Aryl hydrocarbon receptor signaling plays a significant role in mediating benzo[a]pyrene- and cigarette smoke condensate-induced cytogenetic damage in vivo

Stephen D. Dertinger1, Daniel A. Nazarenko1, Allen E. Silverstone2 and Thomas A. Gasiewicz1,3

1 Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, NY 14642, USA and
2 Department of Microbiology and Immunology, State University of New York, Upstate Medical University, Syracuse, NY 13210, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This laboratory has previously reported data suggesting that aryl hydrocarbon receptor (AhR) signaling may have a net potentiating effect on the DNA damaging potential of cigarette smoke. The experiments described in this report extend these studies by testing whether the potent AhR antagonist 3'-methoxy-4'-nitroflavone (3'M4'NF) can modify the in vivo genetic toxicity of benzo[a]pyrene (B[a]P) and the complex mixture of chemicals in cigarette smoke condensate (CSC). Initial experiments were designed to determine 3'M4'NF doses which can antagonize AhR in vivo but which have little effect on constitutive cytochrome P4501A (CYP1A) activity. These experiments took three forms: (i) zoxazolamine paralysis tests, a functional assay of cytochrome P450 CYP1A activity in 3'M4'NF-treated C57Bl/6J mice; (ii) co-treatment of Ahr null allele mice with 150 mg/kg B[a]P plus a range of 3'M4'NF concentrations in order to evaluate the potential of the flavone to interact with non-AhR targets which may affect B[a]P toxicity; (iii) an evaluation of the in vivo AhR antagonist activity of 3'M4'NF using transgenic mice which carry a dioxin-responsive element-regulated lacZ reporter. Once an appropriate dose range was determined, C57Bl/6J mice were challenged with B[a]P or CSC with and without 3'M4'NF co-treatment. Chromosome damage was measured by scoring the frequency of micronuclei in peripheral blood reticulocytes. Data presented herein suggest that 3'M4'NF can protect mice from B[a]P-induced bone marrow cytotoxicity and genotoxicity. Furthermore, CSC-associated genotoxicity was abolished by the flavonoid. These data add support to our hypothesis that AhR signaling has a net potentiating effect on the genetic toxicity and, presumably, carcinogenicity of cigarette smoke.

Abbreviations: AhR, aryl hydrocarbon receptor; B[a]P, benzo[a]pyrene; CSC, cigarette smoke condensate; DMSO, dimethyl sulfoxide; DRE, dioxin-responsive element; ß-gal, ß-galactosidase; 3'M4'NF, 3'-methoxy-4'-nitroflavone; MN-RET, micronucleated reticulocyte; PI, propidium iodide; RET, reticulocyte; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aryl hydrocarbon receptor (AhR) signaling is known to be an important pathway by which enzymes are induced in response to cigarette smoke. Löfroth and Rannug (1) have shown that uncharacterized chemicals contained in cigarette smoke condensate compete with radiolabeled 2,3,7,8-tetracholordibenzo-p-dioxin (TCDD) for receptor binding and Gebremichael et al. (2) have demonstrated that these chemicals are capable of transforming the receptor to an active transcription factor in vitro. Both phase I enzymes (most notably CYP1A1, 1A2 and 1B1) and phase II enzymes are regulated by the AhR and are known to either bioactivate procarcinogens in tobacco smoke or to sequester and detoxify reactive electrophiles (35). It is therefore plausible that AhR-induced enzyme activity has a measurable and biologically significant effect on the carcinogenicity of cigarette smoke. Whether it mediates a net increase or decrease in tumorigenicity is difficult to predict. However, some indirect evidence suggests that AhR signaling may result in a net potentiation of cigarette smoke carcinogenicity. For instance, studies have been published which link high aryl hydrocarbon hydroxylase inducibility (a function of CYP1A activity) with increased cancer risk (6,7). Furthermore, this laboratory has demonstrated that mutant AhR cell lines and Ahr null allele mice are relatively resistant to chromosome damage caused by cigarette smoke condensate (CSC) compared with wild-type controls (8). Additionally, a recent report observed that while benzo[a]pyrene (B[a]P), an important constituent of cigarette smoke, induced tumors in wild-type mice, no tumors were observed in Ahr null allele animals (9). Additional evidence is supplied by a cohort of TCDD-exposed individuals (male factory workers). Increased total cancer deaths were observed in individuals who actively smoked as exposure to TCDD increased. High TCDD exposure levels were not observed to enhance total cancer deaths in non-smokers or ex-smokers (10).

