Dipotassium disulphite

Exposure related observations in humans: other data

Administrative data

Endpoint:

exposure-related observations in humans: other data

Type of information:

other: human data

Adequacy of study:

key study

Reliability:

other: not rated acc. to Klimisch

Rationale for reliability incl. deficiencies:

other: Any kind of reliability rating is not considered to be applicable, since human studies/reports are not conducted/reported according to standardised guidelines.

Data source

Reference

Reference Type:

publication

Title:

Alternative pathways of sulfite oxidation in human polymorphonuclear leukocytes

Author:

Constantin, D. et al.

Year:

1994

Bibliographic source:

Pharmacology & Toxicology. 74: 136 - 140.

Materials and methods

Endpoint addressed:

basic toxicokinetics

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In the present study, the oxidation of sulfite in human polymorphonuclear leukocytes was investigated and the sulfite-oxidase catalyzed reaction and the non-enzymatic pathway were compared.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Sodium sulphite

EC Number:

231-821-4

EC Name:

Sodium sulphite

Cas Number:

7757-83-7

Molecular formula:

NA2SO3

IUPAC Name:

disodium sulfite

Details on test material:

- Name of test material (as cited in study report): Sodium sulfite

Method

Details on study design:

PREPARATION OF HUMAN POLYMORPHONUCLEAR LEUKOCYTES:
Human polymorphonuclear leukocytes were isolated from buffy coat (obtained from the Blood Donor center of Sabbatsbergs Hospital, Stockholm, Sweden), essentially using the method described by Trush et al. (1985)(Trush, M.A., J.L. Seed & T. Kensler: Oxidant-dependent metabolic activation of polycyclic aromatic hydrocarbons by phorbol ester-stimulated human polymorphonuclear leukocytes: Possible link between inflammation and cancer. Proc. Natl. Acad. Sci. USA 1985, 82, 5194 - 5198.), with minor modifications (Constantin, D., B. Jernström, I.A. Cotgreave & P. Moldéus: Sodium nitrite-stimulated metabolic activation of benzo(a)pyrene-7,8-dihydrodiol in human polymorphonuclear leukocytes. Carcinogenesis 1991, 12, 777 - 781.).

INITIATION OF THE OXIDATIVE BURST:
To a reaction mixture containing 5 x 10^6 polymorphonuclear leukocytes and 0.2 mg cytochrome C (obtained from Sigma Chemical Company, St. Louis, MO, U.S.A.) in 1 ml phosphate buffer saline (NaCl 8.0 g/l, glucose 1.0 g/l, CaCl2 x 2H2O 0.1 g/l, MgCl2 x 6 H2O 0.1 g/l, KCl 0.2 g/l, KH2PO4 0.2 g/l, Na2 HPO4 2.16 g/l), pH 7.4, 100 nM phorbol myristate acetate (obtained from Sigma Chemical Company, St. Louis, MO, U.S.A.) was
added in order to start the oxidative burst. This initiation of the respiratory burst was also a criterion of the viability of the cells. Formation of superoxide was assayed essentially as described by Green et al. (1987)(Green, M.J., H. Allen, O. Hill & D.G. Tew: The rate of oxygen consumption and superoxide anion formation by stimulated human neutrophils. FEBS Lett. 1987, 216, 31 - 34.). The mixture was supplemented with sulfite (1 mM) in order to see the effect on superoxide formation.

DETERMINATION OF OXYGEN UPTAKE AND SULFITE OXIDASE ACTIVITY IN HUMAN POLYMORPHONUCLEAR LEUKOCYTES:
Oxygen consumption was measured with a Clark-type oxygen electrode. The final reaction volume was 3 ml. Polymorphonuclear leukocytes (5 x 10^6 cells/ml) were suspended in phosphate buffer saline, pH 7.4 and the O2 consumption registered. 1mM sodium sulfite and/or 100 nM phorbol myristate acetate were added to the mixture.

HPLC ANALYSIS OF SULFITE:
Cell suspensions (100 µl) were mixed with 8 mM monobromobimane (obtained from Calbiochem-Behring, La Jolla, U.S.A.) in 50 mM N-ethylmorpholin (obtained from Fluka A.G., Buchs, Switzerland) pH 8 (100 µl). The samples were kept in the dark at room temperature for 5 min. and trichloroacetic acid (10 µl) was then added. The protein was removed by centrifugation at 3000 x g for 5 min. and 25 µl of the supernatant was used for analysis of sulfite by HPLC (Cotgreave, I.A. & P. Moldéus: Methodologies for the application of monobromobimane to the stimultaneous analysis of soluble and protein thiol components of biological systems. J. Biochem. Biophys. Meth. 1986, 13: 231-249.) (Cotgreave, I.A., M. Berggren, T.W. Jones, J. Dawson & P. Moldéus: Gastrointestinal metabolism of N-acetylcysteine in the rat, including an assay for sulfite in biological systems. Biopharmaceutics Drug Disp. 1987, 8: 377-386.)).

