Immunological and enzymatic comparison of hepatic cytochrome P-450 fractions from phenobarbital-, 3-methylcholanthrene-, β-naphtoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats

A comparison of the cytochrome P-450 forms induced in rat liver microsomes by phenobarbital on the one hand, and 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin on the other hand, was performed using specific antibodies: anti-P-450 B₂ PB IG (against the phenobarbital-induced cytochrome P-450) and anti-P-450 B₂ BNF IG (against the β-naphtoflavone-induced cytochrome P-450). On DEAE-cellulose chromatography, four cytochrome P-450 fractions were separated, called P-450 A (non-adsorbed), P-450 Ba, P-450 Bb and P-450 Bc, from control, phenobarbital-, 3-methylcholanthrene, /gb-naphtoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats. Cytochrome P-450 A fractions appeared to be unmodified by the inducers, whereas the specifically induced cytochrome P-450 forms were always recovered in Bb fractions. The P-450 Bb fractions induced by 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibited common antigenic determinants, comparable catalytic activities (benzphetamine, N-demethylase, benzo[a]pyrene hydroxylase) and similar mol. wts. Moreover, the inhibition patterns by the two antibodies of benzphetamine N-demethylase and benzo[a]pyrene hydroxylase activities catalysed by 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin microsomes or by the corresponding P-450 Bb fractions in a reconstituted system were quite identical. By these different criteria, β-naphtoflavone, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin seem to induce a common cytochrome P-450 species in rat liver.     

Modifications of carcinogen metabolism in hepatic microsomes of suckling young by 3-methylcholanthrene or β-naphthoflavone administered to lactating rats

The effects of treating lactating rats with 3-methylcholanthrene (3-MC) or β-naphthoflavone (β-NF) (three i.p. injections of 20 or 40 mg compound/kg of body weight) on hepatic microsomal enzymes of their suckling young were examined. This treatment had no apparent effect on the contents of cytochromes P-450 and b₅ or on the activities of NADH- and NADPH-cytochrome c reductases in hepatic microsomes of the pups. However, these microsomes had 8- and 6-fold increased capacities for hydroxylations of benzo[a]pyrene (B[a]P) and N-2-fluorenylacetamide (2-FAA) respectively. These increases were about 5-fold greater in the hepatic microsomes of the dams, in which they were inhibited by 0.1 mM α-naphthoflavone (α-NF) invitro 72–81 and 89–95% and by 0.1 mM β-NF in vitro 12–41 and 60–76% respectively. In the pups, the induced activities were also inhibited, whereas the basal hydroxylations of B[a]P and 2-FAA were stimulated by α-NF 2.7- and 5.0-fold and by β-NF 1.4- and 2.4-fold respectively. The inhibition of the induced hydroxylations by α-NF and β-NF may be explained by their higher affinities (K_s, 0.14 and 0.28 μM, respectively) than those of B[a]P and 2-FAA (K_s 4.4 to 8.8 and 2.4 to 3.1 μM, respectively) for cytochrome P-450. Whereas β-NF gave a type I binding spectrum, α-NF gave a spectrum composed of type I and reverse-type I elements. Analysis of metabolites of 2-FAA showed differences in their type and amounts formed by hepatic microsomes of β-NF-treated lactating rats and their pups. Thus, in the dams the formation of 1-, 3-, 5-, 7-, 9- and N-hydroxy-2-FAA was increased by 9-, 30-, 40-, 5-, 20- and 5-fold respectively. In the pups, the formation of 1-, 3-, 5-, 7- and N-hydroxy-2-FAA was increased by 2-, 30-, 18-, 4- and 27-fold respectively. All these increased hydroxylations were inhibited by 0.1 mM α-NF in vitro. In the hepatic microsomes of pups from the corn oil-treated dams, α-NF stimulated all ring-hydroxylations, but not N-hydroxylation of 2-FAA. The results support earlier findings that microsomal enzymes differ in immature and mature rat liver and suggest that N-hydroxylation of 2-FAA, the activation required for carcinogenesis, and specific ring-hydroxylations are catalyzed by different cytochrome P-450 isozymes. Our studies showed that 3-MC and β-NF and/or their metabolites were transferred with milk of dams to their suckling pups in which they modified metabolism of carcinogens.     

