Effect of phorbol esters and liposome-delivered phospholipids on the differentiation program of normal and dystrophic satellite cells

Satellite cells, isolated from hind limb of normal C57BL/6J mice, differentiate in culture in the presence of concentrations of phorbol esters which inhibit differentiation of embryonic myoblasts. However, if phosphatidylserine containing liposomes were added to the culture medium together with TPA, differentiation of satellite cells was reversibly inhibited. Under these conditions, the withdrawal of these cells from the cell cycle still occurred as in untreated cells. Phosphatidylserine liposomes alone or liposomes containing phosphatidylcholine (either alone or in combination with TPA) had no effect on satellite cell differentiation. In the case of satellite cells from dystrophic C57BL/6J/dydy mice, TPA addition (0.1 microM) to the culture medium partially (about 70%) inhibited morphological and biochemical differentiation. This effect could be prevented by preincubating dystrophic satellite cells with liposomes containing phosphatidylcholine but not other phospholipids. These data indicate that it is possible to change the sensitivity to TPA of satellite cells by modifying the phospholipid composition of their plasma membrane. Possible relationships of these phenomena with activation of protein kinase C or phosphatidylinositol breakdown have been investigated. The results obtained are discussed with regard to possible modulation of the intracellular response to agonist binding.     

Cutaneous metabolism of benzo[a]pyrene: comparative studies in C57BL/6N and DBA/2N mice and neonatal Sprague--Dawley rats

The metabolism of the polycyclic aromatic hydrocarbon (PAH) carcinogen benzo[a]pyrene (BaP) was studied using microsomes prepared from the skin of the mouse and rat. Topical application of the polychlorinated biphenyl (PCB) Aroclor 1254 or the PAH 3-methylcholanthrene (3-MC) to the skin of the C57BL/6N and DBA/2N mouse and the Sprague-Dawley rat caused statistically significant enhancement of cutaneous microsomal aryl hydrocarbon hydroxylase (AHH) activity in each animal. PCB was a more potent inducer of the enzyme than was 3-MC. BaP metabolism by skin microsomes from the same animals was assessed using high performance liquid chromatography (HPLC). The skin of untreated animals metabolized BaP into 9,10-, 7,8- and 4,5-dihydrodiols, phenols and quinones. Skin application of PCB caused greater than 16–18-fold enhancement of BaP metabolism in the C57BL/6N mouse and the rat and 2–5-fold enhancement in the DBA/2N mouse. Skin application of 3-MC enhanced BaP metabolism 2–8-fold in the C57BL/6N mouse and 5–10-fold in the rat and had no effect in the DBA/2N mouse. The formation of procarcinogenic metabolite BaP-7, 8-diol was greatly enhanced (4–12-fold) by treatment with the PCB and 3-MC in the tumor susceptible C57BL/6N mouse and in the tumor-resistant neonatal Sprague-Dawley rat. In contrast, the formation of BaP-7,8-diol was either slightly enhanced (2-fold) or unaffected by treatment with the PCB or 3-MC in the tumor-resistant DBA/2N mouse. Our data indicate that neither the patterns of metabolism nor the amount of BaP-7,8-diol formation in the skin are reliable predictors of tumor susceptibility to the PAH in rodent skin.     

The induction of ornithine decarboxylase by tumor promoting phorbol ester analogues in Reuber H35 hepatoma cells

The induction of ornithine decarboxylase activity was studied in a rat hepatoma cell line (Reuber H35) incubated with a group of structurally-related phorbol ester analogues. A single application of 1.6 μM of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to H35 cells caused a dramatic increase in the activity of ornithine decarboxylase. The stimulation of the enzyme activity was rapid but transient, peaking at 4 to 5 hr with a value which was 116-fold greater than control and then declining to the basal level after 8 hr. In addition, the increase in ODC activity was dependent upon the concentration of TPA added to the culture medium and the EC₅₀ was estimated to be about 2.63 × 10^?7 M. Our studies of the effect of various phorbol ester analogues on the H35 ODC activity indicated an apparent correlation between the ability of phorbol ester derivatives to induce ODC activity in the H35 cells and their activity to promote papilloma formation in the mouse skin in that the various derivatives possessed the following relative abilities to increase ODC activity: TPA > PDB > PDA > 4 α-P > 4 α-PDD. Concurrent addition of either actinomycin D or cycloheximide abolished the increase in ODC activity after TPA treatment. Changes of intracellular concentrations of polyamines, particularly putrescine, were in good agreement with the increase in ODC activity in response to TPA: a 10-fold increase in putrescine over the control level was observed at 6 hr. Our data suggest that cultured Reuber H35 hepatoma cells exhibit a marked and specific response to the phorbol ester tumor promoters and may be of great value in studying the biochemical mechanism of ODC induction by these agents.     

