The role of the Ah receptor and p38 in benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced apoptosis

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in the environment. Benzo[a]pyrene (B[a]P), a prototypical member of this class of chemicals, affects cellular signal transduction pathways and induces apoptosis. In this study, the proximate carcinogen of B[a]P metabolism, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-dihydrodiol) and the ultimate carcinogen, B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide(+/-) (BPDE-2) were found to induce apoptosis in human HepG2 cells. Apoptosis initiated by B[a]P-7,8-dihydrodiol was linked to activation of the Ah receptor and induction of CYP1A1, an event that can lead to the formation of BPDE-2. With both B[a]P-7,8-dihydrodiol and BPDE-2 treatment, changes in anti- and pro-apoptotic events in the Bcl-2 family of proteins correlated with the release of mitochondrial cytochrome c and caspase activation. The onset of apoptosis as monitored by caspase activation was linked to mitogen-activated protein (MAP) kinases. Utilizing mouse hepa1c1c7 cells and the Arnt-deficient BPRc1 cells, activation of MAP kinase p38 by B[a]P-7,8-dihydrodiol was shown to be Ah receptor-dependent, indicating that metabolic activation by CYP1A1 was required. This was in contrast to p38 activation by BPDE-2, an event that was independent of Ah receptor function. Confirmation that MAP kinases play a critical role in BPDE-2-induced apoptosis was shown by inhibiting caspase activation of poly(ADP-ribose)polymerase 1 (PARP-1) by chemical inhibitors of p38 and ERK1/2. Furthermore, mouse embryo p38-/- fibroblasts were shown to be resistant to the actions of BPDE-2-induced apoptosis as determined by annexin V analysis, cytochrome c release, and cleavage of PARP-1. These results confirm that the Ah receptor plays a critical role in B[a]P-7,8-dihydrodiol-induced apoptosis while p38 MAP kinase links the actions of an electrophilic metabolite like BPDE-2 to the regulation of programmed cell death.     

Sensitivity of two Chinese hamster cell lines to SCE induction by a variety of chemical mutagens

SCE induction in Chinese hamster Don (lung) cells was compared with that in CHO (ovary) cells exposed under identical conditions to 14 known mutagens. Test protocols used for comparison were selected following a study of Don and CHO cell responses to aflatoxin B1 and benzo[a]pyrene. In the absence of added metabolizing enzymes 9-aminoacridine, 4-nitroquinoline 1-oxide, N-methyl-N-nitrosourea, dimethylcarbamoyl chloride, beta-propiolactone, daunomycin, aflatoxin B1 and 2-aminoanthracene were directly active in both cell lines; every substance positive in CHO cells was also positive in Don cells. However, the latter detected cyclophosphamide, hydrazine sulphate, benz[c]acridine, 3-methylcholanthrene and benzo[a]pyrene without addition of S9. CHO cells did not respond equivalently to these mutagens, either in the presence or absence of S9. Other differences between the cell lines depended on chemical exposure time, S9 pre-incubation or co-incubation conditions. For example, the ability of CHO cells to detect SCEs due to 2-aminoanthracene was acutely dependent on exposure time. In addition, Don cells exhibited lower background SCE values which were less variable than those of CHO cells under the same culture conditions. Although incapable of detecting 4-dimethylaminoazobenzene (butter yellow) and not as sensitive to cyclophosphamide as certain cell lines of liver origin, the pseudodiploid Don cell line possesses other desirable characteristics required for in vitro SCE assays, particularly with regard to intrinsic metabolic activation of polycyclic aromatic hydrocarbons and related substances.     

Induction of cytotoxicity and mutagenesis is facilitated by fatty acid hydroperoxidase activity in Chinese hamster lung fibroblasts (V79 cells)