The studies outlined above suggest that induction of the AhR gene battery may favor bioactivation of tobacco smoke mutagens relative to detoxification processes. Even so, it is clear that no direct evidence is available which describes the consequence of AhR-mediated signal transduction on the genotoxic and carcinogenic potential of cigarette smoke. For instance, even experiments with Ahr null allele mice do not provide formal proof that under physiologically relevant circumstances AhR signaling significantly affects cigarette smoke toxicity. Since knockout animals express a low basal level of CYP1A2 activity relative to controls, reduced by ~90% (11), it is not clear whether differential sensitivity to cigarette carcinogens are related to altered constitutive enzyme expression profiles or the lack of AhR-dependent induction processes. For the work described herein we utilized a chemical AhR antagonist, which could be used to temporally block signaling, to discriminate between these two possibilities.

Results from cell-free and cell culture studies indicate that 3'-methoxy-4'-nitroflavone (3'M4'NF) is a potent AhR antagonist with little or no agonist activity (1214). Furthermore, we have demonstrated that 3'M4'NF is able to modify the in vivo genotoxicity of B[a]P (15). However, these same in vivo data also suggested that at the single high concentration of antagonist tested significant non-AhR targets are affected. A likely secondary target is inhibition of CYP1A enzyme activity (15). Thus, these studies did not clearly determine whether the attenuation of B[a]P genotoxicity was due to inhibition of the AhR signaling pathway or CYP1A activity. The experiments described in the current report were performed to test whether 3'M4'NF can attenuate B[a]P- and CSC-induced genotoxicity at concentrations which do not significantly affect basal CYP1A activity.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents
Absolute methanol was purchased from Fisher Scientific (Springfield, NJ). Sodium heparin, propidium iodide (PI), RNase A, B[a]P, Triton X-100, phenylmethylsulfonyl fluoride, leupeptin and dithiothreitol were obtained from Sigma (St Louis, MO). 3'M4'NF was synthesized and purified (>98%) by procedures previously described (16). TCDD was purchased from Cambridge Isotopes (Cambridge, MA). Anti-CD71–FITC was purchased from BioDesign (clone no. R17217.1.4; Kennebunk, ME). Fixed malaria-infected mouse erythrocytes were purchased from Litron Laboratories (Rochester, NY). CSC was generated from University of Kentucky reference cigarettes, type 1R4F, as described previously (8). For injection, B[a]P, zoxazolamine and TCDD were prepared in olive oil. CSC stock [100 mg/ml dimethyl sulfoxide (DMSO)] was diluted in olive oil to provide 2.5 mg/ml for injection. In early studies 3'M4'NF was prepared in DMSO. We switched to olive oil after it became apparent that precipitates periodically formed in the intraperitoneal cavity of treated mice when DMSO was the vehicle.

Animals
Male wild-type C57Bl/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME). Male Ahr null allele mice (Ahr–/–, exon 1 targeted) were bred at SUNY (Upstate Medical University, Syracuse, NY). The founder mice of this colony were originally developed by and received from P.Fernandez-Salguero and F.Gonzalez (National Cancer Institute, Bethesda, MD) (17). Transgenic mice (DRE–lacZ) used in these studies have been described previously (18). Briefly, the DREs in this reporter are bona fide sites from the CYP1A1 promoter. The transgenic mice were developed by microinjecting DNA containing the DRE–lacZ construct into pronuclei of recently fertilized C57Bl/6JxSJL ova. A PCR screening method was used to identify heterogzygous, transgene-positive mice (18). Note that at the time of the current experiments the transgene-positive line had been backcrossed to the C57Bl/6J background more than nine times. Purina Mills Rodent Chow 5001 and water were available to mice ad libitum. For all experiments mice were allowed to acclimatize for at least 1 week.

