Induction of chromosomal aberrations and spindle disturbances in Chinese hamster epithelial liver cells in culture by pyrene and benzo[a]pyrene quinones

Regioisomers of pyrene and benzo[a]pyrene quinones were testedfor their ability to induce structural and numerical aberrationsand spindle disturbance in Chinese hamster epithelial liver(CHEL) cells in culture. All quinones tested were clastogenicPyrene-1,8-quinone (P-1,8-Q) and benzo[a]pyrene–3,6–quinone(BP-3,6-Q) induced strikingly high levels of triradials. Inaddition, dicentrics and ring chromosomes were very common inBP-3,6-Q-treated cultures. Isomers of these compounds, pyrene-1,6-quinone(P-1,6-Q) and benzo[a]pyrene-1,6-quinone (BP-1,6-Q), inducedunobtrusive patterns of chromosomal aberrations. We suspectthat the P-1,8-Q and BP-3,6-Q moieties bound to the DNA werestill reactive, and formed crosslinks and/or underwent redoxcycling leading to high local concentrations of reactive oxygenspecies. In addition, P-1,8-Q and BP-3,6-Q induced c-mitoses,hyperdiploidies and polyploidies, in particular endoreduplications.These effects were not seen with the other two test compounds,or they were only detected at the highest concentrations used,which were strongly cytotoxic (c-mitoses with P-1,6-Q, polyploidieswith BP-1,6-Q). ⁶To whom correspondence should be addressed at: European Centre for the Validation of Alternative Methods (ECVAM), Joint Research Centre (JRC), TP58O, 1–21020 Ispra, Italy     

Metabolic activation to a mutagen of 3-hydroxy-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, a secondary metabolite of benzo[a]pyrene

3-Hydroxy-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (3-OH-BP-7,8-diol)wag isolated from arylsulfatase/ß-glucuronidase-treatedbile of rats to which 3-hydroxybenzo[a]pyrene (3-OH-BP) hasbeen administered. This triol was investigated for mutagenicityin Salmonella typhimurium (reversion to histidine prototrophyof strains TA 97, TA 98, TA 100 and TA 1537) and in V79 Chinesehamster cells (acquisition of resistance to 6-thioguanine).When no exogenous metabolizing system was added the triol wasinactive, while 3-OH-BP showed weak mutagenic effects with allfour bacterial strains. In the presence of NADPH-fortified postmitochondrialsupernatant fraction (S9 mix) of liver homogenate from Aroclor1254-treated rats, the mutagenicity of 3-OH-BP was potentiated,and the triol was activated to a mutagen(s). In the presenceof S9 mix, the triol was 5—18 times more mutagenic than3-OH-BP in strains TA 97, TA 100 and TA 1537, but both compoundsshowed similar mutagenic potencies with strain TA 98. Thesestrain differences strongly suggest that the mutagenicity of3-OH-BP in the S9 mix-mediated test was not exclusively dueto metabolites of 3-OH-BP-7, 8-diol. Trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BP-7,8-diol), like the triol, showed mutagenic effects onlyin the presence of S9 mix. Strain TA 1537 was reverted by thetriol but not by the diol. In the other bacterial strains thediol was more mutagenic than the triol, the difference in potencybeing largest in strain TA 100 (2.5-to 10-fold, depending onthe experimental conditions). In V79 cells, the diol was a potentmutagen, while the triol showed only very weak mutagenic effects.However the triol was more cytotoxic than the diol. High cytotoxicityof the triol was observed even in the absence of S9 mix. Theresults of the present study demonstrate that metabolites of3-OH-BP-7, 8-diol) are biologically-active derivatives of benzo[a]pyrene.Comparison of the mutagenic effectiveness in different bacterialstrains also reveals that metabolites of 3-OH-BP-7, 8-diol andof BP-7, 8-diol substantially differ in the kind of geneticalterations they evoke.     