EPR SPECTROSCOPY:
EPR spectra were recorded using a Bruker 200 D EPR spectrometer with the center field set at 3390 G, microwave power of 10 mW, modulation amplitude 1 G, field with 100 G and microwave frequency 9.14 GHz. The samples (100 µl) contained non-stimulated or phorbol myristate acetate-stimulated polymorphonuclear leukocytes (10 x 10^6/ml), 200 µM DETAPAC (obtained from Aldrich, W-7924 Steinheim, Germany), 50 mM DMPO (obtained from Sigma Chemical Company, St. Louis, MO, U.S.A.) and 1 mM sodium sulfite in phosphate buffer saline, pH 7.4 and were placed directly in a micro flat cell. The measurements were carried out at 35° for 30 min.

SPECTROPHOTMETRIC ASSAY OF SULFATE:
Sulfate formation was measured at 360 nm as described by Krijgsheld et al. (1979) (Krijgsheld, K. R., H. Frankene, E. Scholtens, J. Zweens & G. J. Mulder: Absorption, serum levels and urinary excretion of inorganic sulfate after oral administration of sodium sulfate in the conscious rat. Biochim. Biophys. Acta 1979, 5861, 492 -497.) using a Shimadzu UV-260 spectrophotometer.

MEASUREMENT OF PROTEIN IN HUMAN POLYMORPHONUCLEAR LEUKOCYTES:
Polymorphonuclear leukocytes protein was assayed as described by Peterson (1977)(Peterson, G.L.: A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 1977, 83, 346 -356.)

Results and discussion

Results:

Metabolism: two different oxidation routes of sulfite to sulfate have been identified in the human polymorphonuclear leukocytes. 1. Via sulfite oxidase and 2. via an one electron oxidation step with an intermediate formation of sulfur trioxide radicals.

Details on metabolites: addition of sulfite to polymorphonuclear leukocytes significantly stimulated the uptake of oxygen.
The oxygen consumption varied substantially between cells from different donors and were divided in those with low (0 -200 nmol O2/ml/min.) and high (>200 nmol O2/ml/min.) capacity.
The interindividual difference in oxygen uptake was also reflected in the rates of sulfite disappearance and sulfate formation, the correlation between these two parameters being fairly good. The correlation was not affected by varying the concentration of sulfite added to the leukocytes. It is assumed that the variation in oxygen consumption mainly reflects the cells capacity to oxidize sulfite direct to sulfate, thus the activity of sulfite oxidase.
Only 30 % to 40% of the sulfite added to cells with low sulfite oxidase activity was oxidized to sulfate after 30 min. incubation whereas on average about 60% was oxidized in cells with high activity.
In the presence of sulfite, addition of phorbol myristate acetate to cells with low sulfite oxidase activity increased the O2 consumption substantially (up to 600 nmol/ml/ min.) In cells with high enzyme activity an inhibitory effect of phorbol myristate acetate on oxygen consumption was observed.
The effect of phorbol myristate acetate can also be seen on the oxidation of sulfite to sulfate. In cells with low sulfite oxidase activity the addition of phorbol myriastate acetate increases the rate of sulfate formation whereas in cells with high activity phorbol myriastate acetate has an inhibitory effect.
The EPR spectrum shows signals consistent with the presence of sulfur trioxide radicals formed during autooxidation of sulfite. A similar spectrum is observed after addition of sulfite to non-phorbol myristate acetate stimulated human polymorphonuclear leukocytes.
When phorbol myristate was added to polymorphonuclear leukocytes and sulfite, an EPR spectrum compatible with the presence of sulfur trioxide radicals as well as hydroxyl adducts with DMPO is observed.

Any other information on results incl. tables

The interaction of sulfite and the superoxide radical anion (O2^-) formed during the oxidative burst in the leukocytes was also investigated. The formation of O2^- was estimated by measuring the reduction of cytochrome C. Sulfite efficiently inhibited the reduction of cytochrome C by cells with low sulfite oxidase activity. For instance in the presence of 1 mM sulfite the inhibition was more than 80%. The reason is possible an interaction between O2^- and sulfite leading to sulfur trioxide radical formation. The effect on cytochrome C reduction by sulfite in leukocytes with high sulfite oxidase activity is minor. Due to the very rapid enzymatic oxidation of sulfite to sulfate only limited interction between sulfite and O2^- occurred.

Applicant's summary and conclusion

Conclusions:

Two different oxidation routes of sulfite to sulfate have been identified in the human polymorphonuclear leukocytes. Besides the pathway via sulfite oxidase another route of oxidation via an one electron oxidation step with an intermediate formation of sulfur trioxide radicals has been identified.
The contribution of the different pathways is expected to vary substantially due to the great interindividual variation in sulfite oxidase activity. The contribution of the trioxide radical pathway is expected to be high in individuals with low sulfite oxidase activity.