The identification and repair of DNA adducts induced by waterborne benzo[a]pyrene in developing Xenopus laevis larvae

We report on the formation and subsequent repair of benzo[a]pyrene-inducedDNA adducts in Xenopus laevis larvae in vivo, as monitored by³²P-post labelling. In vivo benzo[a]pyrene is metabolized bythe cytochrome P450 family of enzymes to metabolites, of whichthe 7,8-diol-9,10-epoxides have been implicated as causing potentiallytumourigenic lesions. Larvae were exposed to waterborne benzo[a]pyrene(0.01, 0.05 and 0.1 mg/1) for 24 h at stages 38, 45 and 50 ofdevelopment (24 h, 5 days and 2 weeks post-hatching, respectively)and allowed to recover for up to 6 days. A wide range of adductlesions were observed at stage 50, three of which were observedat all stages investigated. Adduct repair was biphasic, withan initial rapid repair over the first 24 h post exposure, followedby a much slower decline, resulting in persistence of adductsfor at least 6 days post exposure. The individual lesions wererepaired at different rates, with some being almost completelyrepaired after 6 days recovery, whereas one of the main adductsshowed restricted repair at stage 50 and another no repair atall. Identification of some adducts has been achieved, by theinclusion of isomeric standards of (+)- or (–)-anti-benzo[a]pyrenediol epoxide reacted with deoxyguanosine and adenosine 3'-monophosphatesprepared in vitro. The non-repairable lesion at stage 50 hasbeen shown to be the (+)-trans-anti-benzo[a]pyrene diol epoxide-N²-guanineadduct. This adduct was observed at all stages, but was onlymaximally repaired at stages 38 and 45. ⁵To whom correspondence should be addressed     

Moderate inhibition of mutagenicity and carcinogenicity of benzo[a]pyrene, 1,6-dinitropyrene and 3,9-dinitrofluoranthene by Chinese medicinal herbs

The activity of six Chinese medicinal herbs against the environmentalmutagens and carcinogens benzo[a]pyrene (B[a]P), 1,6-dinitropyrene(1,6-diNP) and 3,9-dinitrofluoranthene (3,9-diNF) was determined.Samples of Prunella spica, Rheum palmatum, Polygonum multiflorum,Agrimonia pilosa, Ephedra sinica and Teitoutou were tested inan in vitro system. Antimutagenic activity against B[a]P wasmarked in the presence of extracts (boiled for 2 h in a waterbath) whereas that against 1,6-diNP and 3,9-diNF varied from20 to 86%. The differences in inhibition might be due to inactivationof metabolic enzymes. An extract of P.multiflorum was dividedinto ether, ethyl acetate and water soluble fractions, whichwere tested for antimutagenic activity against B[a]P. The antimutagenicaction of the ethyl acetate soluble fraction was substantialand dose-dependent. Tannins and related compounds were the majorcomponents of the extract, of which epigallocatechin, epigallocatechingallate, epicatechin gallate and tannic acid strongly inhibitedthe mutagenicity of B[a]P (2.5 µg/plate) in Salmonellatyphimurium TA98 with S9 mix. To confirm the results of thein vitro test system, F344/DuCrj male rats were given a subcutaneousinjection of B[a]P. Thereafter, they received water extractsof the six Chinese medicinal herbs for 50 weeks and were examinedfor tumors. The P.multiflorum extract significantly reducedthe tumor incidence. ³To whom correspondence should be addressed     

Induction of SCE by indirect mutagens in cultured rat hepatoma cells and in Chinese hamster V79 cells co-cultivated with hepatocyte primary cultures