Effects of disulfiram on mixed function oxidase system and trace element concentration in the liver of rats

Disulfiram (DSF), an inhibitor of chemically induced carcinogenesis, and its metabolite diethyldithiocarbamate (DDTC) have been investigated for their influence on trace element distribution and on certain enzymes of the drug metabolizing system in the livers of phenobarbital (PB) treated rats. Both substances diminished the PB induced enzyme response in liver microsomes, DDTC being more effective (?85%) than DSF (?60%). The copper, cobalt and zinc content of the livers of DSF treated animals were increased by factors of 6, 3 and 1.5 respectively as compared to controls, while DDTC treatment had no influence on liver trace element content. A correlation between enzyme inhibition and enhanced trace element uptake of the liver after DSF administration could not be observed. The change of trace element transport into the liver during DSF treatment is discussed.     

Studies in the metabolism of carcinogenic polycyclic heteroaromatic compounds. I. The hepatic microsomal metabolism of 7-methylbenz[c]acridine

An enzyme assay for the metabolism of the carcinogenic aza-aromatic polycyclic compound 7-methylbenz[c]acridine has been developed using a modification of a radiochemical assay described for the polycyclic aromatic hydrocarbon benzo[a]pyrene by DePierre et al. [J. W. DePierre, M. S. Moron, K. A. M. Johannesen and L. Ernster, Analyt. Biochem. 63, 470 (1975)]and Van Cantfort et al. [J. Van Cantfort, J. DeGraeve and J. E. Gielen, Biochem. biophys. Res. Commun. 79, 505 (1977)]. When the activities of control microsomes and microsomes of phenobarbital-, 3-methylcholanthrene-and 7-methylbenz[c]acridine-pretreated animals were compared, strong similarities were displayed toward oxidation of benzo[a]pyrene and 7-methylbenz[c]acridine. These similarities were seen in turnover numbers, Michaelis constants, and inducibility of both enzyme systems. 7-Methylbenz[c]acridine afforded a type I difference spectrum with 3-methylcholanthrene-pretreated microsomes. It is suggested that 7-methylbenz[c]acridine is oxidized by the same or a similar set of enzymes which is responsible for benzo[a]pyrene metabolism.     

Oxidative activation and inactivation of the carcinogen 2-acetylaminofluorene by various purified cytochromes P-450 from 3-methylcholanthrene pretreated rats

Ring- and N-hydroxylations of 2-acetylaminofluorene (AAF) have been examined in a reconstituted system with 4 purified hepatic microsomal cytochromes P-450 isolated from 3-methylcholanthrene (MC)-pretreated rats. Among these 4 isozymes, P-450D showed the most activity whereas P-450C was devoid of any activity; two other P-450s exhibited moderate activity. These and Ouchterlony's double diffusion analyses suggest involvement of multiple cytochromes P-450 in AAF oxidations.     

Monoclonal antibodies to phenobarbital-induced rat liver cytochrome P-450

Somatic cell hybrids were made between mouse myeloma cells and spleen cells derived from BALB/c female mice immunized with purified phenobarbital-induced rat liver cytochrome P-450 (PB-P-450). Hybridomas were selected in HAT medium, and the monoclonal antibodies (MAbs) produced were screened for binding to the PB-P-450 by radioimmunoassay, for immunoprecipitation of the PB-P-450, and for inhibition of PB-P-450-catalyzed enzyme activity. In two experiments, MAbs of the IgM and IgG1 were produced that bound and, in certain cases, precipitated PB-P-450. None of these MAbs, however, inhibited the PB-P-450-dependent aryl hydrocarbon hydroxylase (AHH) activity. In two other experiments, MAbs to PB-P-450 were produced that bound, precipitated and, in several cases, strongly or completely inhibited the AHH and 7-ethoxycoumarin deethylase (ECD) activities of PB-P-450. These MAbs showed no activity toward the purified 3-methylcholanthrene-induced cytochrome P-450 (MC-P-450), β-naphthoflavone-induced cytochrome P-450 (BNF-P-450) or pregnenolone 16-α-carbonitrile-induced cytochrome P-450 (PCN-P-450) in respect to RIA determined binding, immunoprecipitation, or inhibition of AHH activity. One of the monoclonal antibodies, MAb 2-66-3, inhibited the AHH activity of liver microsomes from PB-treated rats by 43% but did not inhibit the AHH activity of liver microsomes from control, BNF-, or MC-treated rats. The MAb 2-66-3 also inhibited ECD in microsomes from PB-treated rats by 22%. The MAb 2-66-3 showed high cross-reactivity for binding, immunoprecipitation and inhibition of enzyme activity of PB-induced cytochrome P-450 from rabbit liver (PB-P-450_LM2). Two other MAbs, 4-7-1 and 4-29-5, completely inhibited the AHH of the purified PB-P-450. MAbs to different cytochromes P-450 will be of extraordinary usefulness for a variety of studies including phenotyping of individuals, species, and tissues and for the genetic analysis of P-450s as well as for the direct assay, purification, and structure determination of various cytochromes P-450.     

