Effect of para halogen modification of S-3-(phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamides on metabolism and clearance.

The purpose of this study was to better understand why para-halogen modifications of S-3-(4-halophenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethylphenyl) propionamide selective androgen receptor modulators (SARMs) had the opposite of expected effects on total clearance, in which electron-withdrawing groups generally protect benzene ring from hydroxylation. We determined the plasma protein binding of this series of halogen substituted SARMs and characterized the qualitative effects of B-ring halogen substitution on in vivo metabolism. In vivo metabolism of S-9, S-10, and S-11 were determined in rats using LC-MS(n) analysis. Intrinsic clearance was measured by in vitro metabolism using rat liver microsomes. Rat plasma protein binding was measured by equilibrium dialysis and drug concentrations after dialysis were analyzed by LC-MS. The major metabolic pathways of the halogen-substituted SARMs examined were very similar and included three major phase I pathways; (1) hydrolysis of the amide bond, (2) B-ring hydroxylation, and (3) A-ring nitro reduction to an aromatic amine. In plasma protein binding studies, S-1 (F, fu = 0.78 ± 0.17 %) showed the greatest unbound fraction, followed by S-9 (Cl, fu = 0.10 ± 0.04 %), S-10 (Br, fu = 0.03 ± 0.01 %), and S-11 (I, fu = 0.008 ± 0.001 %). The CLint values of S-1, S-9, S-10, and S-11 were 2.4, 2.5, 2.8, and 4.6 µL/min/mg, respectively. These findings suggest that as lipophilicity increased the free fraction was reduced thus compensating for metabolic liability and resulting in the apparent discrepancy between CLint and CL total of halogen-substituted SARMs series.

Determination of haloacetic acids in human urine by headspace gas chromatography-mass spectrometry.

Haloacetic acids (HAAs) are water disinfection byproducts (DBPs) formed by the reaction of chlorine oxidizing compounds with natural organic matter in water containing bromine. HAAs are second to trihalomethanes as the most commonly detected DBPs in surface drinking water and swimming pools. After oral exposure (drinking, showering, bathing and swimming), HAAs are rapidly absorbed from the gastrointestinal tract and excreted in urine. Typical methods used to determine these compounds in urine (mainly from rodents) only deal with one or two HAAs and their sensitivity is inadequate to determine HAA levels in human urine, even those manual sample preparation protocols which are complex, costly, and neither handy nor amenable to automation. In the present communication, we report on a sensitive and straightforward method to determine the nine HAAs in human urine using static headspace (HS) coupled with GC-MS. Important parameters controlling derivatisation and HS extraction were optimised to obtain the highest sensitivity: 120 microl of dimethylsulphate and 100 microl of tetrabutylammonium hydrogen sulphate (derivatisation regents) were selected, along with an excess of Na(2)SO(4) (6 g per 12 ml of urine), an oven temperature of 70 degrees C and an equilibration time of 20 min. The method developed renders an efficient tool for the precise and sensitive determination of the nine HAAs in human urine (RSDs ranging from 6 to 11%, whereas LODs ranged from 0.01 to 0.1 microg/l). The method was applied in the determination of HAAs in urine from swimmers in an indoor swimming pool, as well as in that of non-swimmers. HAAs were not detected in the urine samples from non-swimmers and those of volunteers before their swims; therefore, the concentrations found after exposure were directly related to the swimming activity. The amounts of MCAA, DCAA and TCAA excreted from all swimmers are related to the highest levels in the swimming pool water.

Optimization of a novel procedure for determination of VOCs in water and human urine samples based on SBSE coupled with TD-GC-HRMS.

In this study, stir-bar sorptive extraction and thermal desorption followed by gas chromatography coupled with high resolution mass spectrometry was applied for determination of halo-organic compounds (bromodichloromethane, dibromochloromethane, bromoform, and tetrachloroethylene) in water and human urine samples. Time of extraction and stirring speed were optimized. The results show that the optimum extraction time is 30 min with 600 rpm of stirring speed with Twister of 20 mm in length and 1.0-mm film thickness of PDMS (126 microL). The calibration curves, limits of detection and quantification for all compounds were calculated. This procedure is characterized by very low limits of detection and quantitation: lower than 0.0017 microg/L and good repeatability for all four volatile compounds. This new analytical procedure was identified to be easy, reliable, sensitive, and requires only small amounts of sample. It can constitute a good alternative to well-known procedures based on application of head space and gas chromatography coupled with electron capture detection.

