3 +/- 0.4 mg/m3. The total MTBE uptake during the shift was calculated to be an average of 106 +/- 65 mumol. The mean concentrations of MTBE, TBA, MTAE and TAA detected in the first urine after the work shift were 113 +/- 76, 461 +/- 337, 16 +/- 21, and 40 +/- 38 nmol/l, and those found the next morning, 16 h later, were 18 +/- 12, 322 +/- 213, 9 +/- 10, and 20 +/- 27 nmol/l. The good relationship (r = 0.84) found between MTBE exposure and postshift excretion suggests that urinary MTBE can be used for biological monitoring of exposure, but at the present low level of exposure the corresponding metabolite TBA is not equally reliable. The determination of MTAE and its metabolite TAA in urine is sensitive enough to detect the low degree of exposure to MTAE, but in this study the data were too scarce to allow calculation of the correlations due to very low levels of MTAE exposure.

Biological monitoring of exposure to benzene in the production of benzene and in a cokery.

The purpose of this study was to compare different biological methods in current use to assess benzene exposure. The methods involved in the study were: benzene in blood, urine and exhaled air, and the urinary metabolites t,t-muconic acid (MA) and S-phenylmercapturic acid (S-PMA). Blood, urine and exhaled air samples were collected from workers in a benzene plant (pure benzene exposure) and cokery (mixed exposure, e.g. polycyclic aromatic hydrocarbons--PAHs) in an Estonian shale oil petrochemical plant. The benzene in these samples was analysed with a head-space gas chromatograph, and the metabolites MA and S-PMA with a liquid chromatograph using methods developed from published procedures. Some of the values measured in the Estonian shale oil area were high in comparison with those published during the last few years, whereas the values measured in the control group did not show any exposure to benzene except in the smokers group. The highest median exposure was in the benzene factory, 0.9 cm3/m3 TWA (2.9 mg/m3) and the highest individual value was 15 cm3/m3 TWA (49 mg/m3). All biological measurements in this study gave the same assessment about exposure to benzene and correlated highly significantly with each other and with the air measurements (r = 0.8 or more). In the benzene factory the correlation was good even when calculated from samples with air concentration < 1 cm3/m3 (3.2 mg/m3) in the case of blood benzene and urinary MA. However, for S-PMA it was weak (r = 0.4) and for benzene in urine and exhaled air it did not exist any more. In the cokery, with mixed exposure, the correlation at low levels was weaker even for blood benzene and urinary MA (r = 0.6). According to the results in the benzene factory the exposure to pure benzene at the level 1 cm3/m3 (3.25 mg/m3) TWA gave: the blood benzene value about 110 nmol/l (8.6 micrograms/l), MA 23 mumol/l (3.3 micrograms/l) or 2.0 mg/g creatinine, S-PMA 58 micrograms/g creatinine or 0.4 mumol/l (95.7 micrograms/l), benzene in urine 499 nmol/l (39 micrograms/l), and benzene in the exhaled air 2.8 nmol/l (0.2 microgram/l). In general, the measurement of benzene in blood and in exhaled air, as well as benzene and its metabolites MA and S-PMA in urine, all gave similar results. However, at low exposure level (< 1 cm3/m3) the most reliable analyses were MA in urine and benzene in blood.

Population toxicokinetics of benzene.

In assessing the distribution and metabolism of toxic compounds in the body, measurements are not always feasible for ethical or technical reasons. Computer modeling offers a reasonable alternative, but the variability and complexity of biological systems pose unique challenges in model building and adjustment. Recent tools from population pharmacokinetics, Bayesian statistical inference, and physiological modeling can be brought together to solve these problems. As an example, we modeled the distribution and metabolism of benzene in humans. We derive statistical distributions for the parameters of a physiological model of benzene, on the basis of existing data. The model adequately fits both prior physiological information and experimental data. An estimate of the relationship between benzene exposure (up to 10 ppm) and fraction metabolized in the bone marrow is obtained and is shown to be linear for the subjects studied. Our median population estimate for the fraction of benzene metabolized, independent of exposure levels, is 52% (90% confidence interval, 47-67%). At levels approaching occupational inhalation exposure (continuous 1 ppm exposure), the estimated quantity metabolized in the bone marrow ranges from 2 to 40 mg/day.

Glycophorin A somatic cell mutation frequencies in Finnish reinforced plastics workers exposed to styrene.