Activation of benzylic alcohols to mutagens by human hepatic sulphotransferases

Four primary and five secondary benzylic alcohols derived frompolycyclic aromatic hydrocarbons were tested for mutagenicityin Salmonella typhimurium TA98 in the presence of 3'-phosphoadenosine-5'-phosphosulphate,the cofactor for sulphotransferases, and varying amounts ofhepatic cytosol from three or four different human subjects,a 3-year-old child, an adult female, an adult male and one unknown.All compounds except one, 4H-cyclopenta[def] phenanthren-4-ol,were activated to mutagens. The interindividual variation inthe activities was at most 3-fold and the individual activitiestowards the different substrates were correlated with each other.The same compounds had previously been tested in the presenceof hepatic cytosol from rats and all compounds activated inone species were also activated in the other species. However,there were marked quantitative differences, which were furthercomplicated by the observation of a substantial sex differencein the rat. Male and female rat liver cytosol showed highersulphotransferase activities towards 1-hydroxymethylpyrene,9-hydroxymethylanthracene, 7-hydroxymethyl-12-methylbenz[a]anthraceneand 4H-cyclopenta[def]chrysen-4-ol than human liver cytosol.The largest difference in activity was seen with 7-hydroxymethyl-12-methylbenz[a]anthracene,reaching a factor of     

Inactivation of electrophilic metabolites by glutathione S-transferases and limitation of the system due to subcellular localization

Benzo(a)pyrene was activated to metabolites mutagenic for Salmonella typhimurium TA 98 by liver microsomes from control and phenobarbital treated mice. Under these conditions benzo(a)pyrene 4,5-oxide accounts for most of the mutagenicity. We have therefore investigated (1) the conjugation of benzo(a)pyrene 4,5-oxide with glutathione and (2) the effect of glutathione on the mutagenicity of benzo(a)pyrene.The spontaneous conjugation occurred only very slowly. The rate of this reaction was slightly augmented by microsomes and very greatly augmented by the cytosol fraction of liver homogenate. With respect to the mutagenicity of benzo(a)pyrene, glutathione had only a weak effect when benzo(a)pyrene was activated by microsomes in the absence of the cytosol fraction. In its presence, however, glutathione was able to strongly reduce the mutagenicity. But this reduction depended on the spatial relationship between microsomes and bacteria. The strongest inactivation was found when bacteria and microsomes were in separate agar layers. In contrast, no inactivation was observed when all the microsomes were in direct contact with the bacteria. When the test was performed according to the Ames procedure the topographical situation was intermediate: some microsomes were adsorbed onto the bacteria and some were free. Accordingly, the effect of glutathione was intermediate. When the premutagen trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene was activated in the presence of the cytosol fraction, glutathione again reduced the mutagenicity, when microsomes and bacteria were separated from each other, but did not reduce the mutagenicity, when all the microsomes were bound to the bacteria.Obviously in the situation where a direct diffusion within the lipophilic environment from the site of formation to the target bacteria was physically possible the mutagenic metabolites diffused preferentially directly to the bacteria and not through the hydrophilic environment of the medium. Therefore they could not be inactivated by components of the cytosol fraction. This could be of significance also for the situation in the eucaryotic cell, since the endoplasmic reticulum is in direct contact with other cell structures such as the nuclear envelope. Thus, hydrophobic metabolites generated in the endoplasmic reticulum could reach such sites by lateral diffusion within the membranes. The observation that benzo(a)pyrene 4,5-oxide was a very good substrate for the cytosol localized glutathione S-transferase, but that it was not inactivated by this system when bacteria and microsomes were in direct contact, indicates that a severe limitation for the inactivation of benzo(a)pyrene metabolites by this enzyme is imposed by its localization in the cytosol.Presented at the Symposium Influence of Metabolic Activations and Inactivations on Toxic Effects held at the 18th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section Toxicology, D-6500 Mainz, March 15, 1977     

Sulfotransferase-independent genotoxicity of illudin S and its acylfulvene derivatives in bacterial and mammalian cells

