Environmental Applications


 Dr. LhoŽst G.J.J.

New Metabolites of benzo(a)pyrene


New metabolites of benzo(a)pyrene were isolated from rat and human liver microsomes and they are to be considered as ultimate carcinogenic compounds

1. Results and Discussion

          PAHs are an important class of environmentally prevalent xenobiotics that exert carcinogenic effects .   The carcinogenic and mutagenic actions of PAHs have been shown to be mediated by reactive electrophilic metabolites rather than the parent compound . It has also been proposed that the presence of direct mutagens in ambient particulates may be due in part to the reactions of BaP and other PAHs with O2, NO2 and free radicals present in polluted atmosphere and in exhaust particulates from spark-ignition engines. Pitts & van Cauwenberghe  have demonstrated that PAHs in simulated atmospheric conditions were reported to produce compounds of molecular weight 284 corresponding to the molecular weight of a metabolite of BaP obtained from phenobarbital induced rat liver microsomes fortified with the usual cofactors and described to be 4-hydroxy-5-oxo-4,5-dihydro-benzo(a)pyrene (MW = 284) by Lhoest et al. ( (figure 1).

 Since most probably PAHs ketol derivative may be formed in the atmosphere, it would be advisable to investigate the metabolism of different PAH ketol derivatives in different animal species. Some carcinogenic compounds are generally recognized to produce complex effects on the immune system. Based on this observation, 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 such as the benzo(a)pyrene 7,8-dihydrodiol-9,10-epoxide which is resistant to epoxide hydrolase and may bind to cellular nucleophilic target sites such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and proteins. These bonds are important in mediating an immune response and/or maintaining cellular homeostasis .



Fig. 1 A metabolite of benzo(a)pyrene was isolated by thin layer chromatography on silica gel plates and identified by EI-mass spectrometry





It has been reported that either the addition of BaP or selected metabolites to splenocyte cultures are capable of suppressing the in vitro AFC response to sheep red blood cells (sRBCs). The metabolites BaP-7,8-diol were found to be the most potent immunosuppressive metabolites evaluated, producing a concentration-dependent suppression of the AFC response similar to parent BaP. Other authors have shown that the BaP-7,8-diol undergo further metabolism to form the reactive BaP-7,8- diol- 9,10- epoxide (BPDE). These two last compounds are the proposed ultimate carcinogenic forms of BaP since direct addition of BPDE was shown to decrease the AFC response of cultured splenocytes at concentrations similar to BaP (19). These findings suggest that the in vitro immunosuppressive action of BaP was mediated by the diol epoxides.


EI mass spectra of the 4,5 ketol derivative of benzo(a)pyrene


                                             Fig.2          ESI+  Mass spectra of the main 4,5 ketol tautomers of BaP 

                                                                                                                   Main 4,5 ketol tautomers of Benzo(a)pyrene   

 Common recognized metabolites of BaP



                                                                                                    ESI+/MS of the 4,5-ketol tautomers of Benzo(a)pyrene




 ESI+ mass spectra of three new metabolites of BaP 




















           BaP 7,8-Ketol 9,10 epoxide,

           BaP 4,5 ketol 7,8-dihydrodiol 9,10 epoxide, BaP 4,5-ketol 7,8-epoxide







Fragmentation pathways of the BaP 4,5 ketol 7,8-dihydrodiol 9,10 epoxide



It has been reported in Nature by Sims et al. a few decades ago that the products of the further metabolism of the 7,8-dihydrodiol of benzo(a)pyrene might be involved in the reaction of benzo(a) pyrene with DNA.  The 3H-labelled 7,8-dihydrodiol of benzo(a)pyrene was incubated in vitro with a rat liver microsomal preparation and DNA.  The DNA was hydrolyzed , mixed with a hydrolysate of DNA from cells treated treated with 14C-labelled benzo(a)pyrene and the mixtures chromatographed on  Sephadex columns.  Hydrolysates of DNA that had been incubated with microsomes and 3H-labelled 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene gave elution profiles with two peaks in which the major hydrocarbon-deoxyribonucleoside product eluted in a position similar to that occupied by the DNA product froml 14C-labelled benzo(a)pyrene treated cells.

Two peaks were observed for the 4,5-ketol of benzo(a)pyrene as shown in figure 2 and corresponding to two tautomers, the 4-hydro,4-hydroxy 5-oxo and the 4-oxo,5-hydro,5-hydroxy benzo(a)pyrene as demonstrated by mass spectrometry and NMR spectroscopy.    The 7,8 ketol 9,10 epoxide of benzo(a)pyrene was isolated in our laboratory after incubation of the 7,8-dihydrodiol with fortified human liver microsomes and this new metabolite of BaP exists most probably, as observed for the 4,5-ketol of BaP, under the form of two tautomeric forms.  This strongly suggests , taking into account  that two peaks were observed after the separation of DNA hydrolysates incubated with microsomes and 3H-7,8-dihydrodiol of BaP as published in Nature, that the ultimate carcinogenic compounds of BaP binding to DNA and/or critical proteins are the 7,8-ketol 9,10 epoxide of benzo(a)pyrene, metabolite existing under different tautomeric forms and which is more lipophilic than the 7,8-dihydrodiol epoxide of benzo(a)pyrene.