The metabolic activation of benzo[a]pyrene and 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene was studied in V79 Chinese hamster fibroblasts after supplementations with arachidonic acid or treatments with linoleic acid hydroperoxide. The extent of metabolic activation was estimated using cytotoxicity and mutagenesis as endpoints. Pretreatment of cells with arachidonic acid for 24 h resulted in significant elevations in the content of this fatty acid in cell phospholipids and increased prostaglandin synthesis. Arachidonic acid and linoleic acid hydroperoxide facilitated 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene cytotoxicity and mutagenesis, and to a lesser extent increased the cytotoxicity and mutagenicity of benzo[a]pyrene. No other compounds tested were mutagenic under these conditions, however, linoleic acid hydroperoxide markedly increased their cytotoxicity. Arachidonic acid-facilitated toxicity and mutagenesis was inhibited by indomethacin, whereas no inhibition was seen when linoleic acid hydroperoxide was used. Nordihyroquairaretic acid abolished the cytotoxicity and mutagenesis facilitated by arachidonic acid and linoleic acid hydroperoxide. Our findings demonstrate that induction of cytotoxicity and mutagenesis following treatment of V79 cells with carcinogens may be limited by low levels of arachidonic acid in these cells. A peroxidatic mechanism is proposed, with limited substrate specificity, for the metabolic activation of chemicals in V79 cells.     

Effects of arachidonic acid and indomethacin on sister-chromatid exchange induction by polycyclic aromatic hydrocarbons in mammalian cell lines

Arachidonic acid (AA), a prostaglandin precursor, significantly potentiated sister-chromatid exchange (SCE) induction in vitro by benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) in the aryl hydrocarbon hydroxylase (AHH)-inducible human hepatoma C-HC-4 cells, and to a lesser extent in the non-inducible rat tumor AH66-B and R1 and Chinese hamster Don-6 cells, all of which were less sensitive to these compounds than C-HC-4 cells. Indomethacin (IM), an inhibitor of prostaglandin endoperoxide synthetase (PES), moderately suppressed SCE induction by BP or DMBA in AH66-B and R1 cells, but it exerted no such effect in C-HC-4 and Don-6 cells. In C-HC-4 cells, however, IM completely eliminated the potentiating effect of AA on SCE induction by both BP and DMBA. The above findings suggest that PES in prostaglandin biosynthesis may also be involved in the metabolic activation of polycyclic aromatic hydrocarbons to genotoxic forms capable of inducing SCEs, in addition to AHH system.     

Evaluation of a human hepatoma cell line as a target cell in genetic toxicology

A cell line derived from a human hepatoblastoma, HepG2, was examined for its ability to activate cyclophosphamide (CY) to a genotoxic form. Metabolism of CY to genotoxic product(s) was determined by the induction of sister-chromatid exchanges (SCE). The dose-dependent response pattern in HepG2 was compared to the patterns obtained by three other mammalian cell lines. HepG2 and a rat hepatoma cell line, H4-II-E, show similar dose-dependent increases of induced SCE, whereas non-hepatic-derived fibroblast lines show little or no CY-induced SCE. Microsomal enzyme activities characteristic of cytochromes P450 and P448 and epoxide hydrolase were examined in the two hepatoma cell lines and compared to levels in rat liver microsomal preparations. Although no cultured cell line can be a universal surrogate for in vivo metabolism, we propose that HepG2 may be useful to determine in a qualitative manner whether human cells possess the ability to activate a chemical to a genetically damaging form.     

Constitutive and inducible expression of cytochromes P4501A (CYP1A1 and CYP1A2) in normal prostate and prostate cancer cells

Constitutive and benzo[a]pyrene (B[a]P) inducible expression of CYP1A1 and CYP1A2 in prostate cancer and normal prostate epithelial cells were examined by immunoblotting. Androgen independent prostate cancer cell lines DU145 and PC3 have constitutive expression of CYP1A and CYP1A1 and CYP1A2, respectively. Four micromolar B[a]P did not appear to induce CYP1A1 or CYP1A2 expression in DU145 or PC3 cells. The androgen dependent prostate cancer cell line, LnCap, also has constitutive expression of CYP1A1 and CYP1A2. However, both CYP1A1 and CYP1A2 are induced by treatment of LnCap cells with 4 microM B[a]P. Untreated normal prostate and primary prostate tumor cells have no detectable CYP1A1 expression. Treatment with 4 microM B[a]P induced CYP1A1 expression in both normal and primary tumor prostate cells. Constitutive CYP1A2 expression was detected in normal prostate cells with little or no induction by exposure to 4 microM B[a]P. Primary prostate tumor cells did not show constitutive expression of CYP1A2. However, CYP1A2 was induced by 4 microM B[a]P in primary prostate tumor cells. These observations indicate that hormonal and cancer specific factors affect the expression and induction of the phase I metabolic enzymes, CYP1A1 and CYP1A2 in prostate cells. These observations may be related to the potential smoking-linked higher risk of prostate cancer development and morbidity of prostate cancer patients who smoke.     

