Sulphur Derivative of Hexachlorobenzene J.
To-Figueras,
J.
in Human Urine
Gómez-Catalán, M. Rodamilans & J. Corbella
Toxicology Unit, Facultat de Medicina. Hospital Clinic Villarroel 170, 08036 Barcelona, Spain
i Provincial. Universitat de
Barcelona,
Pentachlorothiophenol, a sulphur derivative of the widespread environmental pollutant hexachlorobenzene (HCB) has been detected and quantified in the urine of a human general population with high body burden of HCB. The sulphur derivative was analysed by GLC-MS as pentachlorothioanisole (PCTA) after hydrolysis and methylation of the respective conjugate and was found in 100% of the samples ( n 40) with a mean =
concentration of 1.85 ± 0.98 ng ml -1(X ± s.d., range 0.58-4.50 ng ml -1 ). No correlation with urinary pentachlorophenol (PCP) and no sex-related differences were found. The derivative may originate from the biotransformation of HCB stored in tissues and may be a useful maker of HCB metabolism in humans. Introduction The presence of diverse chlorophenols in trace amounts in the urine of the general population of industrialized areas has been reported. °2 The
residues generally found are pentachlorophenol (PCP) and diverse tri-and tetra chlorophenols.3 The significance of these compounds in human urine is not completely elucidated. PCP is a widely used biocide’ and residues of that compound are commonly found in fruits and vegetables that are part of the diet of most human populations. Thus, the presence of PCP in human blood, tissues or urine might partly originate from residues found in food. Also, the technical formulations of PCP usually contain impurities of tetrachlorophenol, so the presence of the later in human urine may indicate a common origin with PCP’ . But PCP in mammals is also a major biotransformation product of hexachlorobenzene (HCB), which is a widespread environmental pollutant. HCB originates from agricultural and industrial sources and, due to its resistance to biodegradation and elevated lipophylicity, tends to accumulate in animal and human tissues. Residues of HCB have been found accumulated in the adipose tissue and breast milk of many industrial populations with no known particular exposure to it.5 The evidence of HCB being metabolized in rodents to phenol derivatives like PCP or tetrachlorohydroquinone6°’ raises the question as to what extend PCP found in human urine originates from the metabolism of HCB accumulated in tissues. Koss et al.’ reported a correlation between HCB concentration in adipose tissue and
PCP in 24-h urine, but other studies made in our laboratory showed a lack of correlation between HCB and PCP concentrations in serum or between PCP in urine and HCB in serum.’ Therefore, exogenous PCP is likely to be a confounding factor, if urinary PCP is to be used as a marker of stored HCB metabolism. In this study, the detection and quantification of another HCB metabolite in human urine is reported. Although it had never been detected in humans previously, the sulphur derivative Nacetyl-S-(pentachlorophenyl) cysteine is the main urinary metabolite of HCB in rodents as it is of the soil fungicide pentachloronitrobenzene (PCNB). This metabolite originates from the conjugation of HCB or PCNB with glutathione (Figure 1) and can be analysed as pentachlorothioanisole (PCTA) by GLC after hydrolysis of the conjugate and methylation of the resulting pentachlorothiophenol (PCThP). In search of a reliable marker of HCB metabolism in humans, a general population with high body burden of HCB - assessed by previous studies was chosen, and the urinary levels of this sulphur derivative were estimated.
Material and methods Urine
was collected from healthy volunteers (n 40) between 8 and 10 a.m. and processed immediately. None of the volunteers had occupational exposure to HCB, PCNB, PCP or any other organochlorine pesticide, but they all lived =
Downloaded from het.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 26, 2015
272
diazoethane instead of diazomethane during derivatization in order to analyse the respective ethyl derivatives. Confirmation of PCTA
chromatograph peak by mass spectrometry was achieved by a Hewlett Packard 5971A mass selective detector.
