Quantitation of urinary o-xylene metabolites of rats and human beings by high performance liquid chromatography

Summary A high performance liquid chromatographic method for the determination of urinary o-xylene metabolites of rats, volunteers, and workers was described. In rat urine, the major metabolite, indicated by the glucuronic acid reaction, was separated with thin-layer liquid chromatography and identified as o-toluic acid glucuronide by high performance liquid chromatography. Another rather minor metabolite was demonstrated to be o-methyl hippuric acid. One of the major urinary metabolite in volunteers administered o-xylene orally was demonstrated to be o-methylhippuric acid and another minor metabolite was o-toluic acid glucuronide. In the urine of volunteers exposed to 138 ppm of o-xylene for 3 h in an artificial exposure chamber, o-methylhippuric acid was found to be the major metabolite and a minute amount of o-toluic acid glucuronide was found to be the minor metabolite. In the urine of shipbuilding workers, using a thinner containing toluene and xylenes (o-, m- and p-), hippuric acid and methylhippuric acids (o-, m- and p-) were recognized. Thus, urinary o-methylhippuric acid could be an index of o-xylene exposure of workers.     

The absorption,metabolism, and excretion of xylenes in man

Summary Fifteen exposure experiments were carried out, in which four persons each, were simultaneously exposed to a defined concentration of o-, m-, and p-xylene vapors and also to their mixture at a ratio of 1:1:1. The concentrations were in all cases around 0.2 mg/l (Czechoslovakian MAC) or around double the amount. The period of exposure amounted to exactly 8 h.It was ascertained that the pulmonary retention is practically identical in all persons and in all isomers and amounts to 63.6 ± 4.2%. Its extent does not depend upon inoculation level, nor upon the duration of the exposure. During the period of desaturation the lungs excrete approximately 5% of the total amount retained in the organism. Xylene excretion via urine is quite negligible and amounts to a thousandth of 1%.The chief metabolites are toluic acids which are excreted in conjugated form with glycine as so-called toluric (=methylhippuric) acids. Free toluic acids, toluylglucuronic acids, and hydroxytoluic acids do not occur in the urine of persons exposed to a reasonable concentration of xylene vapors.The excreted amounts of toluric acids reach a maximum in the portion of urine collected at the end of exposure; then they decrease rapidly, but trace amounts can be still observed even after 4–5 days. The excretion takes a similar course in different persons and at different intensity of exposure. During the last 2 h of exposure a mean of 23.6% is excreted and in 8 h of exposure 71.7% of the amount excreted within 24 h.The side metabolites of xylenes are compounds hydroxylated on the aromatic nucleus. After exposure to o-xylene the presence of 2.3- and 3.4-xylenol was observed in urine; after exposure to m-xylene the presence of 2.4 xylenol, and finally after exposure to p-xylene the presence of 2.5-xylenol. The excretion of xylenols reaches a maximum as a rule just after termination of exposure.It was proved by balance calculation that of the total amount of xylene retained in the organism during exposure, more than 95% is excreted in the form of toluric acid (o-97.1; m-99.2; p-95.1%) and only a small part in form of xylenol (o-0.86; m-1.98; p-0.05%).     

Mass spectrometric identification of hemoglobin modifications induced by nitrosobenzene

Aniline and nitrobenzene (NB) are widely used industrial chemicals. Early effects of aniline toxicity include methemoglobin formation and damage to erythrocytes (Jenkins, F.P., 1972. The no-effect dose of anilne in human subjects and a comparison of aniline toxicity in man and rat. Food Cosmet. Toxicol. 10, 671-679; Bus, J.S., Popp, J.A., 1987. Perspectives on the mechanism of action of the splenic toxicity of aniline and structurally-related. Food Chem. Toxicol. 25, 619-627). In this report, we describe an analytical method, based on LC techniques and mass spectrometry, which could help in monitoring the exposure to aniline and NB. In particular, we describe and characterize the formation of specific adducts during an in vitro reaction of nitrosobenzene (NOB), the main metabolite of aniline and NB, and human hemoglobin.     