Sister-chromatid exchange induction by indirect mutagens/carcinogens, aryl hydrocarbon hydroxylase activity and benzo[alpha]pyrene metabolism in cultured human hepatoma cells

2 human hepatoma cell lines (C-HC-4 and C-HC-20), in which aryl hydrocarbon hydroxylase activity was induced with benz[alpha]anthracene in vitro to about 140- and 64-fold of the respective basal levels, yielded an increased frequency of sister-chromatid exchanges (SCEs) when exposed to benzo[alpha]pyrene (BP), 7,12-dimethylbenz[alpha]anthracene and 3-methylcholanthrene in vitro. Analysis of the metabolism of BP by these cells by high-pressure liquid chromatography revealed that both cell lines produced various BP metabolites including the proximate form BP-7,8-dihydrodiol which has been reported to be the most potent inducer of SCEs among the metabolites of BP. In addition, aflatoxin B1 and cyclophosphamide also induced SCEs in these cell lines. The above findings suggest that these cells may be capable of metabolizing a range of indirect mutagens/carcinogens into DNA-active forms. These cells may therefore serve as a useful test system in vitro for the detection of genotoxic agents, without the use of an exogenous activating system.     

Different mechanisms involved in apoptosis following exposure to benzo[a]pyrene in F258 and Hepa1c1c7 cells

The present study compares and elucidates possible mechanisms why B[a]P induces different cell signals and triggers apparently different apoptotic pathways in two rather similar cell lines (hepatic epithelial cells of rodents). The rate and maximal capacity of metabolic activation, as measured by the formation of B[a]P-tetrols and B[a]P-DNA adducts, was much higher in mouse hepatoma Hepa1c1c7 cells than in rat liver epithelial F258 cells due to a higher induced level of cyp1a1. B[a]P increased intracellular pH in both cell lines, but this change modulated the apoptotic process only in F258 cells. In Hepa1c1c7 cells reactive oxygen species (ROS) production appeared to be a consequence of toxicity, unlike F258 cells in which it was an initial event. The increased mitochondrial membrane potential found in F258 cells was not observed in Hepa1c1c7 cells. Surprisingly, F258 cells cultured at low cell density were somewhat more sensitive to low (50nM) B[a]P concentrations than Hepa1c1c7 cells. This could be explained partly by metabolic differences at low B[a]P concentrations. In contrast to the Hepa1c1c7 model, no activation of cell survival signals including p-Akt, p-ERK1/2 and no clear inactivation of pro-apoptotic Bad was observed in the F258 model following exposure to B[a]P. Another important difference between the two cell lines was related to the role of Bax and cytochrome c. In Hepa1c1c7 cells, B[a]P exposure resulted in a "classical" translocation of Bax to the mitochondria and release of cytochrome c, whereas in F258 cells no intracellular translocation of these two proteins was seen. These results suggest that the rate of metabolism of B[a]P and type of reactive metabolites formed influence the resulting balance of pro-apoptotic and anti-apoptotic cell signaling, and hence the mechanisms involved in cell death and the chances of more permanent genetic damage.     

Differences in murine procarcinogen activation enzymes are not accompanied by parallel differences in procarcinogen-induced sister-chromatid exchange

C57B1/6 and DBA/2 mice, strains in which there is marked induction of hepatic monooxygenase activity by phenobarbital, were tested for in vivo sister-chromatid exchange (SCE) formation in response to cyclophosphamide, an agent metabolized by this inducible enzyme system. Baseline SCE frequencies were between 4 and 6 SCEs/cell in regerating liver and bone marrow of both strains of mice. Administration of cyclophosphamide (5 mg/kg) led to an increase of nearly 8 SCEs/cell in both tissues of C57B1/6 mice and an increase of more than 10 SCEs/cell in DBA/2 mice. Prior exposure to phenobarbital induced p-chloromethylaniline demethylase activity in regenerating liver of both mouse strains approx. 6-fold, but the changes in measured SCE frequencies were not significantly different from those obtained in the absence of enzyme induction. These results, together with our previous observation that induction of by 3-methylcholanthrene of benzo[a]pyrene hydroxylase activity in the same mouse strains was not accompanied by a comparable change in benzo[a]pyrene-induced SCE formation, reinforce the impression that simple assays of differences in mixed function oxidase activities may not necessarily be good predictors of hereditary differences in the response to genetic damage by procarcinogens which are presumed to be metabolized by these enzymes.     