The continuous rat hepatoma cell line H4IIEC3/G^– and rathepatocyte primary cultures (hpc) were compared with regardto their capacity to metabolize structurally different promutagens.The sensitivities of both activation systems were evaluatedby comparing the induction of SCE in H4IIEC3/G^– cellsthemselves with that in V79 cells co-cultured with hpc. Of thesix chemicals tested, aflatoxui B₁ (AFB1), cyclo-phosphamide,dimethylnitrosamine and nitrosomorpholine (NM) were shown tobe inducers of SCE in H4HEC3/G^– cells as well as in V79cells with hepatocyte activation. 7,12-Dimethylbenzanthracenegave positive responses in hpc/V79 co-cultures but not in H4IIEC3/G^–cells whereas benzo[a]pyrene was negative in both systems. Theseresults suggest that H4IIEC3/G^– cells retain metabolicactivities to convert different indirect mutagens into theiractive forms and clearly indicate the presence of liver specificcytochrome P-450-dependent mono-oxygenases. However, freshlyisolated hepatocytes are more efficient in metabolizing thetest compounds. Although hpc provide only external activation,the V79 cells system appears to be more sensitive for the detectionof promutagens.     

Suppressive effects of methyl methacrylate on the mutagenicity and DNA adduct formation induced by 1-nitropyrene and benzo[a]pyrene

Methyl methacrylate (MMA) is widely used as a cement in dentistry,orthopaedic surgery and ophthalmology. Studies based on short-termgenotoxicity tests have produced conflicting results in thelast two decades. In the present study, the effects of MMA onthe mutagenicity of 1-nitropyrene (1-NP) and benzo[a]pyrene(B[a]P) were evaluated with the Salmonella typhimurium TA98strain in the absence and presence of S9 mix. The direct-actingmutagenicity of 1-NP was markedly decreased by MMA in a dose-dependentmanner. However, a low inhibitory effect of MMA on the metabolic-actingmutagenicity of B[a]P was observed. MMA did not show mutagenicitywithin the concentrations of 4.7–37.6 µM eitherwith or without S9 mix. The inhibitory effect of MMA was notdue to its cytotoxicity because very low and/or no cytotoxicityof MMA to S.typhimurium TA98 was observed. To confirm the antimutagenicityof MMA against 1-NP and B[a]P, a ³²P-postlabelling method wasused to determine whether MMA modified DNA adduct formationproduced by both compounds in calf thymus DNA. MMA inhibitsthe formation of 1-NP- and B[a]P-DNA adducts in a dose-dependentmanner. The DNA adduct of 1-NP reduced by MMA was greater thanthat of B[a]P. Thus, we suggested that MMA was possibly actingas an inhibitor of chemical carcinogenesis. ¹To whom correspondence should be addressed     

Benzo[a]pyrene site of contact mutagenicity in skin of MutaTMMouse

Benzo[a]pyrene (BP) has been investigated for the ability toinduce mutation at the site of contact. Skin painting treatmentswith BP caused a time-dependant and statistically significantincrease in mutation frequency (MF) in the treated areas ofskin. The MF exceeded 500x10^–6 21 days after either 1x25or 5x5 µg treatments. Increases to >700x10^–6were seen when doses of 1x50 or 5x10 µg were used. Neitherthe liver nor the lung showed any increase in mutation frequencyafter 21 days in animals exposed to the 5x10 µg treatmentregime. It is concluded that following topical administration,BP is able to induce mutation in the skin at the site of application,but not in either the lung or liver. ³To whom correspondence should be addressed. Tel: 01423 500011; Fax: 01423 500803; Email: stephen.dean{at}covance.com     

DNA repair induction by UV irradiation and various mutagens requiring metabolic activation in rat hepatocytes maintained in culture for several days