Interaction of polyhalogenated compounds of appropriate configuration with mammalian or bacterial CYP enzymes. Increased bilirubin and uroporphyrinogen oxidation in vitro

Polyhalogenated compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, are associated with toxic Uroporphyria and cause alleviation of jaundice in the Gunn rat. These effects have been attributed to a microsomal oxidation of uroporphyrinogen and bilirubin for which supportive evidence has been obtained in vitro. CYP1A1 required planar polyhalogenated biphenyls for these oxidative reactions, while CYP1A2 was capable of oxidation in their absence.We have now used rat CYP1A1 and confirmed with the pure enzyme that increased bilirubin oxidation was caused by the addition of 3,4,3',4'-tetrachlorobiphenyl. CYP1A2 was more active than CYP1A1 at oxidizing bilirubin in presence of NADPH alone and reacted to addition of 3,4,3',4'-tetrachlorobiphenyl with a depression rather than a stimulation of bilirubin oxidation.We have also tested a bacterial enzyme, CYP102. Dodecanoic acid and its polyhalogenated analogue (perfluorododecanoic acid) both stimulated NADPH oxidation by CYP102, but only the perfluoro analogue stimulated markedly bilirubin oxidation. The analogue exhibited much greater potency than the normal substrate in stimulating NADPH and bilirubin oxidation and also showed greater affinity for CYP102, as measured by the binding constant, Ks. The molar stoichiometry ratio between NADPH and O(2) consumption was 1 in the case of the substrate, but approximated 2 with the perfluoro analogue. We conclude that halogenated substrate analogues can interact with different CYPs to increase production of oxidative species, probably by an uncoupling mechanism. A role of the ferryl-oxygen intermediate is suggested in the oxidation of biologically important molecules, with possible implications for the therapy of jaundice and for toxic oxidative reactions, such as uroporphyria and cancer.     

Induction of liver microsomal cytochrome P-450 And associated monooxygenases by octachlorostyrene in inbred strains of mice: Lack of correlation with the murinE Ah locus

Single intraperitoneal injections of octachlorostyrene (OCS) and hexachlorobenzene in genetically polycyclic aromatic hydrocarbon ‘responsive’ C57/BL/6 (B6) mice led to a time- and dose-dependent increase in the levels of liver microsomal cytochromes P-450 and b₅ as well as in the activities of NADPH cytochrome P-450 (cytochrome c) reductase, ethylmorphine (EM) N-demethylase, 4-nitroanisole (PNA) O-demethylase and acetanilide 4-hydroxylase (AcA hydroxylase). No, or only a very moderate, increase in the activity of aryl hydrocarbon hydroxylase was seen after OCS and HCB, respectively. Pretreatments with phenobarbital (PB) or 3-methylcholanthrene (MC) both increased AcA hydroxylase activity to a similar degree, whereas pretreatment with polychlorinated biphenyls (Aroclor 1254) had an effect equal to the sum of PB and MC. Judged from sodium dodecylsulfate polyacrylamide gel electrophoresis studies, OCS and HCB predominantly increased a microsomal polypeptide of apparent mol. wt 52,000, similar to PB. A reduced response was seen after OCS or HCB treatment of aromatic hydrocarbon ‘non-responsive’ DBA/2 (D2) mice compared to B6 mice, both with respect to AcA hydroxylase as well as EM demethylase and PNA demethylase activities. OCS treatment of B6D2F1 mice resulted in a doubling of AcA hydroxylase activity, but in mice of the (B6D2)D2 backcross no distinct subgroupings of individual AcA hydroxylase activities were apparent. These results demonstrate that OCS is an inducer of the PB-type in mice and that induction of AcA hydroxylase by OCS is not regulated by the Ah locus.     