Suppression of aryl hydrocarbon hydroxylase activity in human primary lung carcinoma x mouse hepatoma somatic cell hybrids

Variants of the mouse hepatoma cell clone inducible for aryl hydrocarbon (benzo(a)pyrene) hydroxylase (AHH) (EC 1. 14. 14.1) activity and deficient in hypoxanthine guanine phosphoribosyl-transferase (EC 2.4.2.8), and human primary lung carcinoma cell clone noninducible for AHH activity and deficient in thymidine kinase (EC 2.7.1.21) were isolated. The variant lines characterized for AHH inducibility and drug resistant phenotype were utilized to study somatic cell hybrids for the expression of AHH induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In two hybrids AHH activity was not expressed. In view of these results we conclude that aryl hydrocarbon hydroxylase activity is suppressed in AHH noninducible human lung carcinoma x AHH inducible mouse hepatoma cell hybrids.     

N′-Isopropylnornicotine in Burley Tobacco (Nicotiana tabacum)

Allyl sulfides such as diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), typical flavor components of Allium vegetables, have been shown to inhibit benzo[a]pyrene (B[a]P)-induced carcinogenesis in animal models. As a possible mechanism of this inhibition, the effect of these volatile substances on cytochrome P450 (CYP)1 (CYP1A1, 1A2 and 1B1)-mediated bioactivation of B[a]P was investigated using a human hepatoma cell model (HepG2). DADS and DATS inhibited the B[a]P-induced ethoxyresorufin O-deethylase (EROD) activity, a marker enzyme for CYP1, by 30-90% and 70-95% at 100-1,000 μM concentration, respectively. The cell viability, an indicator of the capacity to inhibit B[a]P bioactivation, was increased by treatments of 100-1,000 μM DADS and 10-100 μM DATS. Immunoblot results indicated that the B[a]P inducible CYP1A2 protein was suppressed by 100-1,000 μM of DADS and 10-100 μM of DATS, but CYP1A1 and 1B1 were not detectable in any microsomes. Analysis of B[a]P metabolites revealed that the level of 7,8-diol formed was significantly reduced in the DADS and DATS treated microsomes as compared to the control. The level of 9,10-diol and 4,5-diol formed was also lowered by the allyl sulfide treatments. These results suggest that the protective mechanism of allyl sulfides on B[a]P-induced carcinogenesis is possibly related with the modulation of CYP1-mediated bioactivation of B[a]P.     

Modulation of cytochrome P4501-mediated bioactivation of benzo[a]pyrene by volatile allyl sulfides in human hepatoma cells.

Allyl sulfides such as diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), typical flavor components of Allium vegetables, have been shown to inhibit benzo[a]pyrene (B[a]P)-induced carcinogenesis in animal models. As a possible mechanism of this inhibition, the effect of these volatile substances on cytochrome P450 (CYP)1 (CYP1A1, 1A2 and 1B1)-mediated bioactivation of B[a]P was investigated using a human hepatoma cell model (HepG2). DADS and DATS inhibited the B[a]P-induced ethoxyresorufin O-deethylase (EROD) activity, a marker enzyme for CYP1, by 30-90% and 70-95% at 100-1,000 microM concentration, respectively. The cell viability, an indicator of the capacity to inhibit B[a]P bioactivation, was increased by treatments of 100-1,000 microM DADS and 10-100 microM DATS. Immunoblot results indicated that the B[a]P inducible CYP1A2 protein was suppressed by 100-1,000 microM of DADS and 10-100 microM of DATS, but CYP1A1 and 1B1 were not detectable in any microsomes. Analysis of B[a]P metabolites revealed that the level of 7,8-diol formed was significantly reduced in the DADS and DATS treated microsomes as compared to the control. The level of 9,10-diol and 4,5-diol formed was also lowered by the allyl sulfide treatments. These results suggest that the protective mechanism of allyl sulfides on B[a]P-induced carcinogenesis is possibly related with the modulation of CYP1-mediated bioactivation of B[a]P.     

