Aroclor 1254-induced Microsomes Research Articles

Ex vivo modulation of chemical-induced mutagenesis by subcellular liver fractions of rats treated with rooibos ( Aspalathus linearis) tea, honeybush ( Cyclopia intermedia) tea, as well as green and black ( Camellia sinensis) teas

Male Fischer rats were given unprocessed (not oxidized) and processed (oxidized) rooibos and honeybush teas as well as green and black teas as a sole source of drinking fluid for 10 weeks, and sub cellular liver fractions were prepared. Cytosolic fractions of rats consuming the unprocessed herbal teas, green and black teas significantly ( P<0.05) protected against 2-acetylaminofluorene (2-AAF)-induced mutagenesis in the Salmonella mutagenicity test with strain TA 98, using Aroclor 1254-induced microsomes. A marginal or no protection was obtained with the processed herbal teas. The mutagenic response of aflatoxin B 1 (AFB 1) against Salmonella strain TA 100 was significantly ( P<0.05) inhibited by cytosolic fractions from rats treated with processed and unprocessed herbal teas, while no effect was obtained with the green and black teas. Microsomal fractions prepared from livers of rats treated with both the processed and unprocessed rooibos teas and the unprocessed honeybush tea, significantly ( P<0.05) reduced the activation of AFB 1 while no protection was observed against 2-AAF-induced mutagenesis. In contrast, microsomal fractions from rats treated with the green, black and unprocessed honeybush teas significantly ( P<0.05) enhanced the mutagenic response of 2-AAF. None of the tea treatments significantly affected the concentration of the microsomal liver cytochrome P450.

Microsome-mediated bioactivation of dibenzo[a,l]pyrene and identification of DNA adducts by 32P-postlabeling.

Dibenzo[a,l]pyrene (DBP) is one of the most potent bacterial mutagen and mammary carcinogens. When DBP (50 microM) was incubated with calf thymus DNA (300 microg/ml) in the presence of liver microsomes from beta-naphthoflavone (beta-NF)- or Aroclor 1254-treated rats, at least eight adduct spots were detected as analyzed by nuclease P1-enhanced 32P-postlabeling assay. DNA adduction was enhanced by nearly 20- and 60-fold with beta-NF- and Aroclor 1254-induced microsomes, respectively, as compared with uninduced microsomes, suggesting a possible involvement of CYP1A family in DBP activation. Inclusion of the selective P4501A1 inhibitor, alpha-naphthoflavone (50 microM) in the activation reaction almost completely (>98%) abolished adduct formation further supporting involvement of P4501A in DBP activation. Analysis of DNA and 2'-deoxynucleosides 3'-mononucleotide reacted with anti- and syn-DBP-11,12-diol-13,14-epoxides (DBPDEs) and co-chromatography analyses in multiple solvents showed that the microsomal DBP-DNA adducts were derived by interaction of both anti- and syn-DBPDEs with adenine and guanine in DNA in the following order: anti-DBPDE-dA approximately syn-DBPDE-dG >> anti-DBPDE-dG approximately syn-DBPDE-dA. It is concluded that (i) most or all DBP adducts were P4501A-mediated; (ii) both the anti- and syn-stereoisomers were involved in the DNA adduct formation; and (iii) both adenine and guanine in the DNA contributed equally to the formation of the major and minor adducts.

Interaction with the Aromatic Hydrocarbon Receptor, CYP1A Induction, and Mutagenicity of a Series of Diaminotoluenes: Implications for Their Carcinogenicity

The present study was undertaken to provide a rationale for the marked difference in carcinogenic potential among isomeric diaminotoluenes, in relation to their ability to induce their own bioactivation through CYP1A induction, their genotoxic potential, and their ability to bind to the cytosolic aromatic hydrocarbon (Ah) receptor. Of the four possible diaminotoluenes only the 2,3- and, to a lesser extent, the 2,4-isomer induced CYP1A activity. Similarly, only these two isomers could displace [3H]T-CDD from the hepatic cytosolic Ah receptor. In the presence of Aroclor 1254-induced microsomes, 2,4- and 2,6-diaminotoluene were potent mutagens in the Ames test. Only 2,4-diaminotoluene could autoinduce its activation. Of the four isomers only 2,4-diaminotoluene is an established carcinogen and this is compatible with the present observations that it is the only isomer that stimulates its own activation through CYP1A induction, is metabolically converted to genotoxic intermediates, and binds to the Ah receptor. 2,6-Diaminotoluene is recognized as a mutagenic noncarcinogen and in the present studies it elicited a positive mutagenic response in the Ames test but failed to induce CYP1A activity and its own activation, and could not bind to the Ah receptor even at concentrations as high as 5 × 10−4M. The present findings demonstrate thatin vitrostudies are very useful tools in predicting the carcinogenic potency of isomeric chemicals.