Dose range finding experiment: zoxazolamine paralysis time
The zoxazolamine paralysis test (19) was used to evaluate 3'M4'NF doses for their ability to inhibit CYP1A1/2 activity in vivo. Zoxazolamine is a potent muscle relaxant which is metabolically inactivated by CYP1A1/2 enzyme activity (and to a lesser extent CYP2E1). In addition to non-invasively phenotyping mice as AhR-responsive and non-responsive (20), it has also been used as a functional test to evaluate chemicals for their ability to inhibit CYP1A1/2 enzyme activity in vivo (21,22). Three independent experiments were performed with male C57Bl/6J mice, 6–9 weeks old. Mice were treated with 0, 0.2, 2, 20 or 40 mg/kg 3'M4'NF i.p. Approximately 30 min after the 3'M4'NF injections mice were treated i.p. with zoxazolamine (125–150 mg/kg). Paralysis times were determined as the length of time required for mice to regain their righting reflex. Data are expressed as relative zoxazolamine paralysis times, normalized to the average paralysis time observed for the vehicle-treated animals in the same experiment (set at 1.0). Mean fold induction for each 3'M4'NF exposure group was compared with the vehicle control group by unpaired, two-tailed t-tests (StatView v.4.5). P < 0.05 was considered a significant effect.

Dose range finding experiment: Ahr null allele mice and splenic lesions
Previous experiments have indicated that a high 3'M4'NF concentration can enhance the systemic toxicity of B[a]P (14). The spleen was found to be a particularly sensitive target of the synergistic toxicity observed when mice were co-treated with B[a]P and 3'M4'NF. Interestingly, Ahr null allele mice were found to exhibit the same types of splenic lesions as wild-type animals. This indicates that the synergistic toxicity observed is mediated by the ability of the flavonoid to interact with a non-AhR target(s). We used this same system to evaluate the ability of the flavonoid to affect secondary (non-AhR) targets.

For this experiment male Ahr–/– mice were treated i.p. with 150 mg/kg B[a]P. The flavonoid 3'M4'NF was administered as a split dose in an effort to maintain plasma levels. Specifically, 3'M4'NF was administered i.p. at 0, 0.2, 2 or 20 mg/kg/injection 4 h prior to, concurrently with and 4 h after B[a]P. Forty-eight hours after the B[a]P bolus, spleens were collected and fixed with 10% neutral buffered formalin. The tissues were sectioned and stained with hematoxylin and eosin. Lesions to the spleen were quantified by Image-Pro Plus software (v.3.0; Media Cybernetics, Silver Spring, MD). Specifically, images of lymphocyte follicles were digitized with a Dell computer (400 MHz) and a Hitachi KP-D50 color camera attached to an Olympus BH-2 microscope. The Image-Pro counting module was used to quantify pycnotic lymphocytes, i.e. those lymphocytes exhibiting condensed and deeply stained chromatin. The best scoring accuracy was obtained by adjusting the color range (red) upper threshold and the lower thresholds of the area, density and heterogeneity parameters. For each mouse four fields (four separate follicles) were analyzed. The average numbers of pycnotic cells per field were compared between treatment groups by ANOVA (StatView v.4.5). P < 0.05 was considered a significant effect.