Influence of beta-myrcene on sister-chromatid exchanges induced by mutagens in V79 and HTC cells

The influence of beta-myrcene (MC) on sister-chromatid exchanges (SCE) in V79 cells induced by 4 S9 mix-activated indirect mutagens was studied. The mutagens used were cyclophosphamide (CP), benzo[a]pyrene (BP), aflatoxin B1 (AFB) and 9,10-dimethyl-1,2-benz[a]anthracene (DMBA). MC effectively inhibited SCEs induced by CP and AFB in a dose-dependent manner, but it had no effect on SCE induction by BP and DMBA. MC also reduced CP-induced SCE frequencies in a hepatic tumor cell line (HTC). These cells are metabolically competent and activate CP into its biologically active metabolites. Our results support the suggestion that MC modulates the genotoxicity of indirect-acting mutagens by inhibiting certain forms of the cytochrome P-450 enzymes required for activation of premutagens like CP and AFB.     

Endogenous xenobiotic enzyme levels in mammalian cells.

The response of mammalian cell lines to chemicals depends, in part, on the exogenous activation system used for the induction of a biological response. This could be attributed to differences in the expression of enzymes involved in xenobiotic metabolism. We have measured the activities of benzo[a]pyrene hydroxylase, dimethylaminoazobenzene N-demethylase, catalase, superoxide dismutase, peroxidase and glutathione-S-transferase in human lymphoblast TK6, mouse lymphoma L5178Y, Chinese hamster ovary (CHO) and lung (V79) and mouse C3H10T1/2 cell lines as well as in primary hepatocytes and S9 preparations of liver from male F344 rats. Nitroreductase was also measured in some of these preparations. Human lymphoblast TK6 and mouse C3H10T1/2 cells had the capacity to metabolize dimethylaminoazobenzene and the latter cell line also metabolized benzo[a]pyrene, indicating the presence of constitutive mono-oxygenase activity. Cytochrome P450 could not be detected spectrophotometrically in the cell lines. Western blot analysis indicated that P450 from the P450IIA family is expressed in C3H10T1/2 cells. Reactivity was also observed with an antibody to P450IA2; however, the identity of this protein remains uncertain. Superoxide dismutase, catalase and peroxidase, which protect cells against oxygen radical damage, were found in all the cell lines and in rat hepatocytes and S9. The human lymphoblast TK6 cell line, however, had the least of each of these three enzymes. Glutathione-S-transferase activity was detected at varying levels in all cell types. Nitroreductase activity was high in S9 and Chinese hamster ovary cells and lower in mouse lymphoma and Chinese hamster V79 cells.     

Differential response to benzo[A]pyrene in human lung adenocarcinoma cell lines: the absence of aryl hydrocarbon receptor activation.

Benzo[a]pyrene (B[a]P) has been shown to produce DNA adducts and to initiate pulmonary carcinogenesis in animals. We observed differential susceptibility to B[a]P in two human lung adenocarcinoma cell lines, A427 and CL3. DNA adducts were induced by B[a]P treatment in CL3 cells, however, A427 cells were much less responsive to B[a]P treatment. Cytochrome P450 1A1 (CYP1A1) is involved in bioactivation of B[a]P in nonhepatic tissues. Cotreatment with alpha-naphthoflavone, a CYP1A1 inhibitor, abolished DNA adduct formation by B[a]P in CL3 cells. Nevertheless, CYP1A1 inducer beta-naphthoflavone, enhanced DNA adduct formation by B[a]P in both A427 and CL3 cells. Both enzyme activity and mRNA levels of CYP1A1 were highly induced by 1 or 10 microM B[a]P treatment for 24 hr in CL3 cells but not in A427 cells. Protein levels of AhR and aryl hydrocarbon receptor nuclear translocator (Arnt) were similar in A427 and CL3 cells before B[a]P treatment. However, B[a]P induced a retarded band with the [32P]-dioxin responsive element in CL3 cells, but not in A427 cells. This study demonstrated that variation in AhR-mediated CYP1A1 induction contributes to differential susceptibility to B[a]P-DNA adduct formation in human lung cells. Since AhR and/or Arnt function is impaired in A427 cells, this cell line offers a model for investigating the repression mechanisms of CYP1A1 induction by B[a]P in lung cells.     

