Immunosuppressive activity of two main tautomers of BaP

In vitro immunosuppressive activity of the two main tautomers of the 4,5-ketol derivative of benzo(a)pyrene.

R. Diedena, D. Latinneb, R. Hertsensa,  C. Van Halluwijn c, G. J. J.  Lhosta

Department of Pharmaceutical Sciences -UCL, Pharmacokinetics and Metabolism Unit (PMNT), Laboratory of  Mass 

    Spectrometry, Av. E. Mounier  7246   B-1200    Brussels Belgium

Experimental Immunology Unit UCL , Clos Chapelle aux Champs  3056, B-1200   Brussels, Belgium.

c Department of Botany and Mycology - Univ-Lille2, Rue du Professeur Laguesse, Lille   France.

 

a Corresponding author (e-mail  georges.lhoest@skynet.be)

___________________________________________________________________________________________________

Abstract

The two main tautomers of an environmental contaminant, the 4,5-ketol derivative of benzo(a)pyrene (BaP) were separated by HPLC, identified by NMR spectroscopy and by electrospray mass spectrometry.  The in vitro immunosuppressive activity of the two tautomeric forms 4,5- H-4-hydroxy 5-oxo benzo(a)pyrene  and  4-5-H-5-hydroxy-4-oxo benzo(a)pyrene were found less immunosuppressive than the mixture of the two tautomeric forms  retaining a more lipophilic character most probably due to the existence of  stacked configurations linked by hydrogen bonds. 

Keywords :  benzo(a)pyrene, NMR, Electrospray mass spectrometry, immunosuppressive activity

Running title:  BaP ketol derivative

1. Introduction

    Many PAHs including benzo(a)pyrene (BaP) and 7,12-dimethyl benzo(a)anthracene (DMBA) are capable of suppressing immune responses of animals in vivo and of human and animal lymphocytes in vitro, as well as decreasing host resistance to tumors in various animal models 1-13.

It has been postulated that the immunotoxicity produced by PAHs is also mediated by reactive metabolites 11 because metabolic activation of PAHs in immune tissue could result in the generation of active metabolites which may then bind to cellular nucleophilic target sites such as deoxyribonucleic acid (DNA) and proteins that are important in mediating an immune response.  In vitro cultures of murine splenocytes have little ability to generate BaP/DNA adducts, suggesting that hepatic bioactivation is an important mediator of BaP-induced immunotoxicity.

The goal of the present article is to evaluate the in vitro immunosuppressive activity of the two main tautomers of the 4,5-ketol derivative of BaP obtained by chemical synthesis, separated by reverse-phase high performance liquid chromatography (HPLC), identified by electrospray mass spectrometry (ESI+/MS) and NMR spectroscopy as the 4,5- H-4-hydroxy 5-oxo benzo(a)pyrene  and the 4-5-H-5-hydroxy-4-oxo benzo(a)pyrene which are environmental contaminants.

 

2. Experimental

2.1  Chemicals and reagents

Spectrograde solvents (acetonitrile, methanol, dichloromethane ) used in extraction or analytical procedures were purchased from Aldrich (Bornem, Belgium). Pyridine and sodium hydrogen sulphite were from Merck (Darmstadt, Germany) and used in synthetic procedures.

Osmium tetroxide and chromium oxide were purchased from Aldrich (Bornem, Belgium).

Demineralized and filtered water (Milli-Q water purification system; Millipore, Bedford, MA,USA) was used.  All cell culture reagents were obtained from Gibco Laboratories (Paisley, UK).

2.2 Synthetic procedures

Synthesis of cis benzo(a)pyrene 4,5 - dihydrodiol 

Osmium tetroxide (1 mM) dissolved in 2.5 mL of dry pyridine was added to an Erlenmeyer flask containing a solution of 1 mM of benzo(a)pyrene dissolved in 10 mL of dry pyridine.