Quantitative analysis of urinary glycine conjugates by high performance liquid chromatography: excretion of hippuric acid and methylhippuric acids in the urine of subjects exposed to vapours of toluene and xylenes

Summary A new method for the direct determination of hippuric acid (HA) and o-, m- and p-methylhippuric acids (MHAs) in the urine, metabolites of toluene and o-, m- and p-xylenes by high performance liquid chromatography (HPLC) is described. A stainless-steel column packed with silica gel having dinitrophenyl residue and a mixed solution of methanol/water/acetic acid (80/20/0.2) containing tetra-n-butylammonium bromide (0.2% w/v) as mobile phase was used. Concentrations of HA and MHAs were estimated from their peak height at a wave length of 225 nm. Urine can be analyzed directly without solvent extraction or pretreatment to obtain complete separation of HA and o-, m- and p-MHAs. Urine samples from male workers exposed to toluene or xylenes were analyzed for HA or MHAs. The urinary levels of HA and MHAs increased by exposure to toluene and xylenes in proportion to the environmental concentrations of the solvents, although there is a considerable variation in metabolite concentrations. The slope of regression line between toluene and HA and that between m-xylene and m-MHA were similar. The urinary concentrations of HA and MHAs corresponding to 100 ppm (TLV) of toluene was 2.35 g/g creatinine and that of m-MHA corresponding to 100 ppm (TLV) of m-xylene was 2.05 g/g creatinine. The warning levels of the urinary metabolite concentrations of a group of workers and that of an individual worker corresponding to TLV of organic solvent concentration is discussed.     

Effect of repeated exposure to aniline, nitrobenzene, and benzene on liver microsomal metabolism in the rat.

Exposure of rats to aniline at daily doses of 50 mg/kg of body weight over a month stimulated the microsomal metabolism as manifested by (1) acceleration of p-hydroxylation of anilin and N-demethylation of aminopyrine in 9-000 times g postmitochondrial supernatant of the liver, (2) shortening the sleeping time after hexobarbital, and (3) reduction of the antipyretic effect of phenacetin. In the rats exposed to nitrobenzene in a similar manner to aniline, nitroreduction of nitrobenzene and p-hydroxylation of aniline remained unaffected; the antipyretic effect of phenacetin was decreased, whereas hexobarbital sleeping time remained unchanged. Exposure of rats to benzene (50 mg/kg of body weight daily for a month) had no effect on the rate of hydroxylation of benzene and N-demethylation of aminopyrine. In benzene-exposed rats hexobarbital sleeping time was prolonged whereas the antipyretic effect of phenacetin was unaffected. Microsomal metabolism of aniline, nitrobenzene, and benzene was stimulated and inhibited when the rats were pretreated with phenobarbital and SKF 525-A, respectively.     

Quantitation of urinary metabolites of toluene,xylene, styrene,ethylbenzene, benzene and phenol by automated high performance liquid chromatography

Summary An automated high performance liquid chromatographic method (HPLC) for the direct determination of urinary concentrations of hippuric acid (HA), and o-, m- and p-methyl hippuric acids (MHAs), metabolites of toluene and o-, m-, and p-xylenes, and of urinary phenyl glyoxylic acid (PGA) and mandelic acid (MA), metabolites of styrene or ethylbenzene, is described. Methanol was added to urine, the mixture was centrifuged and the supernatant was injected into HPLC. A stainless-steel column packed with octadecyl silanized silicate was used and the mobile phase was a mixed solution of 5 mM potassium phosphate monobasic/acetonitrile (90/10). The method is simple and specific. Urine can be analyzed without solvent extraction. Analysis can be performed satisfactorily within 45 min for samples containing HA, MHAs, PGA and MA, and within 15 min for those containing HA, PGA and MA. Another automated HPLC method for the determination of urinary concentrations of phenylsulfate (PhS) and phenylglucuronide (PhG), metabolites of benzene and phenol, is also described. Urine was centrifuged and the supernatant was injected into HPLC. A column packed with octadecyl silicate and a mobile phase of 50 mM of potassium phosphate monobasic/acetonitrile (85/15) were used. The whole analyses and quantitative determination can be performed within 15 min for samples containing PhS and PhG in the worker's urine with a simple mobile phase. The accuracy and precision in the present methods by the use of automated HPLC were satisfactory.     

