Polycyclic Aromatic Hydrocarbons (PAH)

PAHs Metabolites of BaP

In vitro immunosuppressive activity of a metabolite of the 4,5-ketol derivative of benzo(a)pyrene isolated from human liver microsomes and identified by electrospray mass spectrometry.

G.J.J.  Lhoëst and D.  Latinne

 Abstract:  It has been hypothesized that the immunotoxicity produced by PAHs is also mediated by reactive metabolites because metabolic activation of a PAH 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.

The in vitro immunosuppressive activity , as measured in the mixed lymphocyte reaction test (MLR), of a metabolite of an environmental contaminant, the 4,5-ketol derivative of benzo(a)pyrene, isolated from human liver microsomes , identified by electrospray (ESI+) and by atmospheric pressure chemical ionization tandem mass spectrometry (APCI+), is discussed.


Key words: Benzo(a)pyrene metabolism, mass spectrometry, immunosuppressive activity


Résumé: L’hypothèse que l’immunotoxicité des hydrocarbures aromatiques polycycliques (HAP) pourrait être due à des métabolites réactifs a souvent été soulevée en raison du fait que l’activation métabolique d’un HAP dans le tissu immunitaire pourrait générer des metabolites actifs.  Ces derniers ont dès lors des chances de se lier à des cibles nucléophiles au niveau cellulaire telles l’acide deoxyribonucléique (ADN) ou des protéines jouant un rôle important dans la médiation d’une réponse immunitaire.

L’activité immunosuppressive in vitro, mesurée dans la réaction lymphocytaire mixte, d’un métabolite du benzo(a)pyrene 4,5-cétol isolé à partir de microsomes de foie humain et identifié par spectrométrie de masse, est discutée.


Mots clés : Métabolisme du benzo(a)pyrene, spectrométrie de masse, activité immunosuppressive


G. J. J. Lhoëst1  Department of Pharmaceutical Sciences -UCL, Pharmacokinetics and Metabolism Unit (PMNT), Laboratory of  Mass Spectrometry, Av. E. Mounier  7246

B-1200    Brussels – Belgium

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

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



Benzo(a)pyrene has always been a much-studied polycyclic aromatic hydrocarbon (PAH) and the phase I metabolism of this xenobiotic is mediated by enzymes such as the cytochrome P-450 dependent mixed function oxygenase system and the epoxide hydratase.  The metabolites commonly reported (1-4) and resulting from cell or tissue preparations are the 1,3,7,9 phenol derivatives, the 4,5 and 7,8-dihydrodiols , quinone derivatives, the 4,5-epoxide and 7,8-dihydrodiol-9,10-epoxide of benzo(a)pyrene.

 Many PAHs including BaP and DMBA are capable of suppressing immune responses of animals in vivo and human and animal lymphocytes in vitro, as well as decreasing host resistance to tumors in various animal models (5-18).

Several mechanisms of PAH-induced immunosuppression have been postulated such as interaction with the Ah receptor (19), membrane perturbation effects (20), altered interleukin production (21), disruption of intracellular calcium (Ca++) mobilization (22).

Moreover, it has been hypothesized that the immunotoxicity produced by PAHs is also mediated by reactive metabolites 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. 

We are reporting in this article the in vitro immunosuppressive activity of a new metabolite of benzo(a)pyrene resulting from the biotransformation of the 4,5-ketol derivative of BaP by human liver microsomes and which was identified by electrospray (ESI+) mass spectrometry and atmospheric pressure chemical ionization (APCI+) tandem mass spectrometry.


 2. Materials and methods

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).


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 µM) dissolved in 0.75 mL of a mixture of benzene - pyridine (1:1) was added lead tetraacetate (4.4 mg , 10 µM) 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 µL of an aqueous solution of  KCN ( 14mg/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.

Human liver

A piece of liver from a healthy donor who died in a traffic accident was a gift from a Belgian hospital surgical department.

Preparation of human liver microsomes. 