The resulting solution was stirred under nitrogen for 4 days at room temperature.  Sodium hydrogen sulphite (1 g) dissolved in 15 mL of water was added to the reaction mixture which was left for 3 h under stirring.  The crude benzo(a)pyrene 4,5 - dihydrodiol was precipitated by addition of 20 mL of water , filtered and washed with 10 mL of water.  Yield of crude product was 69 %.  The crude product was purified by reverse phase high performance liquid chromatography. 

Synthesis of 4-5 ketol derivative of benzo(a)pyrene

To cis benzo(a)pyrene 4,5 - dihydrodiol (2 mg, 8 M) dissolved in 0.75 mL of a mixture of benzene - pyridine (1:1) was added lead tetraacetate (4.4 mg , 10 M) dissolved in 0.75 mL of the same solvent mixture.  The reaction mixture was stirred for 3 h at room temperature and the dark - brown colour of the solution became lighter after two hours. The reaction mixture was transferred to a separation funnel containing 10 mL of water and 10 mL of benzene.  After liquid - liquid extraction, the organic phase was evaporated to dryness under vacuum.  To 2 mg of the crude benzo(a)pyrene dialdehyde dissolved in 3 mL of ethanol was added 500 L of an aqueous solution of KCN ( 14 mg/mL).  The reaction mixture was heated under reflux for 1hr.  To the reaction mixture cooled at room temperature, 10 mL of water were added resulting in the precipitation of a crude compound which was extracted two times with 10 mL of dichloromethane. The organic phase was evaporated to dryness under vacuum and the dry residue was submitted to HPLC analysis.

2.3  HPLC Separation

      The HPLC system consisted of two Shimadzu LC10AD pumps, a Waters U6K injector, a variable - wavelength Waters 2487 LC/UV detector (Waters, Brussels, Belgium) connected to an AST computer loaded with a Softron PC integration pack ( Kontron, Zrich, Switzerland). Cis-benzo(a)pyrene 4,5 - dihydrodiol was separated on a Nova-Pak HRC18 (Waters, Brussels, Belgium) column (6 m, 300 x 7.8 mm i.d.). The mobile phase was methanol - water (90 : 10) the flow - rate and UV detector settings were 2.5 mL min -1 and 290 nm, respectively.

Pure cis benzo(a)pyrene 4,5 -dihydrodiol and unchanged benzo(a)pyrene were collected at  retention times of 4.4 min and 22 min, respectively.  Reverse -  phase HPLC of the residue resulting from the synthesis of the ketol derivative of benzo(a)pyrene was performed using a methanol - water mobile phase (75 : 25) and the same flow - rate and detector settings as described for the cis-4,5-dihydrodiol of benzo(a)pyrene.  Under those experimental conditions, a  chromatographic peak was collected at a  retention time of  13.3 min identified by mass spectrometry and NMR spectroscopy as the 4,5 ketol derivative of benzo(a)pyrene.

The 4,5-ketol derivative of BaP was separated in its two main tautomeric forms using the same Nova-Pak HCR18 column with a 52:48 acetonitrile-water mobile phase, the flow rate and detector settings being 2.75 mL/min and 290 nm, respectively.

2.4  Electrospray mass spectrometry

ESI+ full mass spectra were obtained with a Jeol (Tokyo, Japan) Lcmate benchtop LC/MS system.  The source voltage was set at 2.5 kV, the ESI/needle voltage at 2.0 kV, the needle current at 1.9 A, the desolvation plate temperature at 200 C, the ion guide at 2.9 V, the ring lens at 130 V , orifice at 30 V and the flow rate of dry gas ( N2 ) at 7 L min-1.  The compounds (50 g ) were dissolved in acetonitrile - 7.5 mM ammonium acetate ( 50 : 50, 100 ng L-1 ) and the solution was infused with the aid of a syringe pump at a flow rate of 10 L min -1