Freshly isolated rat hepatocytes cultivated in Williams MediumE (WME) quickly lose their cytochrome P-450-dependent metabolicactivities. A significantly better maintenance of cytochromeP-450 was achieved by supplementing WME with hormones and nutrientsand 2% DMSO and 10% fetal calf serum. After 72-h cultivationin this medium the cytochrome P-450 content was still 72% ofthe amount measured in freshly isolated cells. Over the sameperiod of time the activity of 7-ethoxycoumarin-O-deethylasedropped to 55% of its initial value. DNA repair induction byUV irradiation, hydrogen peroxide, benzo[a]pyrene and 2-acetylaminofluorenewas measured. A clear dose-dependent increase of the incorporationof [³H]thymidine was found with all agents tested, regardlessof whether the treatment of the hepatocytes began 4 or 52 hafter cell isolation. In the case of UV light and hydrogen peroxidethe maximum effects were slightly lower after prolonged cultivation,whereas little change or even a slight increase was found withbenzo[a]pyrene and 2-acetylaminofluorene. These results indicatedthat cell cultivation led to a slight loss of DNA repair activitybut did not, despite the loss of cytochrome P-450, cause a decreasein DNA-damaging intermediates produced from the two indirectmutagens. The absolute amounts of [³H]thymidine incorporatedby the solvent control cells of the 52-h groups was consistentlyfound to be twice the level measured in the corresponding 4-hcontrol groups. The incorporation of deoxy[³H]cytidine was notchanged when tested in parallel. The increase in [³H]thymidineincorporation can therefore be explained by the assumption ofa diminution of the thymidine pool during cell cultivation. ¹To whom correspondence should be addressed     

Quantitative analysis of the metabolism of benzo(a)pyrene by transformable C3H10T1/2CL8 mouse embryo fibroblasts

The metabolism of benzo(a)pyrene [B(a)P] to organic soluble and water soluble metabolites by transformable C3H10T1/2CL8 mouse embryo fibroblasts was studied as a function of time, B(a)P concentration, and cell density. The total formation of organic-soluble and water-soluble metabolites increased with incubation time from 4 to 48 h and with B(a)P concentration from 4 to 40 microM. As cell density increased, the metabolic rate decreased for organic-soluble and water-soluble products between 6,300 and 54,000 cells/cm2 probably due to decreases in B(a)P concentrations to values below saturation. Specific organic-soluble metabolites identified were B(a)P-pre-9,10-diols, B(a)P-9,10-diol, B(a)P-7,8-diol, B(a)P-3,6-quinone, B(a)P-3-phenol, and B(a)P-9-phenol. Water-soluble metabolites were subjected to enzymatic hydrolysis with beta-glucuronidase and aryl sulfatase to identify specific conjugated products. The sulfate conjugated metabolites identified were B(a)P-7,8-diol, B(a)P-pre-9,10-diols, B(a)P-9,10-diol, and B(a)P-3,6-quinone. The beta-glucuronic acid metabolites identified were B(a)P-pre-9,10-diols, B(a)P-3,6-quinone, and B(a)P-3-phenol. Patterns of metabolite formation rates are discussed as to their possible effect on morphological transformation rates in C3H10T1/2 cells with respect to incubation time and cell density.     

Aneuploidy induction by benzo[a]pyrene and polyploidy induction by 7,12-dimethylbenz[a]anthracene in Chinese hamster cell lines V79-MZ and V79

We found that different ploidy effects were induced in fourChinese hamster-derived cell lines (V79-MZ, V79, CHL and CHO-K1)treated through two cell cycles with polycyclic aromatic hydrocarbonsin the absence of a metabolic activation system. 5-Bromodeoxyuridinewas used to investigate cell cycle delay and sister chromatidexchanges (SCE) induced by the chemicals. Benzo[a]pyrene (BP)induced aneuploidy at 2.5–10 µg/ml in V79-MZ cells.7,12-Dimethylbenz[a]anthracene (DMBA) induced polyploidy at3.125–6.25 and 6.25–12.5 µg/ml in V79-MZ andV79 cells respectively. Higher concentrations caused cell cycledelay and, therefore, did not affect ploidy. BP and DMBA didnot induce a significant increase in SCE frequency at the abovedoses. 3-Methylcholanthrene tested up to its solubility limit(10 µg/ml) did not induce numerical aberrations in anycell line. The clastogen mitomycin C, tested up to 0.01 µg/ml,did not produce numerical aberrations but did significantlyincrease SCE frequency in all cell lines. The spindle poisoncolchicine, tested up to 0.1 µg/ml, induced ploidy changesin the four cell lines that showed different sensitivities.Four cell lines showed no arylhydrocarbon hydroxylase activity,and V79-MZ, but not the other cells lines, showed high glutathioneS-transferase activity. Aneuploidy induction by BP and polyploidyinduction by DMBA in the absence of S9 mix in vitro have notbeen described before, and the finding might be due to the effecton tubulin. Due to their specificity and high sensitivity, theV79-MZ and V79 cell lines might be good systems for detectinganeuploidogens ³To whom correspondence should be addressed     