Divalent cation requirements for mounting a respiratory burst in response to phorbol diesters by macrophages from SENCAR and C57BL/6 mice

Oxygen radicals are thought to play an important role in the promotion phase of carcinogenesis and the action of phorbol esters. Inflammatory cells are an abundant source of reactive oxygen intermediates (ROI) in the body and release large quantities of ROI when exposed to phorbol esters. Both protein kinase C (PKC), the receptor for phorbol esters, and the NADPH oxidase which generates ROI are Ca2+- and Mg2+-dependent. We investigated the requirements for Ca2+ and Mg2+ of macrophages from strains of mice sensitive and resistant to the promotion of tumors by phorbol esters. Macrophages from SENCAR mice, which are sensitive to phorbol ester promotion, required much lower levels of Ca2+ or Mg2+ to mount a full respiratory burst, as measured by the release of H2O2 in response to phorbol ester stimulation, than macrophages from C57BL/6 mice, which are resistant to promotion by phorbol esters. Conversely, when the particulate stimulus zymosan was used, there was little difference between macrophages from the two strains in requirements for Ca2+ and Mg2+ to release H2O2. Lowering the concentration of either cation in the absence of the other was more inhibitory than in the presence of the other cation. The studies demonstrate that differences in sensitivity to divalent cations by macrophages from these two strains is selective for phorbol ester stimulation and that lower requirements for Ca2+ and Mg2+ for ROI release correlates with sensitivity to the promotion of tumors by phorbol esters.     

Only a subset of 12-O-tetradecanoylphorbol-13-acetate-promoted mouse skin papillomas are promotable by benzoyl peroxide

The two-stage skin carcinogenesis model of initiation and promotion in Carcinogenesis-susceptible (Car-S) mice has been used to investigate the pathways of promotional activity of 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester tumor promoter, and benzoyl peroxide (BzPo), a free radical-generating compound. To test whether distinct populations of 9,10-dimethyl-1,2-benzanthracene (DMBA)-initiated epidermal keratinocytes are responsive to the two promoters, tandem experiments were performed. DMBA-initiated Car-S mice were promoted twice weekly with maximal promoting doses of TPA or BzPo. When the number of papillomas/mouse reached a plateau, promotion in the TPA and BzPo groups was switched to BzPo or TPA, respectively, until achievement of a new plateau. Mice promoted with BzPo developed 11.0 +/- 1.3 papillomas/mouse and subsequent TPA promotion induced 13.8 additional papillomas, for a total of 24.8 +/- 2.1 papillomas/mouse. TPA-promoted mice developed 23.3 +/- 1.1 papillomas/mouse, and subsequent BzPo promotion for 91 days did not promote additional papillomas. Our results show a less than additive tumor response after sequential promotion with BzPo and TPA, or vice versa, indicating that the pathways of promotional activity of TPA and BzPo are interacting. While the final papilloma yield was similar at the end of the two tandem promotion experiments independently of promoter sequence, the percentage of mice developing carcinomas was significantly higher in mice that were promoted with BzPo in the first stage. No significant differences in the frequency and type of c-Ha-ras mutations were observed in TPA- and BzPo-promoted tumors, suggesting that promotion of DMBA-initiated cells by BzPo requires introduction of additional molecular alterations compared to TPA.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

Antibody response against hamster red blood cells (H-RBC) was examined in inbred strains of C57BL/6, AKR, C3H/He, DDD and SL mice, and outbred CF1 mice. 1) There were strain differences in antibody response after a primary intravenous injection of H-RBC. DDD, SL and CF1 mice belonged to high-responder strains, while C57BL/6, AKR and C3H/He to low-responder strains. In the spleens of immunized CF1 and SL, 40 to 70 times as many plaque-forming cells (PFC) as those in C57BL/6 mice were detected. The magnitudes of the response were: CF1 ≒ SL>DDD>>C3H/He ? AKR>C57BL/6. 2) 2-mercaptoethanol resistant (MER) antibody was detected in neither low- nor high-responders after a primary intravenous antigen-injection. 3) After a secondary intravenous antigen-injection, MER antibody was detected in all the SL mice, but only in 30 to 50% of AKR and C57BL/6 mice. 4) A subcutaneous injection of H-RBC in Freund's complete adjuvant (FCA) did not elicit antibody production within 10 days. When mice pre-sensitized 7 days in advance w^Tith H-RBC in FCA were intravenously injected with H-RBC, enhanced antibody production of the primary type was observed in all the mouse strains. 5) In pre-sensitized mice, the extent of the enhancement of antibody production was the highest in low-responder C57BL/6 mice and the lowest in high-responder SL and CF1 strains. Thus, there was no strain difference in antibody titers or the numbers of PFC after the booster.     