APCI+ - LCMS mass spectra were obtained with a Hewlett Packard series 1100 HPLC system connected to the JEOL LCmate APCI interface.  The 4,5 ketol derivative of BaP  was separated in its main tautomeric forms on a Nova-Pak C18 (Waters, Brussels, Belgium) column ( 150 x 3.9 mm i.d.). The mobile phase was acetonitrile - water (48 : 52), the flow - rate and UV detector settings were 500 L min -1 and 290 nm, respectively. Under those conditions two main peaks were recorded at 16.54 and 17.49 min.  The source voltage of the mass spectrometer was set at 2.5 kV, the APCI/needle voltage at 4.0 kV, the needle current at 3.5 A, the vaporizer temperature at 500 C, the ion guide at 2.9 V, the ring lens at 80 V , orifice 1 at 30 V and the flow rate of dry gas ( N2 ) at 7 L min-1

2.3  NMR spectroscopy

1H NMR spectra were recorded in CD3CN at 300K on a Bruker Avance 400 NMR spectrometer ( Bruker, Rheinstetten, Germany), equipped with an SGI Octane2 data station.  Spectra were referenced versus the residual solvent peak ( d(CD2HCN) = 1.96 ppm. 

Measurement conditions: The 1D 1H spectra were acquired in deuterated acetonitrile

(CD3CN) with 32k data points over a sweep width of 8250 Hz, using a 30 degree pulse, a 3s relaxation delay and quadrature detection.  Prior to Fourier transform, data were zero filled up to 64k real points and apodized with a 0.3 Hz exponential decay window function.

Phase sensitive double quantum-filtered 1H correlation spectra 14 were acquired using a relaxation decay of 1.5 s, a 90 pulse of 6 s, 1k data points in F1 with 128 transients each, quadrature detection, zero filling up to 1k in both dimensions.  Apodization was carried out with a 90 shifted sine bell and a 90 shifted squared sinebell in F2 and F1, respectively.

2.3   Primary mixed lymphocyte culture (MLC)

Bidirectional MLC were conducted by co-culturing 0.1 mL of two cell suspensions at a concentration of 2 x 106 PBMC/ml from two histoincompatible blood donors. All co-cultures were set up in triplicate in 96 U-well microplates (Falcon). The plates were incubated for 6 days at 37C, 5% CO2, pulsed with [3H]thymidine for 6 h, harvested and counted on a b-counter (Beckman, Analis, Belgium). All the results are expressed in c.p.m. as the mean of three wells. SD was always <15% of the mean. Products were added at serial dilutions starting from 5000 ng/mL to 100 ng/mL at the initiation of the incubation. The percentage of MLC inhibition was calculated as the ratio between the c.p.m. obtained after addition of products and the c.p.m. of the culture in the absence of products.

3. Results and Discussion

    The reverse-phase chromatogram (fig. 1) of the 4,5-ketol of BaP shows the presence of  two main peaks which were shown to be the tautomers 1 and 2 of the BaP 4,5- ketol derivative.

 

 

 

 

 

 

 

The electrospray mass spectrum of these two tautomers 1 and 2 (fig. 2) reveals the presence of molecular adducts at m/z = 285 (M + H)+ and 307 (M + Na)+,  of fragment ions at m/z = 279 (M - CO + Na)+, 269 (M - O + H)+, 255 (M - H2 - CO + H)+, 241 (M - O - CO + H)+, as illustrated in the fragmentation pathways of figure 3. 

If  the mass chromatogram of selected ions such as m/z = 285 and 241 is produced (fig.4), it may be observed that these ions possess retention times identical to the one of the TIC trace of the two tautomers 1 and 2.  This indicates that the molecular adduct m/z = 285 and the fragment ion m/z =241 are closely related in the two tautomeric forms.

 

 

 

 

 

 

 

Both tautomers are easily distinguished by 1H NMR spectroscopy, due to the large chemical shift difference for the H6 singlets ( 8.73 and 8.17 ppm for the 5-oxo (Fig. 5 top) and the 4-oxo ( fig. 5 bottom) tautomer, respectively.  Assignments for the different signals of both tautomers are reported in table 1 and 2 and were confirmed by COSY.