Results
Figure 1 Biotransformation of HCB in humans and urine major metabolites. Abrevations: GA (glucuronic acid); GSH (reduced glutathione); NAC (N-acetyl cysteine); PCP (pentachlorophenol); PCThP (pentachlorothiophenol) ; PCTA (pentachlorothioanisole). in the city of Barcelona (Spain) where HCB body burden in the overall population is considerable (mean concentration in adipose tissue 2.42 ± 1.34 ppm ; range 0. 15-5.68).9 Age, sex, tobacco smoking and dietary habits were recorded. The sulphur derivatives along with PCP
analysed by GLC-MS following an extraction procedure initially described by Koss et al.’ Urine (4 ml), 2M NaOH (2ml), and ascorbic acid (10 mg) were mixed and heated at 70 ° C under N2 for 3 h to hydrolyse the conjugates. After cooling, concentrated HCI was added until pH 1 was reached. Then the metabolites were extracted twice with toluene (5 ml). The solvent extracts were concentrated to 0.5 ml, and 0.25 ml of diazomethane in diethyl ether were added and left for 30 min in the dark. After derivatization, excess of diazomethane was withdrawn under nitrogen stream, and the clean-up procedure of Veierov and Aharonson&dquo; with H2SO4 was applied. Previously, it had been assayed and proved that neither PCTA nor methylated PCP were affected by H2SOI treatment. The organic phase was separated and concentrated to aprox 50 ~.1, and an internal standard (aldrin) was added. The analyses were performed in a Hewlett Packard 5890 gas chromatograph with splitless injection, 63Ni electron capture detector and a fused silica capillary column (50 m x 0.2 mm i.d.) with 100% dimethylpolysiloxane fluid (HP-101 ) as stationary phase. An alternative chromatograph separation with a 5% diphenyl-95% dimethylpolysiloxane HP-ultra 2, 25 m x 0.2 mm i.d.) capillary column was carried out using were
=
The mass spectrum of PCTA isolated from human urine after hydrolysis and methylation of the respective conjugate is shown in Figure 2. The sulphur derivative appeared in 100% of the samples with a mean concentration of 1.85 ± 0.98 ng ml -‘ (X ± s.d.) and range of 0.58 - 4.50 ng ml -’ (Figure 3). If derivatization wtih diazomethane was omitted PCTA was not detected. PCP was also present in all the samples with a mean concentration of 18.0 ± 9.1 ng ml -’I (X±s.d.) and a range of 6.3-39.5 ng ml-’, but no correlation was found between PCTA and PCP levels. Sex - or age-related differences did not appear and no relation was found between PCTA or PCP concentration and dietary or tobacco smoking habits. Other metabolites of HCB or PCNB, like tetrachlorohydroquinone (TCHQ) or
pentachloronaniline (PCA), normally found
in several animal species, above the 0.1 ng ml -’ level, with H,S04 was omitted.
Discussion
were even
not detected if the clean-up
’
The significance of this sulphur derivative in human urine is uncertain. Although the toxicokinetics and the metabolism of HCB in rodents is well known, it is not so in humans. In the population under study, HCB levels in adipose tissue and breast milk are well
Figure 2 Mass spectra of PCTA isolated from human urine.
Downloaded from het.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 26, 2015
273
diet. The urinary sulphur conjugate, which after hydrolysis and methylation gives PCTA, is probably N-acetyl-S-(pentachlorophenyl) cysteine, as
3 Frequency distribution of pentachlorothioanisole (PCTA) levels in human urine. PCTA 1.85 ± 0.98 ng ml -’ (X ± s.d.) [0.58-4.50].