Recent studies on the biofunctions and biotransformations of curcumin

Curcumin is a major component of Curcuma species, which is commonly used as a yellow coloring and flavoring agent in foods. Curcumin has shown anti-carcinogenic activity in animals as indicated by its ability to block colon tumor initiation by azoxymethane and skin tumor promotion induced by phorbol ester TPA. Curcumin possesses anti-inflammatory activity and is a potent inhibitor of reactive oxygen-generating enzymes such as lipoxygenase/cyclooxygenase, xanthine dehydrogenase/oxidase and inducible nitric oxide synthase. Curcumin is also a potent inhibitor of protein kinase C, EGF-receptor tyrosine kinase and IkappaB kinase. Subsequently, curcumin inhibits the activation of NFkappaB and the expressions of c-jun, c-fos, c-myc and iNOS. It is proposed that curcumin may suppress tumor promotion through blocking signal transduction pathways in the target cells. Curcumin was first biotransformed to dihydrocurcumin and tetrahydrocurcumin and that these compounds subsequently were converted to monoglucuronide conjugates. These results suggest that curcumin-glucuronide, dihydro-curcumin-glucuronide, tetrahydrocurcumin-glucuronide and tetrahydrocurcumin are major metabolites of curcumin in mice.     

Chalcone tetramers, lophirachalcone and alatachalcone, from Lophira alata as possible anti-tumor promoters.

Two chalcone tetramers were isolated as inhibitors of Epstein-Barr virus (EBV)-activation induced by a tumor promoter, teleocidin B-4, from a medicinal plant in tropical west Africa, Lophira alata (Ochnaceae). One of them was identified as lophirachalcone. The other, named alatachalcone, was new, and the structure was determined by spectral properties. Both compounds also showed potent inhibitory activities against teleocidin B-4-induced inflammation on mouse ear. In an initiation-promotion experiment on mouse skin, alatachalcone (16 nmol) significantly inhibited tumor promotion caused by 12-O-tetradecanoylphorbol-13-acetate (TPA, 1.6 nmol).     

Possible involvement of microfilaments in protein kinase C translocation

We investigated the role of microfilaments in stimulus-induced translocation of protein kinase C (PKC) in polymorphonuclear leukocytes (PMNs) from C57BL/6 mice. Cytochalasin B and dihydrocytochalasin B almost completely inhibited PKC translocation induced by either TPA or Ca2+ ionophore after pretreatment of cells for 30 min. In addition, ML-9, a potent inhibitor of Ca2+/calmodulin-dependent myosin light chain kinase which regulate microfilament contraction, and a calmodulin antagonist W-7, also inhibited PKC translocation. These findings suggest the possibility that microfilaments are involved in the translocation of PKC.     

Age- and strain-related differences in murine spleen cell responses to different activation signals.

The effect of age on the response of splenocytes to activation with anti-CD3 mAb and a combination of anti-CD3 mAb and TPA, as evidenced by interleukin-2 (IL-2) and interleukin-4 (IL-4) production and cell proliferation, was examined in the C57BL/6 and DBA/2 murine strains. Depending on the mode of activation, there were age and strain differences in IL-2 and IL-4 production. With all modes of activation, cells from the old C57BL/6 mice produced less IL-2 than their young counterparts. In DBA/2 mice there was no age-related difference in IL-2 production with anti-CD3 mAb activation alone, whereas when the same cell population was activated with anti-CD3 mAb and TPA an age-associated decrease in IL-2 production occurred. In both strains, there was an age-related increase in IL-4 production with anti-CD3 mAb activation. After addition of TPA, however, there was an age-related decrease in IL-4 production. An age-related decline in the proliferation occurred with all modes of activation in both mouse strains. There were also strain-related differences in proliferation after the addition of forskolin, an inhibitor of Th1-cell function. While forskolin inhibited the proliferation of cells from the young C57BL/6 mice only, in the DBA/2 mice proliferation of cells was inhibited in both age groups. There were no strain-related differences in inhibition by anti-transferrin receptor (TrfR) mAb, although cells from the old mice were slightly more sensitive to this inhibition.     