Table 1: 1H NMR assignements for 4,5-H-4-hydroxy-5-oxo benzo(a)pyrene

 

Assignment

Chemical shift, d [ppm]

Multiplicity* (J [ppm])

1

8.18

D(5)

2,3

7.87

D(5)

4

5.47

bs

5

-

-

6

8.73

S

7

8.25

Dd (8, 7, 1)

8

7.81

Ddd (8,7,1)

9

7.92

Ddd (8,7,1)

10

8.90

Dd (8,1)

11

8.82

D (8.5)

12

8.18

D (8.5)

 

 *: (b:  broad, d: doublet,  s: singlet)

 

 

Table 2 : 1H NMR assignments for 4,5-H-5-hydroxy-4-oxo benzo(a)pyrene

Assignment

Chemical shift, d [ppm]

Multiplicity*  (J [ppm])

1

8.40

Dd (1.5,7)

2

8.37

Dd (1.5,7)

3

8.92

Dd (1,8)

4

-

-

5

5.54

bs

6

8.17

S

7

8.17

Dd(1.5,8)

8

7.81

Ddd(1.5,7,8)

9

7.87

Ddd(1.5,7,8)

10

8.90

Dd(1.5,7)

11

8.87

D(9)

12

8.24

D(9)

 

 *: (b:  broad, d: doublet,  s: singlet) 

If  the electrospray mass spectra of the two tautomers are compared , it may be observed that one tautomeric form (fig. 2 bottom) gives rise more easily to the fragment ion m/z = 241, resulting from the loss of 44 Daltons (O + CO) from m/z = 285 as demonstrated by linked scan mass spectrometry.  Since the two tautomers were structurally identified by NMR spectroscopy, the differences in abundance observed between the two fragment ions at m/z = 241 may be used henceforth as a spectroscopic peculiarity leading to structural identification.

The in vitro immunosuppressive activities (fig. 6) of the individual tautomeric forms 1 and 2, as measured by the mixed lymphocyte reaction (MLR), were found similar to each other but the mixture of the two tautomers was observed to be more immunosuppressive than the individual tautomers in the higher concentration range.

This may be the result of inter-molecular interaction taking place between hydroxy and carbonyl groups of the two individual main tautomeric forms giving rise to more predominant stacked configurations with hydrogen bonds between the hydroxy and carbonyl groups as also  strongly suggested by the electrospray mass spectrum of the mixture of the two tautomeric forms as illustrated in figure 7 where [2M + Na]+ is observed.  

As a consequence, the lipophilic character of the mixture of the 2 tautomers should be relatively increased mimicking certain reported non oxidized  PAH-induced mode of  immunosuppression  such as interaction with the Ah receptor 15 or membrane perturbation effects 16

 

 

 

 Legend to figures

Fig. 1  HPLC separation of two tautomers of the 4,5-ketol of benzo(a)pyrene

Fig. 2  Electrospray mass spectrum (ESI+/MS) of the two tautomers 1 (fig. 2 top)

           and 2 (fig. 2 bottom).

Fig. 3  Fragmentation pathways of the 4,5 ketol of benzo(a)pyrene.

Fig. 4  Mass chromatogram of selected ions m/z = 285 and 241.

Fig. 5  1H NMR spectra of the two tautomers 1 and 2 identified as the 4,5- H-4-hydroxy 5-

            oxo benzo(a)pyrene  and  4-5-H-5-hydroxy-4-oxo benzo(a)pyrene, respectively.

Table 1  NMR assignements for 4,5-H-4-hydroxy-5-oxo benzo(a)pyrene.

Table 2  NMR assignements for 4,5-H-5-hydroxy-4-oxo benzo(a)pyrene.

Fig. 6   In vitro immunosuppressive activity, as measured by the mixed lymphocyte reaction

          (MLR), of the mixture and the individual tautomeric forms of the 4,5-ketol derivative of

           benzo(a)pyrene.

Fig. 7  Illustration of stacked configurations for the mixture of the two ketol tautomers of

           benzo(a)pyrene.

 

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