Biological monitoring of low level occupational xylene exposure and the role of recent exposure

The correlation between low level time-weighted average (TWA) atmospheric xylene exposure (p.p.m.) and urinary methylhippuric acid (MHA) expressed per gram of creatinine was examined. Subjects were recruited from workplaces that utilized xylene. Ambient monitoring of o-, m- and p-xylene isomers was carried out using passive diffusion vapour monitors. Adjusted (post-shift minus pre-shift) and post-shift urinary levels of xylene metabolites (2-, 3- and 4-MHA) were determined by GC-MS. Twenty subjects were recruited into the study. Total xylene TWA exposures were 3.36 +/- 3.63 p.p.m. (mean +/- SD) with a range of 0.03-14.44 p.p.m. The r(2) values for the regression equations between xylene exposure and individual and total adjusted MHA isomers were 0.390, 0.709, 0.677 and 0.631 for o-, m-, p- and total xylenes, respectively, which was greater than the respective correlations between non-adjusted samples. In conclusion, biological monitoring of occupational xylene exposure at levels <15 p.p.m. using urinary MHA showed a good correlation with atmospheric levels and is a valid complement to ambient monitoring. Even though occupational xylene exposure in the workplaces studied was generally low, MHA was found in the pre-shift urine of all workers and the use of adjusted values showed modest improvements in correlations. Recent exposure prior to sampling, either from occupational or non-occupational sources, should be considered when biological monitoring of xylene is undertaken. Extrapolation of data from this study predicted a MHA concentration in post-shift urine of 1.3 g/g creatinine after exposure to a TWA of 100 p.p.m. xylene.     

In vitro and in vivo estrogenicity and toxicity of o-, m-, and p-dichlorobenzene

The estrogenicity of o-, m-, and p-dichlorobenzene (DCB) was evaluated with a yeast estrogen screen (YES) and zebrafish (Danio rerio) vitellogenin (VTG) assays. With the YES, p-DCB and m-DCB were found to be estrogenic in a concentration-responsive manner. The relative potency measured with the YES (relative to 17beta-estradiol) was 2.2 x 10(-7) for p-DCB and 1.04 x 10(-8) for m-DCB. Following acute toxicity tests with the zebrafish, plasma VTG production was measured to examine the in vivo estrogenic activity of the three compounds after a 14-d exposure. Adult zebrafish were exposed to different concentrations of o-, m- and p-DCB, ranging from 0.1 to 32 mg/L; ethynylestradiol ([EE2]; 5 ng/L, 10 ng/L, 50 ng/L, and 100 ng/L) was used as a positive control. After exposure, blood samples were taken and protein electrophoresis was performed to determine the relative VTG content. Gonadosomatic indices (GSI) and condition factors (CF) were also calculated. Elevated VTG levels and decreased female GSIs were found in fish exposed to > or = 5 ng EE2/L and in fish exposed to > or = 10 mg p-DCB/L. Low GSIs coincided with high levels of VTG in the blood of female zebrafish. This relation was not only found in fish exposed to EE2 but also in controls and fish exposed to DCB. Therefore, a direct or indirect effect of VTG on the GSI is suggested rather than a direct toxic effect of the tested compounds on the gonads.     

A field survey on the health status of workers in dye-producing factories

Summary Health status of the workers in dye-producing plants were examined repeatedly in winter and in summer. The items studied cover 1) diazo-positive metabolites in urine 2) methemoglobinaemia, Heinz bodies, methemoglobin reductases and anaemia, and 3) liver function. The urinary diazo-positive metabolite level in the exposed was higher than that in the non-exposed and marked a conspicuous increase in summer as compared in winter, indicating significant intake of the aromatic nitro-amino compounds, while no significant methemoglobinaemia, Heinz bodies, anaemia nor disturbed liver function was observed in any group of the examinees. The results were discussed in connection with the dose-effect relationship, and compared with the experiences in the past.Copies should be requested to: Prof. Masayuki Ikeda, Department of Environmental Health, Tohoku University School of Medicine, Sendai 980, JapanThis work was supported by research grants from the Ministry of Labour, the Government of Japan     

The simultaneous determination of hippuric acid, o-, m-, p-methylhippuric acids, mandelic acid and phenylglyoxylic acid in urine by HPLC