Effects of diverse intracellular thiol delivery agents on glutathione peroxidase activity, the ratio of reduced/oxidized glutathione, and ornithine decarboxylase induction in isolated mouse epidermal cells treated with 12-O-tetradecanoylphorbol-13-acetate

Since the enhancement of the activity of the natural glutathione (GSH)-dependent antioxidant protective system of the epidermal cells appears to inhibit the oxidative challenge presumably linked to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA), we have compared the effectiveness of diverse intracellular thiol delivery agents as inhibitors of the effects of TPA on GSH metabolism and ornithine decarboxylase (ODC; L-ornithine carboxylase, EC 4.1.1.17) induction in isolated mouse epidermal cells. Here we report at a 2-mM concentration, the monoethyl and monomethyl esters of GSH, N-acetyl-L-cysteine, and L-2-oxothiazolidine-4-carboxylate are all significantly more effective than GSH in inhibiting the sharp decline in the intracellular ratio of reduced GSH/oxidized glutathione (GSSG), the prolonged decrease in GSH peroxidase (GSH:H2O2 oxidoreductase, EC 1.11.1.9) activity, and the induction of ODC activity caused by 1 microM TPA. Moreover, diethyldithiocarbamate prevents totally the initial drop in the GSH/GSSG ratio of TPA-treated cells and is the most potent inhibitor of TPA-decreased GSH peroxidase activity in relation with its remarkable 98% inhibition of TPA-induced ODC activity, suggesting that the potential antitumor-promoting activity of this compound in mouse skin may be far superior to that previously demonstrated by GSH in the initiation-promotion protocol.     

Mice deficient in tumor necrosis factor-alpha are resistant to skin carcinogenesis.

Given the associations between chronic inflammation and epithelial cancer, we studied susceptibility to skin carcinogenesis in mice deficient for the pro-inflammatory cytokine TNF-alpha (refs. 5,6). TNF-alpha(-/-) mice were resistant to development of benign and malignant skin tumors, whether induced by initiation with DMBA and promotion with TPA or by repeated dosing with DMBA. TNF-alpha(-/-) mice developed 5-10% the number of tumors developed by wild-type mice during initiation/promotion and 25% of those in wild-type mice after repeated carcinogen treatment. TNF-alpha could influence tumor and stromal cells during tumor development. The early stages of TPA promotion are characterized by keratinocyte hyperproliferation and inflammation. These were diminished in TNF-alpha(-/-) mice. TNF-alpha was extensively induced in the epidermis, but not the dermis, in TPA-treated wild-type skin, indicating that dermal inflammation is controlled by keratinocyte TNF-alpha production. Deletion of a TNF-alpha inducible chemokine also conferred some resistance to skin tumor development. TNF-alpha has little influence on later stages of carcinogenesis, as tumors in wild-type and TNF-alpha(-/-) mice had similar rates of malignant progression. These data provide evidence that a pro-inflammatory cytokine is required for de novo carcinogenesis and that TNF-alpha is important to the early stages of tumor promotion. Strategies that neutralize TNF-alpha production may be useful in cancer treatment and prevention.     

Effect of genotype and day or night time of testing on mice behavior in the light-dark box and the open-field tests

The light-dark box (LDB) and the open-field (OF) tests are widespread experimental models for studying locomotion and anxiety in laboratory rats and mice. The fact that rodents are nocturnal animals and more active at night raises a critical question of whether behavioral experiments carried out in the light phase are methodologically correct. Parameters of behavior of four mouse strains (C57BL/6J, DBA2/J, AKR/J and CBA/LacJ) in the light-dark box and open-field tests in the light and dark phases were compared. No significant influence of the phase of testing on anxiety in LDB and OF tests was revealed. In the OF test CBA mice showed increased locomotor activity, whereas AKR and C57BL/6 mice showed increased defecation in the dark phase. It was concluded that: 1) the phase of testing is not crucial for the expression of anxiety in LDB and OF; 2) the sensitivity to the phase of testing depends on the genotype; 3) the indices of behavior in the genotypes sensitive to the phase of testing (locomotion in the CBA and defecation in the AKR and C57BL/6 mouse strains) are increased in the dark phase.     