Dose range finding experiment: in vivo AhR antagonist activity of 3'M4'NF
To investigate the ability of 3'M4'NF to antagonize the AhR in vivo, we utilized male mice heterozygous for a DRE–lacZ transgene (aged 7–12 weeks). These mice express ß-galactosidase (ß-gal) in response to AhR agonist treatment (18). Animals were divided into four treatment groups and were injected i.p. with vehicle, 15 µg/kg TCDD or 15 µg/kg TCDD plus 0.6 or 6 mg/kg 3'M4'NF. Note that 3'M4'NF was administered as a split dose: one treatment 4 h prior to TCDD, a second concurrently with TCDD and a third 4 h after TCDD (i.e. 0, 0.2 or 2 mg/kg/injection). The vehicle control group comprised three mice and each of the other three treatment groups comprised four mice. Sixteen hours after TCDD exposure mice were killed by CO2 asphyxiation and liver and lung sections were collected for ß-gal measurements. Specifically, tissue sections (~10x10x5 mm) were finely minced and transferred to hypotonic ammonium chloride solution (ACK buffer) to lyse red blood cells. Minced tissue was subsequently rinsed in phosphate-buffered saline and transferred to 1 ml of tissue lysis solution containing 100 mM potassium phosphate, pH 7.8, and 0.2% Triton X-100, supplemented with phenylmethylsulfonyl fluoride (0.2 mM), leupeptin (1 µg/ml) and dithiothreitol (1 mM) immediately before use. Tissue was homogenized between the ends of frosted glass slides and transferred to microcentrifuge tubes. All extracts were centrifuged at 12 000 g for 5 min and supernatants were held at 48°C for 60 min to inactivate endogenous ß-gal (23). Samples were stored at –20°C until analysis. Transgene-encoded bacterial ß-gal activity was measured with a commercial luminescence assay system (Galacto Light Plus; Tropix). Activities were normalized to protein concentration (NanoOrange protein quantitation kit; Molecular Probes). The effect of treatment on TCDD-induced ß-gal activity was evaluated by ANOVA. P < 0.05 was considered a significant effect.

Effect of 3'M4'NF on B[a]P and CSC genotoxicity
Twenty wild-type C57Bl/6J mice, age 7–8 weeks, were randomly assigned to five treatment groups: vehicle control and B[a]P plus 0, 0.2, 2 or 20 mg/kg/injection 3'M4'NF. B[a]P was delivered as a bolus of 150 mg/kg i.p. and 3'M4'NF was administered according to our standard split dose schedule, 4 h prior to, concurrently with and 4 h after the B[a]P challenge. Fifty-four hours post-B[a]P treatment peripheral blood samples were collected from the tail vein of each mouse. Mice were killed by CO2 overdose and spleens were weighed and fixed in 10% neutral buffered formalin. Blood cells were collected and processed for flow cytometric analysis of genotoxicity as described below. Spleens were embedded, sectioned and stained for analysis of pycnotic lymphocytes as described.

In a second experiment, 15 wild-type C57Bl/6J mice (age 7–8 weeks) were divided among three treatment groups: vehicle control, CSC and CSC plus 3'M4'NF. CSC was administered at 24 h intervals for 3 consecutive days, 25 mg/kg/injection. 3'M4'NF was administered according to the split dose schedule described above, 4 h prior to, concurrently with and 4 h after each CSC challenge. The concentration of 3'M4'NF that we chose was the lowest in vivo concentration that was studied in the DRE–lacZ and B[a]P co-exposure models, 0.2 mg/kg/injection (i.e. 0.6 mg/kg/day). Twenty-four hours after the last CSC treatment peripheral blood and spleen tissues were prepared as described above.

Genotoxicity measurements
The frequency of micronuclei in peripheral blood reticulocytes was measured to index cytogenetic damage. Micronuclei arise as a consequence of clastogenic or aneugenic action and this end-point is widely used to evaluate the carcinogenic potential of test agents (24,25). For the current study a high throughput flow cytometric technique was used to quantify the incidence of micronuclei. Heparinized blood from each animal was fixed with ultra-cold methanol and prepared for analysis as described previously (26). Briefly, cells were collected by centrifugation and incubated in flow cytometry tubes with 80 µl of working RNase solution plus anti-CD71–FITC (1 mg RNase and 10 µl stock anti-CD71–FITC per ml bicarbonate-buffered saline). Cells were maintained at 4°C for 30 min, moved to room temperature for 30 min and then kept on ice until analysis (the same day). Immediately before each sample was analyzed 1 ml of ice-cold PI solution was added (1.25 µg/ml PI in bicarbonate-buffered saline).