Volatile organohalogen compounds in human urine: the effect of environmental exposure.

The paper presents the results of determination of volatile organohalogen compounds (VOX) in urine samples from subjects exposed to these compounds in their workplaces and through consumption of chlorinated tap water. The analytes were isolated and preconcentrated from the complex urine samples using the thin layer headspace (TLHS) technique with autogenous generation of the liquid sorbent. Final gas chromatographic determination was carried out by direct aqueous injection with electron capture detection (DAI-ECD). The results indicate that only a small fraction (<4%) of the VOX input is excreted with urine in the non-metabolized form. A positive correlation was found between the occupational levels of VOX in the workplace and their levels in urine. VOX levels in the urine of subjects not exposed to them in the workplace were significantly lower. Their presence in the organisms was most probably related to consumption of tap water produced by chlorination of surface waters.

Relationship between volatile organohalogen compounds in drinking water and human urine in Poland.

Selected volatile organohalogen compounds (VOX) were investigated in urine samples from people living in different areas of the Gdansk-Sopot-Gdynia TriCity (Poland). The analytes were isolated and preconcentrated using the so-called thin layer headspace technique with autogenous generation of the liquid sorbent. Final gas chromatographic determination was carried out by direct aqueous injection with electron capture detection. Analyte concentrations in drinking water ranged from not detected to approximately 8 microg/l (chloroform), depending on the source of drinking water in a given part of the TriCity (underground, surface or mixed). The corresponding urine levels were typically lower by about an order of magnitude. VOX levels in urine of people living in the parts of the TriCity supplied with drinking water containing elevated levels of the analytes were higher than the levels in urine of people whose drinking water originated from deep underground wells. The linear correlation coefficients for the relationships between total VOX and chloroform levels in drinking water and in urine were r2=0.65 and 0.88, respectively. The fraction of VOX excreted with urine in unchanged form did not exceed 20%.

Determination of volatile organohalogen compounds in human urine.

A variant of thin layer headspace (TLHS) analysis with autogenous generation of the liquid sorbent was applied for the determination of volatile organohalogen compounds in urine. The analytes were isolated from the matrix at an elevated temperature in a TLHS column. They were then transported by a stream of purified air to a second TLHS column kept at sub-ambient temperature. Water vapor contained in the air condensed in the second column together with the analytes. The effluent from the second column was analyzed by direct aqueous injection gas chromatography with electron capture detection (DAI-GC-ECD). It has been established that the complex urine matrix did not affect the results at a 95% confidence level. Volatile organohalogen compounds were detected in urine of employees of a chemical laboratory where halogenated solvents were used. The urine of people who were not exposed contained insignificant or undetectable levels of these compounds. Urine levels of target analytes were the higher, the longer a person was exposed to them. The results obtained confirm that the urinary system participates in excretion of volatile organohalogen compounds.

Exposure estimates to disinfection by-products of chlorinated drinking water.

Exposure to disinfection by-products (DBPs) of drinking water is multiroute and occurs in households serviced by municipal water treatment facilities that disinfect the water as a necessary step to halt the spread of waterborne infectious diseases. Biomarkers of the two most abundant groups of DBPs of chlorination, exhaled breath levels of trihalomethanes (THMs) and urinary levels of two haloacetic acids, were compared to exposure estimates calculated from in-home tap water concentrations and responses to a questionnaire related to water usage. Background THM breath concentrations were uniformly low. Strong relationships were identified between the THM breath concentrations collected after a shower and both the THM water concentration and the THM exposure from a shower, after adjusting for the postshower delay time in collecting the breath sample. Urinary haloacetic acid excretion rates were not correlated to water concentrations. Urinary trichloroacetic acid excretion rates were correlated with ingestion exposure, and that correlation was stronger in a subset of individuals who consumed beverages primarily within their home where the concentration measurements were made. No correlation was observed between an average 48-hr exposure estimate and the urinary dichloroacetic acid excretion rate, presumably because of its short biological half-life. Valid biomarkers were identified for DBP exposures, but the time between the exposure and sample collection should be considered to account for different metabolic rates among the DBPs. Further, using water concentration as an exposure estimate can introduce misclassification of exposure for DBPs whose primary route is ingestion due to the great variability in the amount of water ingested across a population.