Activation of P53 in HepG2 cells as surrogate to detect mutagens and promutagens in vitro

The current genotoxicity tests of the standard in vitro battery, especially those using mammalian cells, are limited by their low specificity and highlight the importance of new in vitro tools. This study aimed to evaluate the suitability of HepG2 cells for assaying mutagens and promutagens. We determined P53 activity as surrogate genotoxicity endpoint in HepG2 cells. Our results revealed a significant P53-induction by actinomycin D, methyl methanesulfonate and etoposide. Prior to the investigation of promutagens we characterized HepG2 cells by analyzing the expression of 45 genes involved in xenobiotic metabolism and measuring the activity of selected Cytochrome-P450 (CYP) enzymes. We determined a limited metabolic capacity prompting us to employ a co-treatment with rat liver S9 as metabolic activation system (MAS) for promutagens. While cyclophosphamide showed an elevation of activated P53 in the presence of S9, 7,12-dimethylbenz[a]anthracene and aflatoxin B₁ responded without the MAS. Inhibition of cellular CYP3A4 or CYP1A/1B suppressed the aflatoxin B₁- and dimethylbenz[a]anthracene-mediated P53 response, respectively, indicating that HepG2 cells are capable of metabolizing these compounds in a CYP1A/B/3A4-dependent manner. In summary, our results indicate that P53 activation in HepG2 cells combined with a MAS can be used for the identification of new (pro)genotoxicants.     

The mechanism of cytotoxicity and DNA adduct formation by the anticancer drug ellipticine in human neuroblastoma cells

Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of covalent DNA adducts mediated by cytochromes P450 and peroxidases. Here, the molecular mechanism of DNA-mediated ellipticine action in human neuroblastoma IMR-32, UKF-NB-3 and UKF-NB-4 cancer cell lines was investigated. Treatment of neuroblastoma cells with ellipticine resulted in apoptosis induction, which was verified by the appearance of DNA fragmentation, and in inhibition of cell growth. These effects were associated with formation of two covalent ellipticine-derived DNA adducts, identical to those formed by the cytochrome P450- and peroxidase-mediated ellipticine metabolites, 13-hydroxy- and 12-hydroxyellipticine. The expression of these enzymes at mRNA and protein levels and their ability to generate ellipticine-DNA adducts in neuroblastoma cells were proven, using the real-time polymerase chain reaction, Western blotting analyses and by analyzing ellipticine-DNA adducts in incubations of this drug with neuroblastoma S9 fractions, enzyme cofactors and DNA. The levels of DNA adducts correlated with toxicity of ellipticine to IMR-32 and UKF-NB-4 cells, but not with that to UKF-NB-3 cells. In addition, hypoxic cell culture conditions resulted in a decrease in ellipticine toxicity to IMR-32 and UKF-NB-4 cells and this correlated with lower levels of DNA adducts. Both these cell lines accumulated in S phase, suggesting that ellipticine-DNA adducts interfere with DNA replication. The results demonstrate that among the multiple modes of ellipticine antitumor action, formation of covalent DNA adducts by ellipticine is the predominant mechanism of cytotoxicity to IMR-32 and UKF-NB-4 neuroblastoma cells.     

The relationship between DNA adduct formation by benzo[a]pyrene and expression of its activation enzyme cytochrome P450 1A1 in rat

Benzo[a]pyrene (BaP) is a human carcinogen requiring metabolic activation prior to reaction with DNA. Cytochrome P450 (CYP) 1A1 is the most important hepatic and intestinal enzyme in both BaP activation and detoxification. CYP1A2 is also capable of oxidizing BaP, but to a lesser extent. The induction of CYP1A1/2 by BaP and/or β-naphthoflavone in liver and small intestine of rats was investigated. Both BaP and β-naphthoflavone induced CYP1A expression and increased enzyme activities in both organs. Moreover, the induction of CYP1A enzyme activities resulted in an increase in formation of BaP–DNA adducts detected by ³²P-postlabeling in rat liver and in the distal part of small intestine in vivo. The increases in CYP1A enzyme activity were also associated with bioactivation of BaP and elevated BaP–DNA adduct levels in ex vivo incubations of microsomes of both organs with DNA and BaP. These findings indicate a stimulating effect of both compounds on BaP-induced carcinogenesis.