Smoking,lung cancers and their TP53 mutations

The TP53 suppressor gene possesses several properties that havefacilitated its use as a reporter of genotoxic exposure (Biggset al., 1993). In particular, it is mutated in a high proportionof many types of human cancer and it sustains a broad spectrumof mutations that can vary in both their position and type.In lung tumours from smokers, the high frequency of GT transversions(30% compared with 10% in cancers without tobacco aetiology)has been attributed to DNA adducts of polycyclic aromatic hydrocarbons(PAHs), such as benzo[a]pyrene, that are present in tobaccosmoke (Hainaut and Pfeifer, 2001). Benzo[a]pyrene is activatedto form benzo[a]pyrene diol epoxide (BPDE), which reacts withDNA predominantly at the N²-position of guanine, and both invitro and in     

Rat nasal tissue activation of benzo(a)pyrene and 2-aminoanthracene to mutagens in Salmonella typhimurium

Cytochrome P-450-dependent monooxygenase activity has been measured in the nasal turbinates of dogs and rats. The capacity of male Fischer-344 rat nasal tissue to bioactivate benzo(a)pyrene (BaP) and 2-aminoanthracene (2-AA) to mutagens in Salmonella typhimurium was investigated. 2-AA was mutagenic in strains TA98 and TA100 when nasal tissue S-9 was utilized as the activating enzyme system and BaP was mutagenic in strain TA100. At all doses and protein concentrations tested, 2-AA displayed nearly 500-1000 times greater bacterial mutagenicity than BaP. In strain TA-100, nasal tissue S-9 was approximately twice as active toward 2-AA as lung S-9 and 75% as active as liver S-9. Aryl hydrocarbon hydroxylase activity was detected in rat nasal tissue when 14C-BaP was used as a substrate. Rat nasal tissue metabolized BaP to several oxidized metabolites which included dihydrodiols, quinones, and phenols. 3-Hydroxybenzo(a)pyrene and BaP-3, 6-quinone were the major metabolites detected (150 pmoles/mg protein/30 min). These results indicate that rat nasal tissue can metabolize promutagens to reactive species which may play an important role in xenobiotic-induced nasal tumors.     

Mutagenicity, clastogenicity and cytotoxicity of procarcinogens in a rat hepatoma cell line competent for xenobiotic metabolism

The present studies were aimed at evaluating the suitabilityof the differentiated Reuber hepatoma cells H4DEC3/G^–for monitoring permanent damage to the DNA caused by hepato-trophicchemicals. First we determined the profile of xenobiotic metabolizingenzymes. The cells expressed various cytochrome P-450-dependentmonooxygenases, UDP-glucu-ronosyl-, phenol sulpho- and glutathioneS-transferase, cytochrome c (P-450) reductase and carboxylesterases.We then established the conditions for genotoxicity testingin H4IIEC/G^– cells. Induction of resistance against 6-thio-guanineand appearance of micronuclei served as indicators for mutagenicityand clastogenicity, respectively. 6-Thio-guanine-resistant H4HEC3/G^–cells were phenotypically stable for at least 30 cell cycles;recovery of 6-thioguanine-resistant cells was not significantlyaffected by the number of cells seeded for mutant selectionup to at least 10⁶ cells/100-mm dish; expression time of chemicallyinduced mutants was 12–15 days; a period of 24 h aftertreatment appeared to be sufficient to allow for the formationof micro-nuclei. Finally we tested the genotoxic effects ofpromutagens which are typically activated or inactivated inliver. Aflatoxin B₁, N-nitrosodiethylamine and cyclophosphamidewere genotoxic to H4IIEC3/G^– cells at concentrations of10–30 nM, 2–20 mM and 1 mM, respectively. N-Nitroso-dimethylamineand benzo[a]pyrene were not or only weakly cytotoxic and genotoxicto the cells, but this appears most likely to be due to protectivemechanisms rather than to lack of metabolic activation. Theresults indicate that differentiated hepatoma cells such asH4HEC3/G^– offer a means of studying the potential of chemicalsfor inducing permanent DNA damage in liver cells. ¹To whom correspondence should be addressed     