[Trends of the biological monitoring measurements from 1991 to 1995].

Partial amendments to the Japanese Regulation on the Prevention of Lead Poisoning and that of Organic Solvent Poisoning were made in 1989. As a result, the measurement of blood lead and urinary delta-aminolevulinic acid (delta-ALA) became indispensable items of the occupational health examination for workers who handle lead. Also, the measurement of urinary metabolites of workers who handle eight kinds of organic solvents (xylene, N,N-dimethylformamide, styrene, tetrachloroethylene, 1,1,1-trichloroethane, trichloroethylene, toluene, and normal-hexane) became mandatory. The results of the biological monitoring mentioned above are classified into one of three categories, that is, distribution 1, 2 and 3, according to the concentration of the determinants. In this paper, the incidence of distribution 1, 2 and 3 of each determinant is reported and its change from 1991 to 1995 is discussed. The incidence of distribution 3 was 0.1-5.0% in each determinant. Although the ratio of distribution 1, 2 and 3 seems to have been almost the same for 5 years some determinants decreased their percentage of distribution 3. It is important to utilize the biological monitoring results for the improvement of working environments and working styles, and health management.

Effect of subchronic corn oil gavage on the acute toxicity of orally administered bromodichloromethane.

Bromodichloromethane (BDCM) is a by-product of water chlorination and is the second most common trihalomethane (THM) in finished drinking water. It has been reported that delivery of THMs in corn oil can influence the site and magnitude of toxic and carcinogenic responses in rodents, perhaps by inducing metabolizing enzymes or altering tissue composition. To determine if corn oil influences the acute toxicity of BDCM, adult male F-344 rats were pretreated 5 days/week for 6 weeks with oral doses of corn oil or water at a volume of 5 ml/kg. Following pretreatment, animals were gavaged with a single dose of 0, 200 or 400 mg BDCM/kg in 10% Emulphor. Urine was collected at timed intervals over a 48-h period following BDCM administration. Rats were sacrificed at this time and organs and blood removed. Urine and serum were analyzed for indicators of toxicity. Corn oil pretreatment did not enhance the acute hepato- or nephrotoxicity of BDCM, suggesting that vehicle effects noted in previous THM toxicity and carcinogenicity studies are more likely due to pharmacokinetic differences between administration in corn oil and aqueous gavage vehicles than to altered tissue composition or physiological changes.

Inhalation toxicity of an isomeric mixture of hydrochlorofluorocarbon (HCFC) 225 in male rats.

A blend of the hydrochlorofluorocarbon isomers HCFC-225ca and HCFC-225cb has been proposed as a potential substitute for CFC-113, an important solvent and cleaning agent. The toxicity following repeated inhalation of an HCFC-225 isomer mixture was assessed in male Crl:CDBR rats. Three groups of 10 male rats were exposed to the test compound in air at design concentrations of 500, 5000, and 13,000 ppm. Rats were exposed 6 hr/day, 5 days/week for 2 weeks. A control group of 10 male rats was exposed to air only. Decreased serum cholesterol, triglycerides, and glucose; dose-related increased mean absolute and relative liver weights; and microscopic hepatocellular hypertrophy were present at all exposure concentrations. Hepatocellular hypertrophy correlated ultrastructurally to proliferation of peroxisomes. Clinical chemical parameters and organ weight and morphologic changes in the liver were reversible following 14 days of recovery.

Measurement of organic halogen compounds in urine as an indicator of exposure.

The report describes the measurement of urinary organic halogen compounds. The method is an application of the adsorbable organic halogen assay which is widely used for the analysis of industrial waste water and drinking water. It was found that this assay can be applied to human urine if the urine is pretreated to hydrolyze the mucins so as to cleave the neuraminic acid residues responsible for the high viscosity of these slimy proteins. The method was found to be sensitive down to 1 microgram of organic halogen/100 ml of urine. Fifty to 260 micrograms of organic halogen was measured in the night urine of healthy, occupationally unexposed volunteers. Since many toxic chemicals to which man may be exposed environmentally or occupationally are, in fact, halogen compounds, this assay may be used to monitor for human exposure.