A high-performance liquid chromatographic method is described for the simultaneous determination of six metabolites of aromatic hydrocarbons: hippuric acid (HA) from toluene; o-, m-, p-methylhippuric acids (o-, m-, p-MHA) from xylene; mandelic acid (MA) and phenylglyoxylic acid (PGA) from styrene and ethylbenzene. Metabolites were first extracted from urine by solid phase extraction with anion exchange resin, then isocratically separated on a C8 column with 3 microns particle size, 10 cm length and 3 mm internal diameter. Mobile phase was prepared diluting 16 mL of tetrahydrofuran, 14 mL of acetronitrile and 5 mL of methanol to 500 mL with phosphoric acid/potassium dihydrogen phosphate buffer 0.01 M (pH 2.7). The internal standard was 3-hydroxybenzoic acid. Chromatographic runs were completed in about 21 min. The accuracy and reproducibility obtained make this method useful for the biological monitoring of occupational exposure to toluene, xylene, styrene and ethylbenzene.     

Measurement by gas chromatography of urinary hippuric acid and methylhippuric acid as indices of toluene and xylene exposure.

A gas chromatographic method was applied to the determination of the urinary glycine conjugates, hippuric, o-, m- and p-methylhippuric acids. These were extracted with ethyl acetate from urine after acidification with hydrochloric acid. The internal standard solution (heptadecanoic acid methanol solution) was added before extraction and a diazomethane-ether-ethanol solution was subsequently added to the dried extracts. The methylated residues were dissolved in methanol and injected into a gas chromatograph as described by Buchet and Lauwerys (1973). By the combined use of gas chromatography and mass spectrometry the methyl esters of hippuric acid and m-methylhippuric acid were identified in the urine of a volunteer who had been exposed to toluene and m-xylene vapours. When the urine specimen contained salicyluric acid (a urinary metabolite of salicylic acid) two sharp peaks were observed. The faster peak coincided with m- or p-methylhippuric acid. The upper limit of urinary hippuric acid concentration in healthy subjects with no occupational exposure was calculated by this method to be 1.026 microgram/ml (fiducial limit 5%) after correction to 1.024 for variation in urinary density.     

Quantitative determination of urinary hippuric acid and m- or p-Methylhippuric Acid as Indices of toluene and m- or p-Xylene exposure by high performance liquid chromatography

Summary A high performance liquid chromatographic method for the determination of hippuric and m-(p-)methylhippuric acids in urine, metabolites of toluene and m-(p-)xylene, is described. A stainless-steel column packed with octadecyl silanized silica gel was used and the mobile phase was a mixed solution of methanol/water/acetic acid (20/80/0.2). The method is simple and specific. Urine can be analyzed directly, without solvent extraction or pretreatment. The method has a lower detection limit of 0.2 g on column. All the analysis and quantitative determination can be performed within about 30 min. for samples containing m-(p-)methylhippuric acid.     

Urine analysis for the evaluation of environmental exposures to aromatic hydrocarbons

The authors have developed a dynamic headspace (purge-and-trap) gas chromatographic method, with photoionization detection, for the determination of benzene (C6H6), toluene (C7H8), ethylbenzene (C8H10), and isomeric (o-, m-, p-) xylenes (C8H10) (BTEX) in urine. Detection limits ranged between 15 and 35 ng/l, relative standard deviations between 0.2 and 10%, and accuracy between 80 and 100%. The primary objective of this study was to use this new method to establish baseline concentration data for BTEX in the urine of the general population of Zagreb, Croatia. A second objective was to evaluate the effect of cigarette smoking on those baseline values. BTEX were analyzed in the urine of 72 subjects (36 nonsmokers and 36 smokers) without occupational exposure to BTEX. The nonsmokers had measurable BTEX in their urine, except for ethylbenzene in 13 and o-xylene in 15 of the samples. Values for BTEX were markedly higher among smokers than nonsmokers. Because the sources of BTEX exposure are commonly derived (i.e., vehicle exhausts and smoking), their values in subjects' urine were significantly intercorrelated. Levels of toluene and o-xylene were correlated significantly with the number of cigarettes smoked per day. The use of purge-and-trap gas chromatography with photoionization detection to determine BTEX in urine offers a convenient approach for biological monitoring of the general population. Study data provide referent values for BTEX in urine, which can be used as biomarkers for environmental exposures. Smoking contributes significantly to the urinary concentration of BTEX.     

Simultaneous determination of toluene and xylene metabolites in urine by gas chromatography.