Flow cytometric analysis was accomplished with a Becton Dickinson FacStarPlus tuned to provide 488 nm excitation. The FL1 detector was configured with a bandpass filter to collect emissions between 520 and 560 nm. PI-associated fluorescence was collected in the FL2 channel with a 580 long pass filter. Before scoring samples the instrument was calibrated with anti-CD71 and PI stained malaria-infected erythrocytes, which guide instrument settings including photomultiplier tube voltages and compensation values (27). After calibration each sample was scored for reticulocyte (RET) and micronucleated reticulocyte (MN-RET) frequency upon acquisition of 20 000 total RETs (CellQuest v.3.0.1 software; Becton Dickinson). ANOVA was performed to evaluate the effect of treatment on mean RET and MN-RET frequencies. P < 0.05 was considered a significant effect.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inhibition of CYP1A activity in vivo
Initial experiments were designed to define in vivo doses of 3'M4'NF that are relatively specific for AhR antagonist activity and included a functional assay of CYP1A inhibition, the zoxazolamine paralysis test. Paralysis times, which are inversely related to CYP1A activity, are presented in Table IGo. Pre-treatment of mice with 3'M4'NF at 20 and 40 mg/kg significantly increased mean paralysis time. Conversely, no measurable effect was observed at 0.2 and 2 mg/kg 3'M4'NF relative to vehicle controls. These results are consistent with previous observations that this flavonoid inhibits EROD activity in vitro (12,15) and suggest that low concentrations of 3'M4'NF have no significant effect on the basal level of CYP1A1/2 enzyme activity in vivo.


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Table I. Effect of 3'M4'NF on zoxazolamine paralysis time
 
Ahr null allele mice and splenic lesions
In a further effort to assess 3'M4'NF for its impact on non-AhR targets, male Ahr null allele mice were treated with vehicle, 150 mg/kg B[a]P or 150 mg/kg B[a]P plus 0.2, 2 or 20 mg/kg/injection 3'M4'NF (three injections). We utilized Ahr null allele mice in this experiment because they are more sensitive to the combined toxicity of B[a]P and 3'M4'NF (8). Furthermore, the toxicity evident in these animals would by definition be related to non-AhR targets. After 48 h spleens were collected, fixed, sectioned and stained with hematoxylin and eosin. Consistent with previous experiments (15), mice co-treated with B[a]P and 20 mg/kg/injection 3'M4'NF (three injections) exhibited small spleens with reduced red pulp. Additionally, the incidence of pycnotic spleen lymphocytes was highly elevated (Figure 1Go). The number of pycnotic lymphocytes per high power field were scored with Image-Pro Plus software and the results are presented in Figure 2Go. A marked increase in spleen lesions was found in the B[a]P plus high 3'M4'NF treatment group. On the other hand, this toxicity was not evident at lower concentrations of flavonoid. Importantly, these results correlate with data obtained with zoxazolamine paralysis times. While 20 mg/kg 3'M4'NF inhibits CYP1A activity, the lower concentrations of 0.2 and 2 mg/kg do not result in a measurable effect. These data suggest that the splenic toxicity of B[a]P plus 3'M4'NF is a result of inhibition of constitutive CYP1A activity resulting in either a change in deposition, clearance and/or bioactivation of the procarcinogen.



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Fig. 1. Section of spleen from a male Ahr–/– mouse treated with 150 mg/kg B[a]P (A) or co-treated with 150 mg/kg B[a]P plus 20 mg/kg/injection 3'M4'NF, with three injections (B). Note the high percentage of lymphocytes which exhibit condensed, pycnotic chromatin in B. This is a lesion which is not observed in mice treated with 3'M4'NF alone or even B[a]P alone at this relatively well-tolerated dose.