Figure
documented9
in serum, liver, kidbut the effectiveness of hepatic biotransformation or renal elimination of the metabolites in relation to total body burden remain obscure. In that sense, PCP urinary levels do not seem to be a reliable marker taking into account that there is an important intake of exogenous PCP through the as
ney, and other
they
are
tissues, 12,13
References
& Corbella J. Pentachlorophenol and hexachlorobenzene in serum and urine of the population of Barcelona. Human Toxicology 1987; 6: 397-400. 2 Bevenue A, Wilson J, Casarett JJ & Klemmer HW. A survey of pentachlorophenol content in human urine. Bulletin of Environmental Contamination and Toxicology 2: 319-33. 1967; 3 Lores EM, Edgerton TR & Moseman RF. Method for the confirmation of chlorophenols in human urine by LC with electrochemical detector. Journal of Chromatography Science 1981; 19: 466-9. 4 Williams PL. Pentachlorophenol, an assessment of the occupational hazard. American Industrial Hygiene Association Journal 1982; 43: 799-810. 5 Bertram HP, Kemper FH & Muller C. Hexachlorobenzene content in human blood and adipose tissue: experiences in environmental specimen banking. In: Hexachlorobenzene: Proceedings of an International Symposium, ed. Morris & Cabral, pp. 173-82. Lyon: IARC, Scientific publication n°
7
77, 1986. Engst R, Macholz
’
Acknowledgements Supported by
a
..
FISS grant (91/0170).
’
1 Gómez-Catalán J, To-Figueras J, Planas J, Rodamilans M
6
it is in rodents. This compound is a metabolite of HCB in several species but also may originate from the endogenous biotransformation of the fungicide pentachloronitrobenzene (PCNB). But since other major metabolites of PCNB-(notably pentachloroaniline, PCA) were not detectded above the 0.1 ng ml -’ level in the urines under study, the results suggest that this sulphur derivative may be mostly a biotransformation product of HCB recently ingested through the diet and/or accumulated in tissues. The relationship between urinary PCTA levels and HCB concentration in serum, liver or adipose tissue, and the possible usefulness of PCTA as indicator of HCB metabolism in humans is under investigation.
RM & Kujawa M. The metabolism of hexachlorobenzene (HCB) in rats. Bulletin ofEnvironmental Contamination and Toxicology 1976; 16: 248-52. Edgerton TR, Moseman RF, Linder RE & Wright LH. Multi-residue method for the determination of chlorinated phenol metabolites in urine. Journal of Chromatography 1979; 179: 331-42.
8 Koss G, Reuter
A & Koransky W. Excretion of metabolites of hexachlorobenzene in the rat and the man. In: Hexachlorobenzene: Proceedings of an International Symposium, ed. Morris & Cabral, pp. 261-6. Lyon: IARC, Scientific publication n° 77, 1986. 9 Planas J, Gómez-catalán J, To-Figueras J, Sabroso M, Camps M & Corbella J. Residuos de hexaclorobenceno en el tejido adiposo de la población de Catalunya (1986-87). In: Hexaclorobenceno (las Jornadas Nacionales), pp. 14959., Barcelona: PPU, 1990. 10 Koss G, Koransky W & Steinbach K. Studies on the toxicology of hexachlorobenzene. IV. Sulphur containing metabolites. Archives of Toxicology 1979; 42: 19-31. 11 Veierov D & Aharonson N. Improved clean-up of large lipid samples for electron capture gas chromatographic quantitation and gas chromatographic-mass spectrometric confirmation of organochlorine residues. Journal of the Association of Official Analytical Chemists 1980; 63: 202-7. 12 To-Figueras J, Gómez-Catalán J, Rodamilans J et al. Evaluation of hexachlorobenzene risk in a human population. Spain (1981-1988). The Toxicologist (Abstracts of the 28th Annual Meeting). 1989; 9: 226. 13 To-figueras J, Rodamilans M, Gómez-Catalán J & Corbella J. Hexachlorobenzene residues in the general population of Barcelona (Spain). In Hexachlorobenzene: Proceedings of an International Symposium, ed. Morris & Cabral, pp 147-48. Lyon: IRAC. scientific publication n° 77, 1986.