A gas chromatographic method for simultaneous determination of hippuric and o-, m-, and p-methylhippuric acids (metabolites of toluene and xylene) in urine is described. The analytical procedure is based on the extraction of the aromatic metabolites with ethyl acetate containing the internal standard and on a methylation with 3-methyl-1-p-tolyltriazene. With this method, which does not require much time and handling, the different acids can be satisfactorily determined with high sensitivity and specificity. A statistical study shows a good reproducibility for the determination of hippuric and o-, m-, and p-methylhippuric acids. The coefficient of variation for 10 determinations in all cases was less than 5%.     

In vitro evaluation on contact sensitivity from DNCB and DNBS using lymphocyte blastogenesis response test.

To assess the contact allergic potential of sensitizers, an in vitro predictive model was developed. First of all, guinea pigs were sensitized by 1-chloro-2-4,dinitrobenzene (DNCB), and lymph node cells from sensitized and control guinea pigs were cultured in the presence of DNCB, 2,4-dinitrobenzene sulfonic acid sodium salt (DNBS), DNCB plus epidermal cell (DNCB + EC), DNBS + EC and epidermal cell modified hapten (dinitrophenyl-EC, DNP-EC) at 3 x 10(5), 5 x 10(5) and 8 x 10(5) cells/well, respectively. Lymphocyte blastogenesis responses were assessed by uptake of 5H-thymidine. The results indicated that lymphocytes from sensitized guinea pigs responded to the foregoing antigens in vitro to a greater degree than those from control guinea pigs and the blastogenesis response by DNBS was the highest among the challenges of DNBS, DNCB, DNCB + EC, DNBS + EC and DNP-EC. When the number of lymphocytes was sufficient, the blastogenesis response of lymphocytes could be in vitro challenged successfully not only by DNBS, DNCB + EC, DNBS + EC and DNP-EC, but also DNCB alone. There was a cross sensitivity between DNCB and DNBS for in vivo and in vitro challenges. Moreover, significant relationships were observed between lymphocyte blastogenesis response and doses of DNBS used in vitro assay at 5 x 10(5) and 8 x 10(5) cells/well. The results indicate that this assay is an useful in vitro predictive model of contact sensitivity and cross sensitivity of sensitizers.     

New bis-N9-(methylphenylmethyl)purine derivatives: synthesis and antitumor activity

A series of ortho-, meta- and para-bis-N9-(methylphenylmethyl)purine derivatives 4-15 were obtained by two-step synthesis from various substituted chloropurines with alpha,alpha'-dichloroxylenes. These bis-N9-(methylphenylmethyl)purines 4-15 were evaluated for the primary cytotoxic activity against a panel of NCI-H460 (lung), MCF-7 (breast) and SF-268 (CNS) cancer cell lines. The 'active' compounds which reduced growth of cancer cells to ca. 32% or less, have been evaluated in a full panel of 60 human cancer cell lines over a 5-log dose range at the National Cancer Institute, Bethesda, MD. In this series, the most activity is correlated to the compounds derived from the 2,6-dichloropurines such as bis-9-[o-(methylphenylmethyl)]2,6-dichloropurine (5), bis-9-[m-(methylphenylmethyl)]2,6-dichloropurine (8), and bis-9-[p-(methylphenylmethyl)]2,6-dichloropurine (11). In particular compound 8 exhibited high sensitivity in leukemia cell lines and compounds 5, 8 and 11 exhibited consistent high sensitivity in many breast cancer cell lines. Compound 11 was the most potent in this series and exhibited GI(50)<0.01 microM sensitivity against non-small lung cancer EKVX, colon cancer HT-29, melanoma SK-MEL-28, renal cancer RXF 393, prostate cancer DU-145 and several breast cancer HS 578T and BT-549 cell lines.     