Modulation of the mutagenic activity of cigarette smoke, cigarette smoke condensate and benzo[a]pyrene in vitro and in vivo

A series of naturally occurring compounds were tested for theability to modulate the mutagenicity induced by cigarette smoke(CS), cigarette smoke condensate (CSC) and benzo[a]pyrene (BP)in the Salmonella/microsome mutagenicity assay and the micronucleustest in mouse bone marrow. Sodium selenite, retinol acetateand     

Inhibition of metabolic activation of the promutagens, benzo[a]pyrene, 2-aminofluorene and 2-aminoanthracene by furanochromones hi Salmonella typhimurium

Khellin, a naturally occurring furanochromone (Ammi visnagafruits), inhibited the mutagenicity of the promutagens benzo[a]pyrene,2-aminofluorene and 2-aminoanthracene in Salmonella typhimuriumTA98.The effect varied greatly and depended on the S9 fractionused.Cytosolic activation of 2-aminoanthracene was also inhibited.Khellin produced no effect or only weak activity against thedirect acting mutagens 2-nitrofluorene, 4-nitro-o-phenylenediamine,1-nitropyrene and ethylmethane sulfonate (in TA100).Daunomycinmutagenicity was inhibited to a greater extent Visnagin wasmore toxic, but showed similar effects.Khellol and its glucosidewere inactive against all the mutagens tested.We conclude thatkhellin acts as an inhibitor of the microsomal cytochrome P450sub-enzymes analogous to the related furanocoumarins and isalso capable of inhibiting cytosolic enzymes.The extract fromAmmi visnaga fruits showed a higher inhibition potency thankhellin alone against 2-aminoanthracene, 1-nitropyrene and daunomycin.Thismight be due to additional inhibitors, e.g. coumarins, or tothe synergistic effects of accompanying compounds. ^*To whom correspondence should be addressed. Tel: +49 9131 85825; Fax: +49 9131 858243.     

Metabolic differences between whole blood and isolated lymphocyte cultures for micronucleus (MN) induction by cyclophosphamide and benzo[a]pyrene

In order to study the metabolic differences between whole bloodand isolated lymphocyte cultures, two indirectly acting mutagenscyclophosphamide (CP) and benzo[a]pyrene (B[a]P) were assessedfor their potential to induce micronuclei (MN) in the presenceand absence of S9 microsomal fractions. In isolated lymphocytecultures supplemented with S9, CP and B[a]P induced a statisticallysignificant increase in MN which was not observed in whole bloodcultures. However, the directacting agent methyl methanesulphonate(which was used as a positive control) showed an increase inMN frequency in a dose-dependent manner in both culture methods.The effect of erythrocytes was then investigated by treatingisolated lymphocyte cultures simultaneously with CP and S9 mixin the presence of purified erythrocyte concentrate (PEC). Aclear reduction in the MN frequency was observed compared tothe frequencies of MN induced in isolated lymphocyte culturestreated with CP and S9 mix in the absence of PEC. Thus, isolatedlymphocyte cultures may represent a more sensitive test systemfor the evaluation of potential indirectacting mutagens. However,whole blood cultures may reflect the ‘real life’situation more accurately as a consequence of the presence oferythrocytes.     