Downloaded from het.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 26, 2015
To-Figueras,
J.
in Human Urine
Gómez-Catalán, M. Rodamilans & J. Corbella
Toxicology Unit, Facultat de Medicina. Hospital Clinic Villarroel 170, 08036 Barcelona, Spain
i Provincial. Universitat de
Barcelona,
Pentachlorothiophenol, a sulphur derivative of the widespread environmental pollutant hexachlorobenzene (HCB) has been detected and quantified in the urine of a human general population with high body burden of HCB. The sulphur derivative was analysed by GLC-MS as pentachlorothioanisole (PCTA) after hydrolysis and methylation of the respective conjugate and was found in 100% of the samples ( n 40) with a mean =
concentration of 1.85 ± 0.98 ng ml -1(X ± s.d., range 0.58-4.50 ng ml -1 ). No correlation with urinary pentachlorophenol (PCP) and no sex-related differences were found. The derivative may originate from the biotransformation of HCB stored in tissues and may be a useful maker of HCB metabolism in humans. Introduction The presence of diverse chlorophenols in trace amounts in the urine of the general population of industrialized areas has been reported. °2 The
residues generally found are pentachlorophenol (PCP) and diverse tri-and tetra chlorophenols.3 The significance of these compounds in human urine is not completely elucidated. PCP is a widely used biocide’ and residues of that compound are commonly found in fruits and vegetables that are part of the diet of most human populations. Thus, the presence of PCP in human blood, tissues or urine might partly originate from residues found in food. Also, the technical formulations of PCP usually contain impurities of tetrachlorophenol, so the presence of the later in human urine may indicate a common origin with PCP’ . But PCP in mammals is also a major biotransformation product of hexachlorobenzene (HCB), which is a widespread environmental pollutant. HCB originates from agricultural and industrial sources and, due to its resistance to biodegradation and elevated lipophylicity, tends to accumulate in animal and human tissues. Residues of HCB have been found accumulated in the adipose tissue and breast milk of many industrial populations with no known particular exposure to it.5 The evidence of HCB being metabolized in rodents to phenol derivatives like PCP or tetrachlorohydroquinone6°’ raises the question as to what extend PCP found in human urine originates from the metabolism of HCB accumulated in tissues. Koss et al.’ reported a correlation between HCB concentration in adipose tissue and
PCP in 24-h urine, but other studies made in our laboratory showed a lack of correlation between HCB and PCP concentrations in serum or between PCP in urine and HCB in serum.’ Therefore, exogenous PCP is likely to be a confounding factor, if urinary PCP is to be used as a marker of stored HCB metabolism. In this study, the detection and quantification of another HCB metabolite in human urine is reported. Although it had never been detected in humans previously, the sulphur derivative Nacetyl-S-(pentachlorophenyl) cysteine is the main urinary metabolite of HCB in rodents as it is of the soil fungicide pentachloronitrobenzene (PCNB). This metabolite originates from the conjugation of HCB or PCNB with glutathione (Figure 1) and can be analysed as pentachlorothioanisole (PCTA) by GLC after hydrolysis of the conjugate and methylation of the resulting pentachlorothiophenol (PCThP). In search of a reliable marker of HCB metabolism in humans, a general population with high body burden of HCB - assessed by previous studies was chosen, and the urinary levels of this sulphur derivative were estimated.
Material and methods Urine
was collected from healthy volunteers (n 40) between 8 and 10 a.m. and processed immediately. None of the volunteers had occupational exposure to HCB, PCNB, PCP or any other organochlorine pesticide, but they all lived =
Downloaded from het.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 26, 2015
272
diazoethane instead of diazomethane during derivatization in order to analyse the respective ethyl derivatives. Confirmation of PCTA
chromatograph peak by mass spectrometry was achieved by a Hewlett Packard 5971A mass selective detector.