Structure-activity relationships of anthraquinones as inhibitors of 7-ethoxycoumarin O-deethylase and mutagenicity of 2-amino-3-methylimidazo[4,5- f]quinoline

The antimutagenicity of 17 natural and synthetic anthraquinones was determined using Salmonella typhimurium TA98 against 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) in the presence of Aroclor 1254-induced rat hepatic S9. In general, the relationship between the chemical structures of anthraquinones and their antimutagenicity was found to contain one or more of the following features: (i) C9 carbonyl group, (ii) hydroxyl group at C1 and C4, (iii) C2 ethyl group, and (iv) C3 methyl group. The inhibitory effect of anthraquinones on 7-ethoxycoumarin O-deethylase (ECD) of Aroclor 1254-induced hepatic microsomes was also examined. In addition, we studied the effect of anthraquinones on the metabolism of IQ by Aroclor 1254-induced microsomes using high-performance liquid chromatography. The antimutagenicity correlated with the inhibition of cytochrome P-450IA2-linked ECD activity in hepatic microsomes, and with the inhibition of N-hydroxy-IQ formation of IQ metabolism by hepatic microsomes. Moreover, we also examined the antimutagenicity of anthraquinones against synthetic N-hydroxy-IQ. Quinizarin and anthraflavic acid were shown to have more effect on the direct mutagenicity of N-hydroxy-IQ than that of the anthraquinones tested. This might explain why both anthraquinones showed higher antimutagenicity, although they inhibited ECD less. These results suggest that there exist at least two mechanisms of action in modifying roles of anthraquinones on the mutagenicity of IQ: (i) mediation through interaction with microsomal activating enzymes to inhibit the major active metabolite of N-hydroxy-IQ formation and (ii) direct interaction with the proximate metabolite of IQ, N-hydroxy-IQ, to block its attack on DNA.

The structure-activity relationships of flavonoids as inhibitors of cytochrome P-450 enzymes in rat liver microsomes and the mutagenicity of 2-amino-3-methyl-imidazo[4,5-f]quinoline.

The antimutagenicity of 19 naturally occurring flavonoids and their derivatives including flavones, flavonols, flavanones, isoflavones and flavanols were determined using Salmonella typhimurium TA98 against 2-amino-3-methylimidazo[4,5-f] quinoline (IQ) in the presence of Aroclor 1254-induced rat hepatic S9. In general, a relationship between the chemical structure of flavonoids and their antimutagenicity was found for compounds containing one or more of the following features: (i) C4 keto group, (ii) aglycone, (iii) double bond at positions C2 and C3, (iv) phenyl group at position C2, and (v) three hydroxy substituents at positions C4', C5 and C7. The inhibitory effects of flavonoids on activities of 7-ethoxycoumarin deethylase (ECD) and 7-ethoxyresorufin deethylase (ESD) of Aroclor 1254-induced hepatic microsomes were also examined. In addition, we studied the effects of flavonoids on the metabolism of IQ by Aroclor 1254-induced microsomes using high-performance liquid chromatography. The antimutagenicity correlated with the inhibition of cytochrome P-450IA1-linked ESD and P-450IA2-linked ECD activity in hepatic microsomes, and with an inhibition of N-hydroxy-IQ formation from IQ metabolism by hepatic microsomes. These results indicated that flavones or flavonols that contain C5, C7 and C4' hydroxyl groups are potent inhibitors of P-450 enzyme activities induced by Aroclor 1254 (P-450IA1 and P-450IA2), and may potentially be useful as chemopreventive agents against heterocyclic amine-induced mutagenesis or carcinogenesis.

Antimutagenicity of Maillard reaction products from amino acid/sugar model systems against 2-amino-3-methylimidazo-[4,5-f]quinoline: the role of pyrazines.

The antimutagenicity of dichloromethane extracts from eight amino acid/sugar model systems was determined using Salmonella typhimurium TA98 against 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) in the presence of Aroclor 1254-induced rat hepatic S9. The Maillard reaction products in the dichloromethane extracts were then quantified and qualified by capillary gas chromatography and gas chromatography-mass spectrometry, respectively. Pyrazines and furans were found to be the major Maillard reaction products yielded in the extracts. Moreover, the antimutagenicity of dichloromethane extracts correlated positively with the total amounts of pyrazines and furans. To elucidate the mechanism of antimutagenicity of dichloromethane extracts, the inhibitory effect of pyrazines on ethoxycoumarin deethylase activity in Aroclor 1254-induced hepatic microsomes was examined. We also studied the effects of pyrazines on IQ metabolism by Aroclor 1254-induced microsomes using high-performance liquid chromatography. The antimutagenicity of pyrazines correlated positively with both the inhibition of cytochrome P-450 IA2-linked ethoxycoumarin deethylase in hepatic microsomes and the inhibition of N-hydroxy-IQ formation from IQ metabolism by hepatic microsomes. Thus we concluded that pyrazines in dichloromethane extracts from eight amino acid/sugar model systems play an important role in the antimutagenicity of IQ. Moreover, we concluded that the modifying effect of pyrazines on the mutagenicity of IQ is mediated through interaction with microsomal activating enzymes to inhibit the major active metabolite in N-hydroxy-IQ formation.