Metabolic activation of the nephrotoxic haloalkene 1,1,2-trichloro-3,3,3-trifluoro-1-propene by glutathione conjugation.

1,1,2-Trichloro-3,3,3-trifluoro-1-propene (TCTFP) is structurally closely related to the stable and non-toxic tetrachloroethylene. However, in TCTFP, the trifluoromethyl group enhances chemical reactivity with nucleophiles. This fact suggested that TCTFP may be metabolized intensively by glutathione (GSH) conjugation and therefore, like hexachlorobutadiene, would be expected to be nephrotoxic. We have investigated the nephrotoxicity and metabolism of TCTFP. Administration of 20 and 40 mg/kg to male rats resulted in a large, dose-dependent increase in urinary excretion of gamma-glutamyl transpeptidase (GGT) indicative of proximal tubular damage. No increase in plasma transaminase concentrations indicative of liver damage was found. In rats, N-acetyl-S-(1,2-dichloro-3,3,3-trifluoro-1-propenyl)-L-cysteine was a major urinary metabolite of TCTFP. TCTFP was transformed by microsomal and cytosolic GSH S-transferases from rat liver to S-(1,2-dichloro-3,3,3-trifluoro-1-propenyl)glutathione (DCTFPG) (identified by NMR and mass spectrometry). DCTFPG was toxic to rat renal cortex cells. Inhibition of GGT and cysteine conjugate beta-lyase blocked DCTFPG cytotoxicity. These results suggest the following TCTFP bioactivation: conjugation with GSH in the liver, catabolism of the GSH S-conjugate to the cysteine S-conjugate and cleavage of the cysteine S-conjugate by beta-lyase with formation of reactive intermediates in the kidney.

Identification of environmental carcinogens utilizing T-cell mediated immunity.

There now exists a very large body of information that directly links the exposure to certain environmental factors with the ultimate development of specific types of human cancer. For this reason, a number of tests possessing varying degrees of biological complexity have been devised with the intent to first identify, and then ultimately reduce the risk of exposure to tumor causing agents. The presently employed chronic whole animal tests generally accepted as measures of human carcinogenicity have certain limitations in that they are lengthy to perform, very expensive, and require complicated pathological examinations of the various tissues. Consequently, there exists a need for short-term whole animal bioassays that can serve to complement such lengthy chronic studies; we are proposing that one such a test can be developed through utilizing procedures that have been designed for evaluating T-cell immunological responses.

Determination of volatile halocarbons in raw and drinking water, human serum, and urine by electron capture GC.

A method for quantitative determination of volatile halocarbons in raw and drinking water, human serum, and urine is presented. Samples of water, serum, or urine were extracted in a single step with petroleum ether, and the extracts are, without further purification, analysed by high resolution gas chromatography (GC) with electron capture detection (ECD). Both external and internal calibrations were used to standardize the analytical system. External calibration is done with pure halocarbons in purified well water. Internal calibration is accomplished by spiking serum and urine samples with reference halocarbons between each of two successive extractions and GC runs. 1-iodobutane is used as internal standard, both in calibrations and actual determinations.

Gas chromatography mass spectrometry computer analysis of volatile halogenated hydrocarbons in man and his environment--A multimedia environmental study.

As part of a study to make a comparative analysis of selected halogenated compounds in man and the environmental media, a quantitative gas chromatography mass spectrometric analysis of the levels of the halogenated compounds found in the breath, blood and urine of an exposed population (Old Love Canal area, Niagara, New York) and their immediate environment (air and water) was undertaken. In addition, levels of halogenated hydrocarbons in air samples taken in the general Buffalo, Niagara Falls area were determined.

Rapid determination of light halogenated hydrocarbons in urine.

We describe a rapid method for determining light halogenated hydrocarbons (LHH) in urine by electron capture gas-liquid chromatography. The hydrocarbons are extracted from urine into pentane. A 2-microliter injection of the pentane extract provides a detection limit of less than 1 microgram/liter.