Results
Figure 1 Biotransformation of HCB in humans and urine major metabolites. Abrevations: GA (glucuronic acid); GSH (reduced glutathione); NAC (N-acetyl cysteine); PCP (pentachlorophenol); PCThP (pentachlorothiophenol) ; PCTA (pentachlorothioanisole). in the city of Barcelona (Spain) where HCB body burden in the overall population is considerable (mean concentration in adipose tissue 2.42 ± 1.34 ppm ; range 0. 15-5.68).9 Age, sex, tobacco smoking and dietary habits were recorded. The sulphur derivatives along with PCP
analysed by GLC-MS following an extraction procedure initially described by Koss et al.’ Urine (4 ml), 2M NaOH (2ml), and ascorbic acid (10 mg) were mixed and heated at 70 ° C under N2 for 3 h to hydrolyse the conjugates. After cooling, concentrated HCI was added until pH 1 was reached. Then the metabolites were extracted twice with toluene (5 ml). The solvent extracts were concentrated to 0.5 ml, and 0.25 ml of diazomethane in diethyl ether were added and left for 30 min in the dark. After derivatization, excess of diazomethane was withdrawn under nitrogen stream, and the clean-up procedure of Veierov and Aharonson&dquo; with H2SO4 was applied. Previously, it had been assayed and proved that neither PCTA nor methylated PCP were affected by H2SOI treatment. The organic phase was separated and concentrated to aprox 50 ~.1, and an internal standard (aldrin) was added. The analyses were performed in a Hewlett Packard 5890 gas chromatograph with splitless injection, 63Ni electron capture detector and a fused silica capillary column (50 m x 0.2 mm i.d.) with 100% dimethylpolysiloxane fluid (HP-101 ) as stationary phase. An alternative chromatograph separation with a 5% diphenyl-95% dimethylpolysiloxane HP-ultra 2, 25 m x 0.2 mm i.d.) capillary column was carried out using were
=
The mass spectrum of PCTA isolated from human urine after hydrolysis and methylation of the respective conjugate is shown in Figure 2. The sulphur derivative appeared in 100% of the samples with a mean concentration of 1.85 ± 0.98 ng ml -‘ (X ± s.d.) and range of 0.58 - 4.50 ng ml -’ (Figure 3). If derivatization wtih diazomethane was omitted PCTA was not detected. PCP was also present in all the samples with a mean concentration of 18.0 ± 9.1 ng ml -’I (X±s.d.) and a range of 6.3-39.5 ng ml-’, but no correlation was found between PCTA and PCP levels. Sex - or age-related differences did not appear and no relation was found between PCTA or PCP concentration and dietary or tobacco smoking habits. Other metabolites of HCB or PCNB, like tetrachlorohydroquinone (TCHQ) or
pentachloronaniline (PCA), normally found
in several animal species, above the 0.1 ng ml -’ level, with H,S04 was omitted.
Discussion
were even
not detected if the clean-up
’
The significance of this sulphur derivative in human urine is uncertain. Although the toxicokinetics and the metabolism of HCB in rodents is well known, it is not so in humans. In the population under study, HCB levels in adipose tissue and breast milk are well
Figure 2 Mass spectra of PCTA isolated from human urine.
Downloaded from het.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 26, 2015
273
diet. The urinary sulphur conjugate, which after hydrolysis and methylation gives PCTA, is probably N-acetyl-S-(pentachlorophenyl) cysteine, as
3 Frequency distribution of pentachlorothioanisole (PCTA) levels in human urine. PCTA 1.85 ± 0.98 ng ml -’ (X ± s.d.) [0.58-4.50].