The metabolism and activation of 15,16-dihydrocyclopental[a]phenanthren-17-one by cytochrome P-450 proteins

The in vitro metabolism and activation to mutagens of 15,16-dihydrocyclopenta[a]phenanthren-17-one (CPP-17-one) were investigated using hepatic preparations from rats pretreated with prototype inducers of the cytochrome P-450-dependent mixed-function oxidases. Aroclor 1254-induced microsomes were the most effective metabolisers of this compound, the major metabolites being oxidation products of the bay region A ring. To a lesser extent hydroxylation of the non-aromatic D ring occurred, the products being the 15- and 16-hydroxyderivatives. Oxidation of the A ring was also achieved with microsomes from benzo[a]pyrene-treated rats but not with those from rats treated with clofibrate, phenobarbitone, isoniazid, dexamethasone and CPP-17-one itself, where the metabolites were primarily the oxidation products of the D ring. When CPP-17-one was used as a promutagen in the Ames test, only microsomes from Aroclor 1254-treated rats could elicit a positive mutagenic response. When 3,4-dihydrodihydroxy-CPP-17-one, the precursor of the ultimate mutagen, was used as the promutagen, a positive response was observed with microsomes from Aroclor 1254- and benzo[a]pyrene-treated rats. It is concluded that (a) CPP-17-one is metabolised through oxidation of the D ring to produce non-mutagenic products and only through A ring oxidation mutagens are produced, (b) only the CYP1A (cytochrome P-450 family 1, subfamily A) family (induced by benzo[a]pyrene and Aroclor 1254) could oxidise the A ring, and (c) Aroclor 1254-induced hepatic microsomes are the most effective catalysts of the metabolism and activation of CPP-17-one because, in addition to the high CYP1A activity, they are also characterised by high levels of microsomal epoxide hydrolase.

Cytochrome P450-dependent metabolism and mutagenicity of 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one and their implications in its carcinogenicity.

Methylation of the non-carcinogen 15,16-dihydrocyclopenta[a]phenanthren-17-one (CPP-17-one) at the bay region to form 11-CH3-CPP-17-one confers carcinogenic potential. In the present study we have investigated the in vitro metabolism and mutagenicity of the methylated compound by hepatic microsomal preparations from rats pretreated with various prototype inducers of cytochrome P450 proteins in order to provide a rationale for this marked difference in carcinogenic activity. The most effective metabolism of 11-CH3-CPP-17-one occurred in the presence of Aroclor 1254-induced microsomes, the principal metabolites being oxidative products of the A- and D-rings and of the methyl substituent. When benzo[a]pyrene-induced microsomes served as the metabolising system, the major A-ring metabolite was the 3,4-diol. A similar metabolic pattern was seen with microsomes from rats treated with 11-CH3-CPP-one itself, but the overall effect of metabolism was lower than that observed with benzo[a]pyrene-treated microsomes but higher than that of control animals. In contrast, microsomes from rats treated with clofibrate, dexamethasone, isoniazid and phenobarbitone failed to enhance the metabolism of 11-CH3-CPP-17-one when compared with control microsomes and the metabolites reflected primarily oxidation of the D-ring. When 11-CH3-CPP-17-one was employed as a promutagen in the Ames test, a mutagenic response was evident only in the presence of microsomes from benzo[a]pyrene-induced rats, but induction with phenobarbitone, isoniazid, dexamethasone, clofibrate and the compound itself, failed to elicit a positive mutagenic response. When 3,4-dihydroxy-11-CH3-CPP-17-one served as the promutagen, a mutagenic response was observed in the presence of benzo[a]pyrene-induced and, to a lesser extent, 11-CH3-CPP-17-one-induced microsomes. Treatment of rats with 11-CH3-CPP-17-one caused a marked increase in the O-deethylation of ethoxyresorufin and, to a much lesser extent in epoxide hydrolase activity. It is concluded that (i) 11-CH3CPP-17-one is an inducer of the CYP1 family; (ii) under the present experimental conditions only the CYP1 family can oxidise the A-ring to form the 3,4-dihydroxy-11-CH3-CPP-17-one, the precursor of the ultimate carcinogen and (iii) only the CYP1 family oxidizes the diol to generate the ultimate carcinogen.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of a metabolic activation system for the frog embryo teratogenesis assay: Xenopus (FETAX).