Role of T-cell-associated lymphocyte function-associated antigen-1 in the pathogenesis of experimental colitis

The ß₂ integrin lymphocyte function-associated antigen-1(LFA-1; CD11a/CD18) is important for lymphocyte traffickingand activation as well as recruitment to sites of tissue inflammation.The objective of this study was to assess the role of ‘T-cell-associated’LFA-1 in the pathogenesis of chronic colitis in vivo. Transferof CD4⁺CD25^– T cells isolated from wild-type (wt) miceinto immunodeficient recipients [recombinase-activating gene-1-deficient(RAG-1^–/–)] produced moderate to severe colitis,whereas RAG-1^–/– mice injected with CD11a-deficient(CD11a^–/–; LFA-1^–/–) donor T cells displayedminimal macroscopic and histological evidence of colitis. Surfaceexpression of L-selectin, ₄, ₄ß₇ and chemokine receptor-7were similar for wt and CD11a^–/– donor T cells.Attenuated disease in the CD11a^–/– RAG-1^–/–animals was associated with decreased numbers of CD4⁺ T cellsin the mesenteric lymph nodes (MLNs), spleen and intestinallamina propria (LP). In addition, significant reductions inT_h1 cytokines were observed following ex vivo stimulation ofmononuclear cells obtained from the MLNs and colonic LP. Interestingly,mononuclear cells obtained from the spleens of CD11a^–/– RAG-1^–/– exhibited enhanced pro-inflammatory cytokineproduction compared with splenocytes obtained from wt RAG-1^–/–colitic mice. Taken together, our data suggest that T-cell-associatedCD11a (LFA-1) expression plays a dual role in the initiationof chronic gut inflammation by facilitating naive T-cell priming/activationand expansion within MLNs and by augmenting pro-inflammatorycytokine production following secondary stimulation by antigen-presentingcells in the colonic interstitium.     

The micronucleus assay in mouse peripheral blood reticulocytes demonstrates the transmission of chromosomal instability induced by mitomycin C and benzo[a]pyrene

The frequency of micronuclei (MN) in mouse peripheral bloodreticulocytes supravitally stained with acridine orange wasassessed at different time intervals after treatment in vivowith two dose levels of mitomycin C (MMC) and benzo[a]pyrene.Increased frequencies were observed for many days after treatment,indicating that MN may be produced as a consequence of chromosomalinstability transmitted by proliferating erythroblasts. Theseresults confirm previous evidence of the persistent cytogeneticeffects of MMC and suggest that clastogenic agents acting bydifferent primary lesions may have a similar ability to inducethis effect.     

Role of DNA repair by (A)BC excinuclease and Ogt alkyltransferase in the final distribution of LacI-d mutations induced by N-butyl-N-nitrosourea in Escherichia coli

In the absence of nucleotide excision repair, the additionaldeficiency of the DNA alkyltransferase (ATase) encoded by theconstitutive ogt gene of Escherichia coli caused a marked increasein mutation induction by N-butyl-N-nitrosourea (BNU). Irrespectiveof the presence or absence of the Ogt ATase, little mutagenicresponse was detected in Uvr⁺ bacteria in the concentrationrange 0–8 mM BNU, indicating that most premutagenic DNAlesions induced at these concentrations are efficiently recognizedand repaired by the nucleotide excision repair system. Increasedsusceptibility to mutagenesis by BNU was detected in Uvr^–Ogt⁺ bacteria, but the Uvr^– Ogt^– double mutant exhibitedmuch higher sensitivity. These data suggest that the Ogt ATasecan replace to a great extent the repair capacity of the (A)BCexcinuclease. Forward mutations induced by 6 mM BNU within theinitial part of the lacI gene of E.coli were recovered fromUvr⁺ Ogt^–, Uvr^– Ogt⁺ and Uvr^– Ogt^– bacteria.A total of 454 independent mutations were characterized by DNAsequence analysis. The BNU-induced spectra were dominated byG:C     