Diurnal variations of food consumption, plasma glucose and plasma insulin concentrations in lean and obese hyperglycaemic mice.

Diurnal variations in food consumption and plasma concentrations of glucose and insulin were determined at 3-hourly intervals in obese hyperglycaemic mice (C57BL/6J ob/ob) and lean mice (C57BL/6J+/+). In lean mice, food consumption and plasma insulin concentrations increased during the light period and were reduced during the dark period, whereas plasma glucose concentrations were maximal at the beginning of the light period and declined to a minimum during the early dark period. In ob/ob mice, the plasma glucose concentration declined temporarily at the beginning of both the light and the dark period and became elevated towards the ends of these periods, but there were no significant diurnal variations of food consumption or plasma insulin concentrations. These observations indicate differences in the diurnal regulation of glucose homeostasis in lean and ob/ob mice.     

Potentiation of tumour promotion by topical application of argemone oil/isolated sanguinarine alkaloid in a model of mouse skin carcinogenesis

Several incidences of adverse effects on human health have been reported in many countries, due to consumption of edible oil adulterated with argemone oil (AO). The clinical manifestation of the disease is commonly referred to as epidemic dropsy. Our prior studies have shown that AO and isolated sanguinarine alkaloid (SANG) possess genotoxic and tumour initiating activity. In this study, the effect of AO/SANG was investigated on the development of tumour formation in mice using 7,12-dimethylbenz (a) anthracene (DMBA) initiated followed by tetradecanoyl phorbol acetate (TPA)-promoted skin tumour protocol. Single application of AO (300 μl) or SANG (4.5 μmol) when used during initiation phase in DMBA/TPA group did not reveal substantial difference in tumourigenic response. However, twice weekly application of AO (100 μl) or SANG (1.5 μmol) during promotion phase (25 weeks) resulted in enhanced tumourigenic response by ≥30% in DMBA/TPA treated group along with significant decrease in dermal tyrosinase (45–49%), histidase (30–32%), superoxide dismutase (53–56%), catalase (41%), GSH reductase (37–40%) and GSH-peroxidase activity (29–33%) compared to control. Furthermore, significant decrease of epidermal GSH (64–66%) content and enhanced formation of lipid peroxides (96–121%) was noticed following AO or SANG treatment during promotion phase to DMBA/TPA induced animals indicating the modified pro-oxidant status in skin. Although dermal biochemical parameters were also altered by AO or SANG when used during initiation phase in DMBA/TPA treated animals, nonetheless, the response in these parameters were relatively more when AO or SANG were used during promotion phase in DMBA/TPA treated animals. These results clearly suggest that AO and SANG have the ability to enhance the tumourigenic response, which may have relevance to its carcinogenic potential.     

Phorbol esters induce synthesis of thromboplastin activity in human monocytes.

12-O-Tetradecanoylphorbol 13-acetate (TPA), phorbol 12,13-diacetate and phorbol 12,13-didecanoate were all potent inducers of thromboplastin activity in human monocytes in vitro, whereas 4 alpha-phorbol 12,13-didecanoate and 4 alpha-phorbol had no such effect. A concomitant increase in titrable apoprotein III antigen was found (apoprotein III is the protein component of thromboplastin). The increase was inhibited by cycloheximide and actinomycin D and partly by alpha-amanitin. The increase of thromboplastin activity was therefore most likely due to synthesis de novo of apoprotein III. The response was approximately halved in the absence of serum or Ca2+. Retinol had a weak inhibitory effect, and retinoic acid was inhibitory only at concentrations that also induced signs of cytotoxicity. TPA caused an initial rise in monocyte cyclic AMP concentration of about 90-120 min duration. No increase in 45Ca2+ influx was induced over 2 h. Good correlation exists between induction of apoprotein III synthesis in monocytes in vitro and mouse skin-tumour promotion in vivo by the various phorbol derivatives. Substances inactive in tumour promotion do not induce the synthesis of apoprotein III. General activating and cytotoxic effects of TPA were monitored by determining release of lysozyme, beta-glucuronidase and lactate dehydrogenase.     