The piece of liver was weighed, washed with ice-cold 0.1 M disodium hydrogen phosphate buffer (pH 7.4) homogenizing medium containing 0.1 M KCl and 10-3 M Na2 EDTA, minced with scissors, and fractionated according to a reported method (23),  to produce a microsomal fraction containing 4.64 mg/mL-1 protein as well as 0.48 nmol of cytochrome P-450 per mg protein determined according to published standard procedures (24,25).


BaP 4,5 - ketol incubation medium and extraction of the metabolites

The NADPH-generating medium (1 ml) containing 1.75 mg of NADPH, 2.54 mg of NAD, 0.2 ml of MgCl2 (0.5 M), 7.5 mg of glucose-6-phosphate and 0.8 ml of Tris (pH 7.4) was preincubated in a Gallenkamp shaking incubator for 15 min at 37°C in small Erlenmeyer flasks.  To this solution was added: 2.5 ml of human liver microsomes, 6 µl of glucose-6-phosphate dehydrogenase and 50 µg of BaP, or of the BaP cis 4,5-dihydrodiol or of the BaP 4,5 - ketol.  This mixture was incubated for 45 min at 37°C.  After the elapsed time and transfer of the incubation medium to a centrifuge tube, 7 ml of dichloromethane was added.  After 2 min of mixing (vortex mixer), the tube was centrifuged for 10 min at 3000 rpm.  The aqueous phase was discarded and the residue, resulting from the evaporation of dichloromethane phase, was dissolved in 1.5 ml of acetonitrile-water (95:1). The resulting solution was subsequently washed with 1.5 ml of hexane ( 2 min of vortex mixing) which was discarded after centrifugation for 5 min at 3000 rpm.  The acetonitrile - water phase was extracted again with 2 ml of dichloromethane.  After 2 min of mixing (vortex mixer) and centrifugation for 10 min at 3000 rpm, the water phase was discarded and the dichloromethane layer was evaporated to dryness under a stream of nitrogen.  The residue was dissolved in 0.8 ml of isopropanol, and the resulting solution was then submitted to HPLC analysis.


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, Zürich, Switzerland). Cis-benzo(a)pyrene 4,5 - dihydrodiol was separated on a Nova-Pak HRC18 (Waters, Brussels, Belgium) column (6 µm, 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 (26).

For the metabolite of the 4,5 ketol of benzo(a)pyrene, a mixture of compounds called peak 3 ( rt = 22 min ) was collected resulting from a purification of the extraction mixture on an Alltech (Gent, Belgium) RSIL column (10 µm, length 250 mm, i.d 10 mm) using hexane/isopropanol (80:20) as the mobile phase.  The flow rate was adjusted at 3 ml/min and UV detector was set at 290 nm.

Peak 3 was rechromatographed on a  Nova-Pak HRC18 (Waters, Belgium) column ( 6 µm, length 300 mm, i.d. 7.8 mm). The mobile phase was a mixture of acetonitrile / water (40/60), the flow rate and UV detector were 2.75 mL/min and 290 nm, respectively.  Under those conditions a main peak RT3.3 was collected at a retention time of 3.3 minutes among other minor peaks which were not collected.


Electrospray - MS

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 µA, the desolvation plate temperature at 200 ° C, the ion guide at 3 V, the ring lens at 133 V , orifice at 33 V and the flow rate of dry gas ( N2 ) at 7 L min-1.  The compounds (50 µg ) were dissolved in acetonitrile - 7.5 mM ammonium acetate ( 50 : 50, 100 ng µL-1 ) and the solution was infused with the aid of a syringe pump at a flow rate of 10 µL min -1

For linked scan mass spectrometry (B/E), helium was used in the collision cell at a pressure of 30 Pa, which corresponds to 80 % of the maximum pressure that may be applied.

Atmospheric pressure chemical ionization (APCI) tandem mass spectra were obtained with a Finnigan LCQ and Msn instrument  (Finnigan, CA, USA).  The Corona voltage was 4.4. kV, the capillary voltage 32 V and the capillary temperature 160 °C.  The octapole offsets 1 et 2 were set at – 3.25 and -6.0 V, respectively, the internal multipole lens voltage was – 18 V.