Figure
documented9
in serum, liver, kidbut the effectiveness of hepatic biotransformation or renal elimination of the metabolites in relation to total body burden remain obscure. In that sense, PCP urinary levels do not seem to be a reliable marker taking into account that there is an important intake of exogenous PCP through the as
ney, and other
they
are
tissues, 12,13
References
& Corbella J. Pentachlorophenol and hexachlorobenzene in serum and urine of the population of Barcelona. Human Toxicology 1987; 6: 397-400. 2 Bevenue A, Wilson J, Casarett JJ & Klemmer HW. A survey of pentachlorophenol content in human urine. Bulletin of Environmental Contamination and Toxicology 2: 319-33. 1967; 3 Lores EM, Edgerton TR & Moseman RF. Method for the confirmation of chlorophenols in human urine by LC with electrochemical detector. Journal of Chromatography Science 1981; 19: 466-9. 4 Williams PL. Pentachlorophenol, an assessment of the occupational hazard. American Industrial Hygiene Association Journal 1982; 43: 799-810. 5 Bertram HP, Kemper FH & Muller C. Hexachlorobenzene content in human blood and adipose tissue: experiences in environmental specimen banking. In: Hexachlorobenzene: Proceedings of an International Symposium, ed. Morris & Cabral, pp. 173-82. Lyon: IARC, Scientific publication n°
7
77, 1986. Engst R, Macholz
’
Acknowledgements Supported by
a
..
FISS grant (91/0170).
’
1 Gómez-Catalán J, To-Figueras J, Planas J, Rodamilans M
6
it is in rodents. This compound is a metabolite of HCB in several species but also may originate from the endogenous biotransformation of the fungicide pentachloronitrobenzene (PCNB). But since other major metabolites of PCNB-(notably pentachloroaniline, PCA) were not detectded above the 0.1 ng ml -’ level in the urines under study, the results suggest that this sulphur derivative may be mostly a biotransformation product of HCB recently ingested through the diet and/or accumulated in tissues. The relationship between urinary PCTA levels and HCB concentration in serum, liver or adipose tissue, and the possible usefulness of PCTA as indicator of HCB metabolism in humans is under investigation.
RM & Kujawa M. The metabolism of hexachlorobenzene (HCB) in rats. Bulletin ofEnvironmental Contamination and Toxicology 1976; 16: 248-52. Edgerton TR, Moseman RF, Linder RE & Wright LH. Multi-residue method for the determination of chlorinated phenol metabolites in urine. Journal of Chromatography 1979; 179: 331-42.
8 Koss G, Reuter
A & Koransky W. Excretion of metabolites of hexachlorobenzene in the rat and the man. In: Hexachlorobenzene: Proceedings of an International Symposium, ed. Morris & Cabral, pp. 261-6. Lyon: IARC, Scientific publication n° 77, 1986. 9 Planas J, Gómez-catalán J, To-Figueras J, Sabroso M, Camps M & Corbella J. Residuos de hexaclorobenceno en el tejido adiposo de la población de Catalunya (1986-87). In: Hexaclorobenceno (las Jornadas Nacionales), pp. 14959., Barcelona: PPU, 1990. 10 Koss G, Koransky W & Steinbach K. Studies on the toxicology of hexachlorobenzene. IV. Sulphur containing metabolites. Archives of Toxicology 1979; 42: 19-31. 11 Veierov D & Aharonson N. Improved clean-up of large lipid samples for electron capture gas chromatographic quantitation and gas chromatographic-mass spectrometric confirmation of organochlorine residues. Journal of the Association of Official Analytical Chemists 1980; 63: 202-7. 12 To-Figueras J, Gómez-Catalán J, Rodamilans J et al. Evaluation of hexachlorobenzene risk in a human population. Spain (1981-1988). The Toxicologist (Abstracts of the 28th Annual Meeting). 1989; 9: 226. 13 To-figueras J, Rodamilans M, Gómez-Catalán J & Corbella J. Hexachlorobenzene residues in the general population of Barcelona (Spain). In Hexachlorobenzene: Proceedings of an International Symposium, ed. Morris & Cabral, pp 147-48. Lyon: IRAC. scientific publication n° 77, 1986.
Downloaded from het.sagepub.com at NORTH DAKOTA STATE UNIV LIB on May 26, 2015