 


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Fig. 2. The mean number of pycnotic lymphocytes per high magnification field are graphed for each of five treatment groups. Standard deviation bars are included. B[a]P, 150 mg/kg; 0.2 mg 3'M4'NF, 0.2 mg/kg/injection, three injections; 2 mg 3'M4'NF, 2 mg/kg/injection, three injections; 20 mg 3'M4'NF, 20 mg/kg/injection, three injections. Scoring was of four randomly chosen follicles per mouse. The Image-Pro count module was used to quantify the number of pycnotic cells per field. The effect of treatment on the mean number of pycnotic cells per field was evaluated by ANOVA and Fisher's PLSD test. The asterisk denotes a statistically significant difference in mean number of pycnotic cells per field compared with each of the other four treatment groups, P < 0.05.

 
In vivo AhR antagonist activity of 3'M4'NF
Data from the zoxazolamine and the Ahr null allele mouse experiments suggest that injections of 3'M4'NF in the range 0.2–2 mg/kg may minimally affect non-AhR target(s), and CYP1A activity in particular. To test the ability of 3'M4'NF to block AhR-mediated signaling in vivo, these concentrations were evaluated in a transgenic mouse model that responds to AhR agonists by the induction of lacZ. Heterozygous mice were treated with 15 µg/kg TCDD and 0, 0.2 or 2 mg/kg 3'M4'NF (three injections). After 16 h liver and lung tissues were processed for ß-gal measurements. These enzyme activities, normalized to protein concentration, are presented in Figure 3Go.



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Fig. 3. Mean ß-gal activity of liver and lung tissue (from DRE–lacZ transgenic mice) is graphed for each of four treatment groups. Standard deviation bars are included. ß-Gal activity induced by 15 µg/kg TCDD is antagonized in a dose-dependent fashion by 3'M4'NF (0.2 mg 3'M4'NF, 0.2 mg/kg/injection, three injections; 2 mg 3'M4'NF, 2 mg/kg/injection, three injections). ANOVA results comparing ß-gal activity among groups treated with TCDD: liver, P = 0.140; lung, P = 0.046.

 
As expected, mean ß-gal activity was markedly enhanced in the TCDD treatment group. Administration of the split dose of 3'M4'NF reduced this activity. Although statistical significance was not achieved in the liver, a clear dose-dependent reduction was observed. In fact, at a total dose of 6 mg/kg 3'M4'NF we saw a nearly complete inhibition of TCDD-induced activity. As observed in other experiments (data not shown), the liver was somewhat variable in terms of TCDD-induced ß-gal activity, and this variation is likely responsible for the lack of statistical significance in this compartment. One possible source of variation is the relatively high hemoglobin content associated with the liver extracts (we have observed that hemoglobin interferes with chemiluminescence-based ß-gal assays; (28). TCDD-induced ß-gal activity was less variable in the lung. Although 3'M4'NF was not shown to reduce transgene expression to as great an extent as in the liver, the dose-dependent reduction was statistically significant (ANOVA, P = 0.046). These data directly demonstrate the in vivo AhR antagonist activity of the flavonoid 3'M4'NF in the concentration range 0.2–2 mg/kg/injection.

Effect of 3'M4'NF on B[a]P and CSC genotoxicity
With a better understanding of the 3'M4'NF concentrations which are more specific for AhR antagonism compared with the one high dose that we initially studied, we were in a better position to evaluate the influence of AhR-dependent signal transduction on the genotoxicity of cigarette smoke mutagens. In one experiment five male C57Bl/6J mice per group were treated with vehicle, B[a]P alone or B[a]P plus 0.6 or 6 mg/kg 3'M4'NF (0.2 or 2 mg/kg, three injections).