Antimutagenic effects of betel leaf extract against the mutagenicity of two tobacco-specific N-nitrosamines

Epidemiological studies have implicated chewing tobacco aloneto be more hazardous than chewing tobacco with betel quid. Experimentalstudies have shown that betel leaf is antimutagenic againststandard mutagens like benzo[a]pyrene and dimethylbenz[a]anthracene.Since the tobacco-specific N-nitrosamines (TSNA) are the onlycarcinogens present in unburnt forms of tobacco, including chewingtobacco, we tested the effect of an extract of betel leaf againstthe mutagenicity of the two important TSNA, viz., N1-nitrosonornicotineand 4-(methylnitrosamino)-1-(3-pyridyl) -1-butanone, using theAmes Salmonella/microsome assay with TA100 +S9 and the in vivomicronucleus test. In both the test systems it was observedthat betel leaf extract suppressed the mutagenic effects ofboth the nitrosamines to a significant extent. ²To whom correspondence should be addressed     

Validation of the in vivo CD1 mouse splenocyte micronucleus test

In order to validate the in vivo micronucleus test in mousesplenocytes using the cytokinesis block method, 14 compoundswith various mechanisms of action were tested: three directalkylating agents (mitomycin C, ethylnitrosourea, ß-propio-lactone),seven indirect alkylating agents (cyclophosphamide, benzo[a]pyrene,diethylnitrosamine, dimethylnitrosamine, 4-aminophenol, 4-aminobiphenyl,1,1-dimethylhydrazine), two intercalating agents (acridine orange,ethidium bromide) and two spindle poisons (vincristine, colchicine).Male mice were dosed once with the compound, and spleen sampleswere taken 2 or 14 days after treatment. A significant increasein the binucleated micronucleated splenocyte rate was observedwith all the alkylating and intercalating agents at at leastone sampling time. In contrast, no increase in the binucleatedmicronucleated splenocyte rate was observed with the spindlepoisons. In conclusion, under these experimental conditions,this in vivo test seems appropriate for the detection of clastogeniccompounds including compounds that cannot be detected hi thebone marrow micronucleus test. The limit of this test, as expected,is the lack of detection of aneugenic compounds. ¹To whom correspondence should be addressed     

Isolation of S9 fractions from mouse and rat with increased enzyme activities after repeated administration of cytochrome P-450 and P-448 inducers

Cytochrome P-450 (cyt P-450), NADPH cytochrome P-450 reductaseand various microsomal monooxygenase activities [e.g. aminopyrineN-demethylase, p-nitroanisole O-demethyl-ase, dinemorphan N-demethylase,ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase(ERD)], were determined in hepatic post-mitochondrial supernatantfrom mice and rats. Experiments were performed on male and femaleanimals treated with a combination of sodium pheno-barbitaland ß-naphthoflavone according to the standard protocolschedule for short-term genotoxicity testing. A second inductivetreatment after 2, 3, 4 or 5 weeks was provided. The increasein cyt P-450 and in all enzymatic activities measured was enhancedin both species by a second induction treatment, particularlywhen given after 4 weeks. ERD activity was the only monooxygenaseactivity which was sex-dependent, being more active in femalethan in male animals. To extend the biochemical data, experimentswere performed with the proposed S9 fractions on styrene, whichpreviously has proved difficult to detect in short-term in vitromutagen-icity tests. Using the new induction conditions positiveresults were obtained with the D7 strain of Saccharomyces cerevisiae.It was concluded that a simple pre-induction of the animals3–4 weeks before the main induction treatment leads toa more active S9 fraction for in vitro genotoxicity studies. ^*This work was presented at the 18th Annual Meeting of the EnvironmentalMutagen Society, April 8–12, 1987, San Francisco, USA.