FETAX (frog embryo teratogenesis assay: Xenopus) is a 96-hr teratogenesis screening assay using embryos of the South African clawed frog, Xenopus laevis. Since Xenopus embryos have limited xenobiotic metabolism through 96 hr of development, we have developed an in vitro metabolic activation system employing Aroclor 1254-induced rat liver microsomes. By adding an exogenous source of mixed functional oxidase (MFO) activity, we may more accurately assess the teratogenic risk of proteratogenic compounds. Xenopus embryos were cocultured with varying concentrations of cyclophosphamide (CP), Aroclor 1254-induced microsomal protein, an NADPH-generating system, and antibiotics in a static renewal fashion for 96 hr. Residual Aroclor 1254 remaining in the microsomes was successfully reduced during purification to levels that had no significant effect on embryo survival and development. The results of three definitive dose-response tests performed with CP revealed that activation reduced the 96 hr LC50 from 8.0 to 1.4 mg/ml (5.7-fold). The 96-hr EC50 (malformation) was reduced from 6.2 to 0.4 mg/ml (15.5-fold). Activation also increased the types and severity of malformation and reduced embryonic growth. Aroclor 1254-induced rat liver microsomes may be used as an acceptable in vitro metabolic activation system for FETAX.

Structure-activity relationships in the inhibitory effects of ellipticines on benzo(a)pyrene hydroxylase activity and 3-methylcholanthrene mutagenicity

The structural features which determine the ability of ellipticine (5,11dimethyl6Hpyrido[4-3b] carbazole) and its derivatives to interact with cytochrome P-450 and to inhibit rat liver microsomal benzo(a)pyrene hydroxylase as well as to inhibit the mutagenicity of 3-methylcholanthrene have been studied. Spectral interactions studies were carried out with either Aroclor 1254-, 3-methylcholanthrene-or phenobarbital-induced microsomes. Inhibitory activities towards benzo(a)pyrene hydroxylase and 3-methylcholanthrene mutagenicity (Ames test), were determined using Aroclor 1254-induced microsomes. It appears that every ellipticine derivative having significant inhibitory effects on hydroxylation of benzo(a)pyrene or mutagenicity of 3-methylcholanthrene also exhibits a very good affinity for microsomal cytochromes P-450. The accessibility of the pyridinic nitrogen of ellipticine derivatives appears as the most important factor for their binding to cytochromes P-450 and the presence of methyl groups in 5 and 11 positions of ellipticine derivatives is an essential condition for the expression of the inhibitory power. Various substitutions in the A ring of ellipticine appear to be of secondary importance. On the other hand the location of the pyridinic ring and consequently the arrangement of the molecule within the hydrophobic pocket of cytochrome P-450 seems also to play an important role in the inhibitory power since isoellipticines are devoid of such properties. These results should help in the design of particularly efficient inhibitors of drug and carcinogen metabolism.

Potentiation of fluroxene (2,2,2-trifluoroethyl vinyl ether) toxicity with polychlorinated biphenyls

The effects of induction of rat hepatic microsomal cytochrome P-450 by commercial mixtures of polychlorinated biphenyls (PCBs) (100 mg/kg/day for 3 days) in potentiating the toxicity of the anesthetic fluroxene (2,2,2-trifluoroethyl vinyl ether) have been determined. The PCB mixtures used were Aroclors 1221, 1016, 1254, and 1260. All Aroclors significantly induced cytochrome P-450 relative to controls (1.4–2.1), and the more highly chlorinated PCB mixtures produced the greatest induction. The cytochrome P-450 composition of the Aroclor-induced microsomes was probed using R warfarin metabolism. Aroclor 1254-induced microsomes comprised mainly cytochrome P-448 of the 3-methylcholanthrene-inducible type, and Aroclor 1260-induced microsomes comprised mainly cytochrome P-450 of the phenobarbital-inducible type, although each contained lesser quantities of the other forms. Only Aroclor 1254 and 1260 induction produced death in rats (12/20 and 11/13 respectively) following ip administration of fluroxene (2.5 g/kg body wt). Fluroxene potentiated the destruction of cytochrome P-450 in vivo and this destruction was enhanced by Aroclor induction. Cytochrome P-448 suffered the greatest destruction in Aroclor 1221- and 1254-induced microsomes, and cytochrome P-450 was most extensively destroyed in Aroclor 1260-induced microsomes. The Aroclor 1254-induced cytochrome P-450 concentration did not return to the corresponding control value 24 hr after clearance of the fluroxene dose which implies that fluroxene metabolism interferes with the biosynthesis of cytochrome P-450. It is concluded that some Aroclors, through induction of cytochrome P-450 of the phenobarbital-inducible type, increase the rates of metabolism of fluroxene, which results in the production of toxic metabolites leading to liver necrosis, destruction of cytochrome P-450, and death.