Observations over the 54 h treatment period suggest that at the concentrations tested 3'M4'NF did not potentiate B[a]P toxicity. The animals did not exhibit any obvious signs of distress, i.e. the mice were able to maintain caloric intake, retain their righting reflex, etc. Furthermore, spleen weight was not affected by B[a]P and 3'M4'NF nor was the frequency of pycnotic lymphocytes enhanced (data not shown). These results are consistent with data from the experiment with the highly sensitive Ahr null allele mice in which 20 but not 2 or 0.2 mg/kg/injection 3'M4'NF potentiated certain B[a]P lesions. Collectively, these data suggest that at the concentrations tested 3'M4'NF does not severely affect that non-AhR target(s) which is responsible for the synergistic toxicity that can result when high concentrations of 3'M4'NF are combined with B[a]P.

The effect of 3'M4'NF treatment on B[a]P genetic toxicity is presented in Figure 4Go. As these data clearly indicate, the flavonoid is capable of attenuating B[a]P-induced chromosome damage. Furthermore, B[a]P-induced stem cell toxicity, as measured by reticulocyte frequency, was abolished by 3'M4'NF co-treatment. Both of these protecting effects were dose dependent and found to be statistically significant (P < 0.05).



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Fig. 4. Attenuation of B[a]P-induced toxicity by 3'M4'NF. Mean RET and MN-RET frequencies are shown. Five animals per group; error bars indicate 1 SD. B[a]P, 150 mg/kg; 0.2 mg 3'M4'NF, 0.2 mg/kg/injection, three injections; 2 mg 3'M4'NF, 2 mg/kg/injection, three injections. Two tailed t-test results: *P < 0.0001, MN-RET (%) significantly different to solvent control; **P < 0.01, RET (%) significantly different to solvent control; {dagger}P < 0.01, MN-RET (%) significantly different to B[a]P only group; {dagger}{dagger}P < 0.01, RET (%) significantly different to B[a]P only group; #P < 0.001, MN-RET (%) significantly different to B[a]P only group; ##P < 0.005, RET (%) significantly different to B[a]P only group.

 
This experiment was followed by a study in which C57Bl/6J mice were treated with CSC with and without 3'M4'NF co-treatment. By substituting the complex mixture of chemicals in CSC for B[a]P we hoped to more realistically model exposure to tobacco smoke. We have previously found that a bolus of 25 mg/kg CSC does not induce a measurable level of DNA damage as measured by micronucleus formation, while a repeat dosing schedule does (data not shown). Therefore, these experiments necessarily involved repeat dosing: CSC administered once a day for 3 days. As with previous experiments, we bracketed each CSC dose with 3'M4'NF injections.

The animals were closely monitored for acute signs of toxicity. No outward signs of distress were evident. Furthermore, spleen weights and the frequency of pycnotic lymphocytes in the spleen compartment were not affected by the CSC or CSC plus 3'M4'NF treatments. Stem cell toxicity and genotoxicity data are provided in Table IIGo. While there have been reports that high concentrations of CSC can arrest mitosis of cultured cells (29), we did not observe any change in erythropoiesis function among treatment groups, i.e. no significant difference was observed in RET frequencies. On the other hand, the incidence of MN-RET was significantly elevated in the CSC treatment group over the vehicle control. This low level of clastogenic activity associated with CSC was abrogated by the AhR antagonist 3'M4'NF.


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Table II. Effect of 3'M4'NF on CSC toxicity
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cigarette smoke contains compounds which can transform the cytosolic AhR to an active transcription factor. However, the consequences of AhR-mediated enzyme induction on the toxicity of cigarette smoke are not clear. For instance, with regard to DNA damaging capacity it is possible that constitutive cytochrome P450 activities dictate the potency of the mixture or that other biochemical processes (e.g. DNA repair) obscure any differences resulting from AhR-mediated gene transcription. Alternately, AhR-dependent events may significantly influence cigarette smoke-induced DNA damage. This information is potentially important, for it may provide the framework for identifying highly sensitive individuals and may represent an approach for attenuating the toxicity of similar types of exposures.