Effect of Nocardia rubra cell-wall skeleton treatment on tumor formation in two-stage chemical carcinogenesis of mouse skin

Summary In two-stage chemical carcinogenesis of mouse skin, Nocardia rubra cell-wall skeleton (N-CWS), a potent immunopotentiator, was injected SC at various times. The dorsal skin of C57BL/6 male mice (about 10 cm²) was painted with 20 g 7,12-dimethylbenz(a)anthracene (DMBA) in 0.1 ml acetone when the animals were 11 weeks old (initiation). Seven weeks later, they were painted with 2.5 g 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in 0.1 ml acetone twice weekly for 30 weeks (promotion). The timing of N-CWS treatment was important. N-CWS treatment before initiation reduced the incidence of skin tumor and the mean number of skin tumors per mouse most effectively. It is speculated that the antitumor activity of N-CWS may be composed of at least two mechanisms, being achieved through the enhancement of immunological surveillance and through changes in the metabolism of chemical carcinogens.     

Mechanisms of cancer chemoprevention by curcumin

Curcumin is a major component of the Curcuma species, which is commonly used as a yellow coloring and flavoring agent in foods. Curcumin has shown anti-carcinogenic activity in animals as indicated by its ability to block colon tumor initiation by azoxymethane and skin tumor promotion induced by phorbol ester TPA. Recently, curcumin has been considered by oncologists as a potential third generation cancer chemopreventive agent, and clinical trials using it have been carried out in several laboratories. Curcumin possesses anti-inflammatory activity and is a potent inhibitor of reactive oxygen-generating enzymes, such as lipoxygenase/cyclooxygenase, xanthine dehydrogenase/oxidase and inducible nitric oxide synthase. Curcumin is also a potent inhibitor of protein kinase C, EGF-receptor tyrosine kinase and IkappaB kinase. In addition, curcumin inhibits the activation of NFkappaB and the expression of c-jun, c-fos, c-myc and iNOS. It is proposed that curcumin may suppress tumor promotion by blocking signal transduction pathways in the target cells. Curcumin was first biotransformed to dihydrocurcumin and tetrahydrocurcumin, and these compounds were subsequently convened into monoglucuronide conjugates. The experimental results suggest that curcumin-glucuronide, dihydrocurcumin-glucuronide, tetrahydrocurcumin-glucuronide and tetrahydrocurcumin are major metabolites of curcumin in mice.     

Immunity to melanoma in mice immunized with transfected allogeneic mouse fibroblasts expressing melanoma-associated antigens

Summary Transfection of genomic DNA from B16 mouse melanoma into LM(TK^–) fibroblasts led to the generation of several clones of transfected cells that strongly expressed B 16 melanoma-associated antigens (MAA). The transfected cells retained their H-2^k markers and served as allogeneic cells with expressive MAA in C57BL/6 mice, syngeneic with the melanoma. The cells were capable of eliciting primary anti-B16 immune responses in vitro in spleen cells from C57BL/6 mice. Immunization of C57BL/6 mice with the transfected cells led to the generation of anti-B16 cytotoxic activity in spleen cells, and C57BL/6 mice immunized with the MAA-positive transfected cells were partially resistant to a lethal challenge with B16 melanoma cells. Under similar conditions, B16 cells were nonimmunogenic. Therefore, transfected allogeneic LM(TK^–) fibroblast cells expressing MAA served as more potent anti-melanoma immunogens than the parental B16 tumor cells themselves.     

Mouse strain differences in the induction of micronuclei by polycyclic aromatic hydrocarbons

The frequency of micronucleated erythrocytes (MNE) in 3 inbred mouse strains and 2 of their hybrids (C57BL/6, BALB/c, DBA/2, BDF1 and CDF1) were examined after polycyclic aromatic hydrocarbons (PAHs; 7,12-dimethylbenz[a]anthracene (DMBA), 3-methylcholanthrene (3-MC), benzo[a]pyrene (BaP), benzo[e]pyrene (BeP) and anthracene (ANT] were injected i.p. PAHs are thought to form active metabolites after being administered to mammals. In mouse strains with inducible PAH activating enzymes, such as C57BL/6 or BALB/c, MNE were significantly induced, as compared to control mice, 48 h after DMBA, BaP, or 3-MC was injected. No increase in the frequency of MNE occurred in the DBA/2 strain which cannot induce the activating enzymes. BeP and ANT did not increase the frequency of MNE in any mouse used. The levels of MNE induction in BDF1 or CDF1 hybrids were similar to those in C57BL/6 or BALB/c. These results support the view that the genetic capacity to metabolize PAHs is strongly associated with micronucleus induction as in the case of PAH carcinogenesis.