The solution was infused with the aid of a syringe pump at a flow rate of 25 µL min-1.

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 37°C, 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 10 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.


Results and Discussion

When the 4,5-ketol derivative of benzo(a)pyrene was incubated in the presence of human liver microsomes, a metabolite RT3.3 (peak 3) of this environmental contaminant  was found to be the 4,5-ketol 7,8-dihydrodiol of benzo(a)pyrene.  The electrospray mass spectrum of this compound (fig. 1) reveals the presence  of a molecular adduct corresponding to (M + H)+ = 319. Fragment ions were also observed at m/z = 301 (M - H2O + H)+, 284 (M - O - H2O)+.,  279 ( M – O - H2O - CO + Na)+ , 273 (M - H2O - CO + H)+, 257 (M - O - H2O - CO + H)+, 245 (M - H2O - 2 CO + H)+ = 245, 239 (M - H2O - 3 CO + Na)+ , 229 (M - O - H2O - 2CO + H)+ = 229 as illustrated in the fragmentation pathways of figure 2.


The product ions of the fragment precursor m/z = 319 (M - O + H)+ were found to be 301 (319 - H2O)+, 285 (319 - H2O - O)+, 279 (319 - O - H2O - CO)+, 275 (319 - O - CO)+, 271 (319 - H2O - H2 - CO)+, 257 (319 - O - H2O - CO)+, 229 (319 - O - H2O - 2 CO)+,  216 (318 - H2O - 3 CO)+., 255 (318 - H2O - 3CO + K)+ confirming that these ions were formed from a 4,5-ketol 7,8-dihydrodiol of benzo(a)pyrene.



These results were also confirmed on a Finnigan LCQ ion trap instrument using the atmospheric pressure chemical ionization interface (APCI) and the product ions of 319 (fig. 3) were found mainly to be 301 (319 - H2O)+ and  257 (319 - O - H2O - CO)+ in good agreement with the presence of a 4,5-ketol 7,8-dihydrodiol of benzo(a)pyrene.  Product ions of the fragment ions m/z = 301 and 257( full ms3 and ms4) were mainly observed to be 257 and 229, respectively, confirming the fragmentation pathway of figure 2.








We were able to demonstrate that an environmental pollutant such as the 4,5- ketol  of benzo(a)pyrene was metabolized by human liver microsomes and by the cytochrome P450 dependent mixed function oxygenase enzymatic system to a BaP  non K region metabolite ,  the 4,5-ketol 7,8-dihydrodiol  of benzo(a)pyrene. 

 The in vitro immunosuppressive activity (fig. 4) of the BaP 4,5-ketol 7,8-dihydrodiol, as measured in the mixed lymphocyte reaction test (MLR), was found to be similar to benzo(a)pyrene but was not found more immunosuppressive than benzo(a)pyrene.  This observation is most probably either the result of partial oxidation of the reported 4,5-ketol 7,8-dihydrodiol of BaP to the corresponding 4,5-ketol 7,8-dihydrodiol 9,10 epoxide by the cytochrome P-450 dependent mixed function oxidase enzymatic system of the T cell giving  rise to covalent adducts of the 4,5-ketol 7,8-dihydrodiol 9,10 epoxide with genetic material (DNA) of the T cells.

PAH-induced immunosuppression may be mediated by reactive metabolites generated within target immune tissue since human peripheral lymphocytes (27) possess cytochrome P450 which are known to metabolize PAHs .  





Legend to figures


Fig. 1   Compared ESI+ mass spectra of the 4,5-ketol and of the 4,5-ketol  7,8-dihydrodiol of benzo(a)pyrene.

Fig. 2   Fragmentation pathways of the 4,5-ketol 7,8-dihydrodiol of benzo(a)pyrene.

Fig. 3   Product ions of the molecular adduct (M + H)+ = 319

Fig. 4   In vitro immunosuppressive activity (MLR) of the 4,5-ketol 7,8-dihydrodiol of

             benzo(a)pyrene compared to benzo(a)pyrene and to its 4,5-ketol derivative.



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