Acylfulvenes are a class of antitumor agents derived from illudin S, a sesquiterpenoid toxin isolated from mushrooms of the genus Omphalotus. Although DNA appears to be their major target, no data concerning mutagenicity of acylfulvenes are available in the literature, and limited data have been published on illudin S. Enzyme-mediated biotransformations have been demonstrated to influence the cytotoxicity of acylfulvenes. Illudin S and some acylfulvenes [e.g., (?)-6-hydroxymethylacylfulvene (HMAF)] are allylic alcohols with potential for enhanced cytotoxicity and genotoxicity by means of metabolic sulfation. Therefore, we studied the influence of various heterologously expressed human sulfotransferases (SULTs) on biological activities of illudin S and HMAF in bacterial and mammalian cells. (?)-Acylfulvene (AF) was tested as a congener lacking an allylic hydroxyl group. We found: (1) all three compounds were mutagenic in standard Salmonella typhimurium strains TA98, TA100 and TA104; (2) they induced gene mutations (at the hypoxanthine phosphoribosyl transferase locus) and sister chromatid exchange (SCE) in Chinese hamster V79 cells; (3) these effects were practically unaffected when human SULTs were expressed in the target bacteria or mammalian cells (using SCE as the endpoint); (4) illudin S demonstrated 40–600 times higher genotoxic activities than the semisynthetic acylfulvenes studied; it was positive in the SCE test even at a concentration of 0.3 nM; (5) genotoxicity in mammalian cells was observed at substantially lower concentrations of the compounds than required for a positive result in the bacterial test (400 nM with illudin S). We conclude that illudin S, HMAF and AF are potent genotoxicants and human SULTs do not play a significant role in their bioactivation.     

The Mechanism of Photoaffinity Labeling

Photoaffinity labeling is a recently introduced method for covalently binding chemical tags to the active sites of protein molecules, which is potentially capable of very great specificities of labeling. A labeling reagent is used that is converted by photolysis to an extremely reactive intermediate. According to the expected mechanism, the reagent molecules that are specifically and reversibly bound to the active site at the instant of photolysis react irreversibly in the site before they can dissociate from the site. In two such reagent-protein systems studied in this paper, however, it is shown that, while by the usual criteria photoaffinity labeling appears to have occurred, the expected mechanism in fact does not hold. This was discovered in experiments with scavengers present in the mixtures that were photolyzed. The general properties of, and criteria for, photoaffinity labeling reactions are discussed in the light of these findings.     

Use of genetically manipulated Salmonella typhimurium strains to evaluate the role of sulfotransferases and acetyltransferases in nitrofen mutagenicity

Nitrofen had been used as a herbicide, until its carcinogenic and teratogenic activity in rodents was detected. A food contamination occurring in 2002 in Germany led to the initiation of new studies in order to better understand the potential risk for humans. Nitrofen is a nitroarene and as such might be activated to a mutagen via reduction to the corresponding hydroxylamine and subsequent formation of a reactive acetic or sulfuric acid ester. Therefore, we have investigated the mutagenicity of nitrofen in Salmonella typhimurium strains engineered for the expression of all human xenobiotic-metabolizing sulfotransferases (SULTs) and acetyltransferases (NATs) identified. Nitrofen was inactive in the parental strains TA1538, TA98 and TA100, but was mutagenic even at low doses when human sulfotransferase SULT1A1 (the major broad-spectrum phenol SULT) was expressed in these strains, but not when it was expressed in a TA1538-derived strain deficient in an endogenous nitroreductase. Several other human SULTs (in particular 1A3 and 1C1) as well as human NAT2 (unlike NAT1) also activated nitrofen, but were markedly less efficient than SULT1A1. Likewise, expression of rat and mouse SULT1A1 led to weaker mutagenic activity of nitrofen than expression of the corresponding human enzyme. An endogenous acetyltransferase only activated nitrofen to a mutagen when it was strongly over-expressed in the TA98-derived strain YG1024. Thus, humans might be more susceptible to the carcinogenic effects of nitrofen than mice and rats, which have been used in long-term studies. The fact that several SULTs show particular high expression in fetal tissues suggests that this activation pathway may also play a role in the teratogenic effects observed.     