We have used the glycophorin A (GPA) in vivo somatic cell mutation assay to assess the genotoxic potential of styrene exposure in 47 reinforced plastics workers occupationally exposed to styrene and 47 unexposed controls matched for age, gender, and active smoking status. GPA variant erythrocyte frequencies (Vf), reflecting GPA allele loss (phi/N) and allele loss and duplication (N/N) somatic mutations arising in vivo in the erythroid progenitor cells of individuals of GPA M/N heterozygous genotype, were flow cytometrically determined in peripheral blood samples from these subjects. Measurements of styrene exposure of the workers at the time of blood sampling showed a mean 8-h time-weighted average (TWA8-h) styrene concentration of 155 mg/m3 (37 ppm) in the breathing zone. Mean urinary concentrations of the styrene metabolites mandelic acid (MA) and mandelic acid plus phenyl glyoxylic acid (MA+PGA) were 4.4 mmol/liter (after workshift) and 2.1 mmol/liter (next morning), respectively. Multivariate analysis of covariance on log-transformed GPA Vf data with models allowing adjustment for age, gender, smoking status, and styrene exposure showed that N/N Vf were nearly significantly increased among all of the exposed workers (adjusted geometric mean, 6.3 per million versus 5.0 in the controls; P = 0.058) and were statistically significantly elevated (adjusted geometric mean, 6.8 versus 5.0 in the controls; P = 0.036) among workers classified into a high-exposure group according to personal TWA8-h concentration of styrene in the breathing zone of > or = 85 mg/m3 (20 ppm; Finnish threshold limit value). Women in this high exposure group showed especially elevated N/N Vf (adjusted geometric mean 8.5 versus 5.3 in control women; P = 0.020); this elevation was also significant if urinary MA+PGA of > or = 1.2 mmol/liter was used as the basis of classification (adjusted geometric mean, 8.3; P = 0.030). The occupational exposure could not be shown to influence phi/N Vf. Cigarette smoking was associated with significantly elevated GPA Vf among active smokers (P = 0.042 for phi/N and P = 0.020 for N/N) and among active and ex-smokers combined (P = 0.014 for N/N). Its influence on phi/N Vf was especially clear among active smokers in the control group (P = 0.005). An effect of smoking, nearly statistically significant, was also observed for the phi/N Vf of control ex-smokers (P = 0.055) and of all active and ex-smokers combined (P = 0.050). Thus, the two characterized chemical exposures experienced by this group of workers and controls appear to produce differential effects on the two independent classes of GPA variants enumerated in the assay. This result suggests that the genotoxicity of these agents is mediated, at least in part, by different genetic mechanisms. Styrene exposure is associated with a specific increase in GPA N/N Vf; these allele loss and duplication variants reflect predominantly somatic recombination mechanisms in erythroid progenitor cells. Tobacco smoke exposure in active and ex-smokers is also associated not only with an increase in N/N Vf but also with an increase in phi/N Vf, reflecting the induction of GPA gene-inactivating mutations, including point mutations and deletions. This finding is consistent with a broad mechanistic spectrum of tobacco smoke genotoxicity associated with this complex mixture of chemical mutagens. Finally, there was no detectable effect of age on phi/N Vf; however, a highly significant (P = 0.0002) increase in N/N Vf with age, even after adjustment for other variables, was observed.

Analysis and stability of phenylglyoxylic and mandelic acids in the urine of styrene-exposed people.

In this work a high-performance liquid chromatographic method is described that is reliable and practical for use in routine biological monitoring of exposure to styrene. The method uses a modern diode array detection technique by which mandelic and phenylglyoxylic acids can be measured simultaneously using different wavelengths. The liquid chromatographic method was compared to a gas chromatographic method developed for the analysis of mandelic, phenylglyoxylic and para-hydroxymandelic acids. The methods gave results consistent with each other. These two methods were then used to check the stability of the main metabolites of styrene, especially of phenylglyoxylic acid, in urine samples stored at +6 degrees C or at -18 degrees C for periods up to 70 days. None of the frozen samples showed any significant decrease in the phenylglyoxylic acid concentration, whereas at 6 degrees C one of the samples showed a reduction of 46% after 1 month.

Biological monitoring of occupational exposure to low levels of benzene.

To obtain reference values for the biological monitoring of benzene, the kinetics of benzene were studied in volunteers. Benzene in blood and expired air could easily be followed until the next morning after a 4-h exposure to a benzene concentration of 10 cm3.m-3. Even after exposure to 1.7 cm3.m-3 the benzene levels in the morning blood and expired air samples differed from those in unexposed subjects. One hour after exposure to 10 and 1.7 cm3.m-3 the mean levels of benzene were 238 and 25 nmol.l-1 in blood and 13.2 and 2.5 mumol.m-3 in exhaled air, respectively. It was concluded that, at high benzene levels (approximately 10 cm3.m-3), samples collected 16 h after exposure reflect the body burden of benzene, while at low exposure (< 1 cm3.m-3) samples collected 1 h after exposure may be used to estimate the exposure over the preceding few hours. Exposure to benzene from smoking is a potential confounder in estimating occupational exposure to low levels of benzene.

Styrene revisited--exposure assessment and risk estimation in reinforced plastics industry.

A survey performed in 32 workshops in reinforced plastics industry showed the mean TWA 8 h concentration of styrene in personal air to be 43 ppm (range 5-182 ppm) among laminators and 11 ppm (range 1-133 ppm) among other workers. The biological measurement of urinary mandelic acid + phenylglyoxylic acid showed mean values of 2.4 mmol/l among laminators without respirators and 1.3 mmol/l when respirators were used. No effects of work related exposure were detected in the cytogenetic parameters, chromosome aberrations, sister chromatid exchanges or micronuclei analyzed in peripheral blood lymphocytes. Grading of the exposure on the basis of lamination method, years of exposure, daily laminating time, air styrene concentration and urinary mandelic acid among laminators, neither revealed any dose dependency.