One of the extreme difficulties associated with the study of host and environmental factors which influence cigarette smoke-induced toxicity and carcinogenicity is that exposure to tobacco smoke is so complex. Approximately 4000 chemicals are found in tobacco smoke and over 50 of these compounds are recognized as known or probable carcinogens (30). Given the multitude of bioactive compounds in smoke, it is clear that investigations hoping to supply new, mechanistic information must employ very refined and specific methods. In studying the influence of AhR-mediated events the availability of AhR mutant cells lines, DRE–reporter cells and animals and Ahr null allele mice are important and necessary tools. Even so, it is clear that these tools alone cannot always discriminate between the effects that agonist-induced up-regulation of DRE-controlled genes have on end-points of toxicity from the consequences that a complete lack of AhR has on setting certain basal enzyme levels such as CYP1A2 (11). For the experiments described herein we successfully employed 3'M4'NF to isolate and study the influence that enzyme induction through AhR signaling has on the genotoxicity of B[a]P and CSC in vivo. This information is potentially important, because it is presumably more relevant to actual exposure conditions compared with models in which signaling has been permanently ablated and constitutive metabolism profiles have therefore been markedly altered.

Collectively, the data presented herein support results from earlier experiments with AhR mutant cell lines and Ahr null allele mice which suggest that AhR signaling may have a net potentiating effect on the toxicity, especially the genotoxicity, of cigarette smoke constituents. Experimentally, we have demonstrated that the flavone derivative 3'M4'NF can be used at low concentrations which do not significantly affect the constitutive in vivo activity of P450 CYP1A but which retain an ability to antagonize AhR signaling. In this same concentration range 3'M4'NF was found to protect mice from B[a]P- and CSC-induced cytogenetic damage. These results therefore strongly suggest that AhR signaling plays an important role in mediating the genetic toxicity of cigarette smoke. A more complete understanding of the significance of AhR signaling on cigarette smoke genotoxicity, and ultimately tumorigenicity, will come from additional studies which incorporate other end-points of DNA damage. Furthermore, if these additional genetic toxicity measurements are obtained in different tissue compartments, for example the lung, added relevance may be achieved.

Although we report here that 3'M4'NF is an effective AhR antagonist in vivo and efficacy can be achieved with some degree of specificity, it is important to realize that other potential biological activities of the synthetic flavonoid have not been fully characterized. For instance, bioflavonoids and various flavone derivatives have been found to exhibit antioxidant activity (31), induce apoptosis (32), suppress cell cycle progression (33) and inhibit mitogen-activated protein kinase kinase (34). Therefore, experiments designed to temporally and specifically block AhR signal transduction may benefit from further characterization of 3'M4'NF and other flavones for other (non-AhR) biological activities which may be partially responsible for the protective effects reported here and elsewhere (3540).


    Notes
 
3 To whom correspondence should be addressed Email: tom_gasiewicz{at}urmc.rochester.edu Back


    Acknowledgments
 
The authors wish to thank the technicians, students and post-doctoral staff in the Gasiewicz laboratory for their critical review of this manuscript. The authors especially want to thank Andrew Kende for synthesizing 3'M4'NF, J. Jeffrey Willey for generating and initially characterizing DRE–lacZ transgenic animals, Frank Gonzalez and Pedro Fernandez-Salguero for supplying the founder Ahr null allele mice, Denise Hahn, Nancy Fiore and Cheryl Hurley for the expert animal care they provided and Litron Laboratories for use of their flow cytometer. This work was supported by NIH Grants ES02515, ES09430, ES04862, ES09702 and ES07216, Center Grant ES01247 and Training Grant ES07026.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Löfroth,G. and Rannug,A. (1988) Ah receptor ligands in tobacco smoke. Toxicol. Lett., 42, 131–136.[ISI][Medline]
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Received August 15, 2000; revised October 17, 2000; accepted October 20, 2000.