Urinary disposition of ethylbenzene and m-xylene in man following separate and combined exposure

Summary Four volunteer subjects were exposed to 150ppm (655 mg/m³) of ethylbenzene and 150ppm (655mg/m³) of m-xylene both separately and in combination. The biotransformation of the solvents was studied on the basis of the metabolites found in the urine. The metabolic conversion of both m-xylene and ethylbenzene proceeded mainly through oxidation of side chains. Ring oxidation seemed to be of minor importance; in the case of ethylbenzene it accounted for 4.0% (combined share of 4-ethylphenol, p- and m-hydroxyacetophenones) and in case of m-xylene for 2.5% (2,4 dimethylphenol), respectively. Mandelic and phenylglyoxylic acids amounted to 90% of the ethylbenzene metabolites, whereas m-xylene were excreted to 97% in the form of m-methylhippuric acid. Almost equimolar amounts in the form of metabolites of both solvents were found in the urine during 24h from the onset of exposure. Most of the ethylbenzene metabolites were excreted at substantially slower rates than those of m-xylene. The combined exposure resulted in a mutual inhibition of the metabolism of ethylbenzene and m-xylene, which was demonstrated by delayed excretion and decreased amounts of metabolites excreted. No sign of alteration in the urinary metabolite patterns of either ethylbenzene or m-xylene could be detected.     

Biological monitoring in workers in a nitrobenzene reduction plant: haemoglobin versus serum albumin adducts

The high priority of monitoring workers exposed to nitrobenzene is a consequence of clear findings of experimental carcinogenicity of nitrobenzene and the associated evaluations by the International Agency for Research on Cancer. Eighty male employees of a nitrobenzene reduction plant, with potential skin contact with nitrobenzene and aniline, participated in a current medical surveillance programme. Blood samples were routinely taken and analysed for aniline, 4-aminodiphenyl (4-ADP) and benzidine adducts of haemoglobin (Hb) and human serum albumin (HSA). Also, levels of methaemoglobin (Met-Hb) and of carbon monoxide haemoglobin (CO-Hb) were monitored. Effects of smoking were straightforward. Using the rank sum test of Wilcoxon, we found that very clear-cut and statistically significant smoking effects (about 3-fold increases) were apparent on CO-Hb (P=0.00085) and on the Hb adduct of 4-ADP (P=0.0006). The mean aniline-Hb adduct level in smokers was 1.5 times higher than in non-smokers; the significance (P=0.05375) was close to the 5% level. The strongest correlation was evident between the Hb and HSA adducts of aniline (r_s=0.846). Less pronounced correlations (but with P values < 0.02) appeared between aniline-Hb and 4-ADP-Hb adducts (r_s=0.388), between 4-ADP and 4-ADP-HSA adducts (r_s=0.373), and between 4-ADP-Hb and aniline-HSA adducts (r_s=0.275). In view of the proposal for additional use of the aniline-HSA adduct for biological monitoring, particularly in cases of acute overexposures or poisonings, the strong correlation of the Hb and HSA conjugates is noteworthy; the ratio aniline-HSA:aniline-Hb was 1:42 for the entire cohort. Received: 2 June 2000 / Accepted: 21 April 2001     

Experimental exposure to toluene: further consideration of cresol formation in man.

In two separate experiments 10 healthy men each were exposed at rest in an exposure chamber to about 200 ppm toluene in the air. Hippuric acid, o-, m-, p-cresol, and phenol in urine were detected by capillary gas chromatography at the beginning and at the end of exposure, and at variable times after the cessation of exposure. In addition toluene in blood was determined at the same intervals. The results indicate that in addition to hippuric acid, o-, m-, p-cresol are metabolites of toluene; the detoxication lasting 24 hours at least.     

Identification of 6-hydroxy-trans,trans-2,4-hexadienoic acid, a novel ring-opened urinary metabolite of benzene.

We studied the in vivo metabolism of benzene in mice to ring-opened compounds excreted in urine. Male CD-1 mice were treated intraperitoneally with benzene (110-440 mg/kg), [14C]benzene (220 mg/kg) or trans, trans-muconaldehyde (MUC; 4 mg/kg), a microsomal, hematotoxic metabolite of benzene. Urine, collected over 24 hr, was extracted and analyzed by HPLC with a diode-array detector and by scintillation counting. In addition to trans,trans-muconic acid, previously the only known ring-opened urinary benzene metabolite, a new metabolite, 6-hydroxy-trans,trans-2,4-hexadienoic acid, was detected in urine of mice treated with either benzene or MUC. We identified the new metabolite based on coelution of metabolites and UV spectral comparison with authentic standards in unmethylated and methylated urine extracts. Results presented here are consistent with the intermediacy of MUC in the in vivo metabolism of benzene to ring-opened metabolites.