Study of the in vitro bioactivation of albendazole in human liver microsomes and hepatoma cell lines

The metabolism of albendazole (ABZ), a benzimidazole anthelminthic, was studied in either microsomal preparations of human liver biopsies or cultured human hepatoma cell lines. Metabolites were analyzed by HPLC. Our data show that microsomes from human biopsies and two human cell lines, HepG2 and Hep3B, oxidize the drug to the sulfoxide very efficiently, whereas the third cell line tested, SK-HEP-1, does not. Both cytochrome P-450 dependent monooxygenases and favin-containing monooxygenases appear to be involved in human ABZ metabolism. Using the cell line displaying the highest ABZ-metabolizing activity, HepG2, the cytotoxic and the inducing effects of the parent drug ABZ and of two primary metabolites, the sulfoxide and the sulfone were studied. These three chemicals provoked a rise in mitotic index resulting from cell division blockage at the prophase or at the metaphase (ABZ metabolites) stage, and ABZ was more cytotoxic than its metabolites. With regard to enzyme-inducing effects, our data clearly demonstrate that the sulfoxide and, to a lesser degree, the sulfone are potent inducers of some drug metabolizing enzymes (i.e., cytochrome P-488 dependent monooxygenases and UDP glucuronyltransferase), whereas ABZ fails to increase and even slightly decreases these enzymatic activities. In conclusion, the HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.Abbreviations ABZ albendazole - B[a]P benzo[a]pyrene - HPLC high-performance liquid chromatography - MC 3-methylcholanthrene - MFO mixed-function oxidase - UDPGT UDP-glucuronyltransferase     

Transitory metabolic disruption and cytotoxicity elicited by benzo[a]pyrene in two cell lines from rainbow trout liver

Two cell lines, RTL-W1 and R1, from rainbow trout liver were used to investigate the effects of benzo[A]pyrene (BaP). BaP induced a catalytic measure of CYP1A, 7-ethoxyresorufin-O-deethylase (EROD) activity, in the rainbow trout liver cell line RTL-W1 but not in R1. Geldanamycin inhibited EROD induction by BaP. Potential BaP metabolites, BaP-7,8-dihydrodiol (BDP) and 6,12-BaP quinone (BQ) also induced EROD activity in RTL-W1. Very low BaP concentrations slightly stimulated cell proliferation in both cell lines. Higher BaP concentrations caused cytotoxicity in RTL-W1 but not in R1. Cytotoxicity was detected in a cell viability assay with 5-carboxyfluorescein diacetate acetoxymethyl ester, and as a decline in cell number. In both cell lines, BaP exposure impaired the reduction of the redox dye, alamar Blue (AB). After BaP removal, AB reduction recovered. Similar results were observed with BQ. As AB monitors metabolic activity, this novel phenomenon was termed transitory metabolic disruption. This decline in AB readings that was caused by BaP was ameliorated in RTL-W1 by alpha-naphthoflavone and geldanamycin, which suggests a role for CYP1A, and in R1 by indomethacin, which suggests involvement of prostaglandin-H-synthase. The significance of the response to BaP that is detected with AB and whether other PAHs cause it will be interesting future questions.     

Rabbit alveolar macrophage-mediated mutagenesis of polycyclic aromatic hydrocarbons in V79 Chinese hamster cells

Rabbit pulmonary alveolar macrophages (PAM) were used as a metabolizing device in combination with V79 Chinese hamster cells as a mutational indicator system. The capacity for metabolic activation of benzo[a]pyrene (B[a]P), its 7,8-diol and 2-aminoanthracene by PAM was investigated. Because of the high variation between different PAM preparations, a statistically significant effect of the 3 compounds could only be demonstrated in a series of 4 or 5 experiments. When the ability of PAM to metabolize B[a]P and the 7,8-diol to mutagenic products was compared with that of primary embryonic fibroblasts from Syrian hamsters, PAM were found to be one-tenth as efficient. Experiments were performed to find out how the phagocytic process could affect the metabolic activation of polycyclic aromatic hydrocarbons. The results showed that PAM-mediated mutagenesis of the 7,8-diol was enhanced 5-10-fold if PAM were fed with opsonized particles. The mechanism by which the phagocytosis of PAM enhanced the mutagenicity of 7,8-diol, as detected in co-cultivated V79 cells, can so far only be a matter for speculation.