Urinary excretion of chlorinated phenols in saw-mill workers.

The excretion and conjugation of chlorophenols were studied in workers exposed to 2,4,6-tri-, 2,3,4,6-tetra-, and pentachlorophenolates, the main components of the chlorophenolate product manufactured by direct chlorination of phenol. The workers were exposed in two different saw mills in which sodium chlorophenolate was used for treatment of lumber during the warm season. Urine specimens were collected at the end of the treatment season as well as at the start of a new treatment period in the spring. Serum specimens were collected towards the end of the treatment period. Total and unconjugated chlorophenols were analyzed with a gas chromatographic method. The maximal concentrations of urinary 2,4,6-tri-, 2,3,4,6-tetra- and pentachlorophenol at the end of the lumber-treatment period were 1-11.8, 3.4-17.3, and 0.2-0.9 mumol/l, respectively, and the average apparent half-times calculated using a one-compartment model were 18 h, 4.3 days and 16 days, respectively. For 2,3,4,6-tetrachlorophenol, the data of some subjects showed a better fit with a two-compartment model; the corresponding half-times were 5.3 and 26 days. During the continuous-exposure period the average serum levels of tetra- and pentachlorophenol were rather similar before and after the working day: 2.79 +/- 1.78 mumol/l for tetrachlorophenol and 0.85 +/- 0.4 mumol/l for pentachlorophenol. Renal clearance values for tetra- and pentachlorophenol were related to urine flow and indicated tubular reabsorption. At low concentrations, sulfate conjugation was dominant. With increasing chlorophenol concentrations the proportion of glucuronide conjugation was increased, especially for pentachlorophenol.

Simultaneous determination of benzene and toluene in the blood using head-space gas chromatography.

A head-space method for the simultaneous determination of benzene and toluene in blood using a gas chromatograph equipped with a photoionization detector was developed. Internal standards for benzene and toluene were fluorobenzene and o-xylene, respectively, and the detection limit was 5 nmol/l for both solvents. This method is sensitive enough for needs of biological monitoring of benzene and toluene in exposed workers. With automation it offers a possibility for routine measurements. An application of the method in monitoring exposed workers in the industry is presented.

Kinetics of 2,4,6-trichlorophenol in different organs of the rat.

The concentration of 2,4,6-trichlorophenol was measured in the blood and various other tissues of the rat after IP administration of the compound at 25 mg per kg body weight. The highest concentration, 329 +/- 117 nmol X g-1, was found in the kidney. Half-times were between 1.4 and 1.8 h in the blood, brain, fat, kidney, liver and muscle. The extent of conjugation of 2,4,6-trichlorophenol was also investigated by measuring total and free chlorophenol in the blood.

Skin absorption as a source of error in biological monitoring.

Concentrations of toluene, tetrachloroethylene, and 1,1,1-trichloroethane were determined in blood collected from both forearms of subjects after one of their hands was soaked for 5 min in the corresponding solvent or in a thinner containing toluene, as a simulation of the washing of hands with solvent after work. The concentrations of toluene, tetrachloroethylene, and 1,1,1-trichloroethane on the soaked side were high, maximally 5.4, 9.0, and 4.0 mumol/l, respectively, and 20-, 130-, and 35-fold, respectively, compared to the contralateral side. Intraindividual differences were very marked, and dramatic changes were detected within a short period of time. It was not until after 3 h with toluene and 5 h with the chlorinated solvents that the difference between the two arms vanished. It is concluded that analyses of solvents in blood specimens drawn during or immediately after the workday may lead to markedly erroneous estimations of exposure.

Natural and acquired resistance of mice to infection by airborne Klebsiella pneumoniae after subchronic intoxication by cadmium administered orally.

Mice daily ingested about 22 mg of cadmium per kg of body weight in drinking water for 30 days. On the 30th day, the liver and kidneys of the mice contained about 18 micrograms of Cd2+ per g of fresh organ. A group of these mice was immunized against Klebsiella pneumoniae using two injections of vaccine, the first on the 7th day and the second on the 14th day of intoxication. On the 28th day, the non-immunized and the immunized mice were infected via a respiratory route by one lethal dose 50% of K. pneumoniae (the LD50 for the immunized mice was 2.4 times higher than the LD50 for the non-immunized mice). Comparison with the non-intoxicated control mice showed that the ingestion of Cd2+ did not significantly modify the natural resistance or the acquired resistance of the mice to the infection by airborne K. pneumoniae.

A simple liquid chromatographic method for the analysis of penta- and tetrachlorophenols in urine of exposed workers.

A liquid chromatographic method was developed for the simultaneous determination of penta- and tetrachlorophenols in urine. The method is more rapid than gas chromatographic methods and does not involve the use of such potentially dangerous compounds as benzene, diazomethane or pyridine, which have been used in several methods described previously.