Activation of 3-nitrobenzanthrone and its metabolites by human acetyltransferases,sulfotransferases and cytochrome P450 expressed in Chinese hamster V79 cells

3-nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and ambient air pollution. 3-aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA) and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) have been identified as 3-NBA metabolites. Recently we found that 3-NBA and its metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) form the same DNA adducts in vivo in rats. In order to investigate whether human cytochrome P450 (CYP) enzymes (i.e., CYP1A2), human N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3-NBA and its metabolites, we developed a panel of Chinese hamster V79MZ-h1A2 derived cell lines expressing human CYP1A2 in conjunction with human NAT1, NAT2, SULT1A1 or SULT1A2, respectively. Cells were treated with 0.01, 0.1 or 1 microM 3-NBA, or its metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA). Using both enrichment versions of the (32)P-postlabeling assay, nuclease P1 digestion and butanol extraction, essentially 4 major and 2 minor DNA adducts were detected in the appropriate cell lines with all 4 compounds. The major ones were identical to those detected in rat tissue; the adducts lack an N-acetyl group. Human CYP1A2 was required for the metabolic activation of 3-ABA and 3-Ac-ABA (probably via N-oxidation) and enhanced the activity of 3-NBA (probably via nitroreduction). The lack of acetylated adducts suggests N-deacetylation of 3-Ac-ABA and N-Ac-N-OH-ABA. Thus, N-hydroxy-3-aminobenzanthrone (N-OH-ABA) appears to be a common intermediate for the formation of the electrophilic arylnitrenium ions capable of reacting with DNA. Human NAT1 and NAT2 as well as human SULT1A1 and SULT1A2 strongly contributed to the high genotoxicity of 3-NBA and its metabolites. Moreover, N,O-acetyltransfer reactions catalyzed by human NATs leading to the corresponding N-acetoxyester may be important in the bioactivation of N-Ac-N-OH-ABA. As human exposure to 3-NBA is likely to occur primarily via the respiratory tract, expression of CYPs, NATs and SULTs in respiratory tissues may contribute significantly and specifically to the metabolic activation of 3-NBA and its metabolites. Consequently, polymorphisms in these genes could be important determinants of lung cancer risk from 3-NBA.     

SULT1C3, an orphan sequence of the human genome,encodes an enzyme activating various promutagens

The human genome contains a sequence that is homologous to genes encoding soluble sulphotransferases (SULTs) based on the nucleotide sequence and possible intron/exon splice sites. The putative coding sequence (termed SULT1C3) was synthesized and integrated into a bacterial expression vector. We used the cDNA-expressed protein for raising an antiserum and studying enzyme activities. No activity was detected with 4-nitrophenol and 1-naphthol, known substrates of all other members of the human SULT1 subfamily. The activity was also negligible with paracetamol, ethanol, 5-hydroxymethylfurfural, 2-hydroxymethylpyrene, 2-(α-hydroxy)ethylpyrene, and corticosterone, compounds for which we have developed sensitive enzyme assays with direct determination of the product by HPLC-UV, HPLC-fluorescence or HPLC-MS/MS. Since diverse sulpho conjugates are chemically reactive – often short-lived and mutagenic – we expressed SULT1C3 in Ames’ Salmonella typhimurium strains TA1538 and TA100, as we had done with many other SULTs previously. The expression level of SULT1C3 protein amounted to 2% of the total cytosolic proteins, which is in the middle range of other SULTs expressed in this model. Using recombinant bacterial tester strains in mutagenicity assays, we observed SULT1C3-mediated activation of several large benzylic alcohols derived from alkylated polycyclic hydrocarbons: 1-hydroxymethylpyrene, both enantiomers of 1-(α-hydroxy)ethylpyrene, 6-hydroxymethylbenzo[a]pyrene and 6-hydroxymethylanthanthrene. 1′-Hydroxysafrole was the smallest molecule activated by SULT1C3 up to date. Our study demonstrates that SULT1C3 has sulphotransferase activity and that it prefers relatively large substrates. The substrates detected were activated to mutagens, which cannot be the regular function of the enzyme. The physiological substrates remain to be identified. Probably, they are relatively large, endogenous or common exogenous, molecules.